WO2006066334A1 - Potassium channels in human heart - Google Patents

Abstract

The present invention relates to a method of modulating cardiac rhythm in a human subject. The method includes the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

Description

POTASSIUM CHANNELS IN HUMAN HEART

This application claims priority from Australian Provisional Patent Application No. 2004907242 filed on 22 December 2004 and Australian Provisional Patent Application No. 2005903110 filed on 15 June 2005, and the contents of both of these applications are to be taken as incorporated herein by this reference.

Field of the Invention

The present invention relates to a method of modulating cardiac rhythm in a human subject by modulating the activity of the potassium ion channels TREK-I and/or TREK-2 in the heart of the subject.

The present invention also relates to a method of preventing and/or treating a disease or condition associated with altered cardiac rhythm in a human subject by modulating the activity of the potassium ion channels TREK-I and/or TREK-2 in the heart of the subject.

The present invention further relates to a method of identifying a compound that modulates cardiac rhythm in a human subject by identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the heart of the subject.

Background of the Invention

Cardiac arrhythmia is a disorder of the rate, rhythm or conduction of electrical impulses within the heart. The disorders include premature contractions (extrasystoles) originating in abnormal foci in atria or ventricles, paroxysmal supraventricular tachycardia, atrial flutter, atrial fibrillation, ventricular fibrillation and ventricular tachycardia. Cardiac arrhythmia is also often associated with coronary artery diseases, such as myocardial infarction and atherosclerotic heart disease.

Anti- arrhythmic drugs are commonly divided into four classes according to their electro-physiological mode of action. Class I agents usually have little or no effect on action potential duration and exert local anaesthetic activity directly on the cardiac cell membrane. Class II agents show little or no effect on the action potential and exert their effects through competitive inhibition of beta-adrenergic receptor sites, thereby reducing sympathetic excitation of the heart. Class III agents are characterized by their ability to lengthen the action potential duration, thereby preventing or ameliorating arrhythmias. Class IV agents are those which have an anti-arrhythmic effect due to their actions as calcium antagonists.

Although Class III agents are generally effective as antiarrhythmic drugs, a major problem that often precludes their use is that undue prolongation of the action potential in normal myocardium, particularly in the ventricles, can be pro-arrhythmic. This has limited the use of these drugs and led to the search for ion channel targets that would not produce this effect. Later drugs such as Ibutilide and dofetilide are examples of drugs designed to target particular ion channels (in this case, IKr). At present, there appear to be few further candidate target ion channels in the heart on which the development of new drugs can be based.

Two pore (2-Pore-4-Transmembrane domain; 2P4TM) potassium channels are a family of recently cloned potassium channels that are characterised by having four transmembrane domains and two pore forming regions in the same subunit. The proteins dimerise to form a potassium pore, in contrast to the voltage dependent and inward rectifier families of potassium channels, which form tetramers. All the two pore potassium channels have the signature sequence of potassium channels, although the hallmark of the 2P4TM channels is that they have two pore sequences on a single subunit (typically being -T-I-G-Y-G- and -T-V-G-F-G-). Since they were discovered, two-pore potassium channels have been of particular interest based on their distinctive physiological and pharmacological properties. Two-pore potassium channels have been cloned in mouse, rat and human.

Members of the two-pore family of potassium channels have a variety of properties, ranging from acid-sensitivity to sensitivity to anaesthetics. TREK-I (Twik-RElated K⁺ channel - 1), TREK-2 (Twik-RElated K⁺ channel - 2) and TRAAK (Twik-RElated Arachidonic Acid-stimulated K⁺ channel) are members of the two-pore family that are activated by a variety of conditions, including membrane stretch, pH, cell swelling, shear stress or negative pressure.

TREK-I, TREK-2 and TRAAK produce outwardly rectifying current in physiological K⁺ gradients, with TREK-I still exhibiting an outward rectification for strong hyperpolarizations. TREK-I, TREK-2 and TRAAK are also activated by unsaturated fatty acids such as arachidonic acid, linoleate, oleate, whereas they are inhibited by saturated fatty acids such as arachidate, stearate, and palmitate.

While no splice variants of TREK-I have yet been described, 3 splice variants have been identified for TREK-2: Variant A (Va; TREK-2a), Variant B (Vb; TREK-2b) and Variant C (Vc; TREK- 2c). These splice variants arise because of alternative splicing in the first exon.

The tissue distribution of two-pore potassium channels is widespread. Previous studies in humans have shown that TREK-I is highly expressed in brain, ovary and small intestine, and is less expressed in kidney, testis, prostate and skeletal muscle. TREK-2 is highly expressed in human brain, pancreas and kidney, and is less expressed in testis, colon and small intestine.

Although these previous studies have demonstrated that TREK-I and TREK-2 are expressed in a number of human tissues, no expression of TREK-I and TREK-2 has been detected in human heart tissue. Other studies have also been conducted to examine the expression of the TREK-2b and TREK-2c splice forms in various human tissues, including the human heart. However, these studies also showed that expression of at least the TREK- 2b and TREK-2c mRNAs is not detectable in human heart.

Despite the absence of detectable expression of TREK-I and TREK-2 in human heart, expression of TREK-I and TREK-2 has been demonstrated in rat heart. These findings have led to the conclusion that TREK-I and TREK-2 do not play a role in human cardiac function, and as such these ion channels have not been the subject of studies to determine their role in human cardiac function. The present invention arises from the unexpected finding that TREK-I and TREK-2 are indeed expressed in human heart tissue. Further, TREK-2 splice variants A and C are only expressed in the human heart. Given that TREK-I and TREK-2 are expressed in human heart, this indicates that these ion channels are potential targets for the treatment and/or prevention of diseases and conditions in humans associated with altered cardiac rhythm or altered cardiac action potential.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

Summary of the Invention

The present invention provides a method of modulating cardiac rhythm in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

The present invention also provides a method of identifying a compound that modulates cardiac rhythm in a human subject, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the heart of a human subject.

The present invention also provides a TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates cardiac rhythm in a human subject.

The present invention also provides a method of preventing and/or treating a disease or condition associated with altered cardiac rhythm in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject. The present invention also provides a method of reducing the risk of a human subject suffering from a disease or condition associated with altered cardiac rhythm, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

The present invention also provides a method of identifying a human subject suffering from, or susceptible to, a disease or condition associated with altered cardiac rhythm, the method including the step of identifying an altered activity of TREK-I and/or TREK-2 in the subject.

The present invention also provides a non-human cardiac cell, the cell expressing human TREK-I and/or TREK-2, or a variant or fragment thereof.

The present invention also provides a non-human animal including cardiac cells expressing human TREK-I and/or TREK-2, or a variant or fragment.

The present invention also provides a method of modulating K⁺ current activity in a human cardiac cell, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the cell.

The present invention also provides a method of identifying a compound that modulates K⁺ current activity in a human cardiac cell, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the cell.

The present invention also provides a TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates K⁺ current activity in a human cardiac cell.

The present invention also provides a method of modulating cardiac K⁺ current activity in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of a human subject. The present invention also provides a method of identifying a compound that modulates cardiac K⁺ current activity in a human subject, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the heart of a human subject.

The present invention also provides a TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates cardiac K⁺ current activity in a human subject.

The present invention also provides a method of preventing and/or treating a disease or condition associated with altered cardiac K⁺ current activity in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

The present invention also provides a method of reducing the risk of a human subject suffering from a disease or condition associated with altered cardiac K⁺ current activity in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

The present invention also provides a method of identifying a human subject suffering from, or susceptible to, a disease or condition associated with altered cardiac K⁺ current activity, the method including the step of identifying an altered activity of TREK-I and/or TREK-2 in the subject.

The present invention also provides a method of modulating cardiac action potential in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

The present invention also provides a method of identifying a compound that modulates cardiac action potential in a human subject, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the heart of a human subject. The present invention also provides a TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates cardiac action potential in a human subject.

The present invention also provides a method of preventing and/or treating a disease or condition associated with altered cardiac action potential in a human subject, the method including the step of modulating the activity of TREK-2 in the heart of the subject.

The present invention also provides a method of reducing the risk of a human subject suffering from a disease or condition associated with altered cardiac action potential in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

The present invention also provides a method of identifying a human subject suffering from, or susceptible to, a disease or condition associated with altered cardiac action potential, the method including the step of identifying an altered activity of TREK-I and/or TREK-2 in the subject.

The present invention also provides a method of modulating the action potential of a human cardiac cell, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the cell.

The present invention also provides a method of identifying a compound that modulates the action potential of a human cardiac cell, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the cell.

The present invention also provides a TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates cardiac action potential in a human cardiac cell. The present invention arises from the finding that both the K⁺ channels TREK-I and TREK-2 are expressed in the human heart, contrary to previous experimental findings that reported that these ion channels are not expressed in the human heart. In particular, it has been demonstrated in the present study that TREK-I is expressed in both ventricle and atrial tissue. TREK-I is expressed in the myocytes and is found in the cell membrane. It has also been demonstrated in the present study that TREK-2 is expressed in human heart, and while TREK-2a and TREK- 2c mRNAs are expressed in both human ventricle and atrial tissue, TREK-2b mRNA is not expressed either in human atrium or ventricle. TREK-2 is also expressed in the myocytes and is also found in the cell membrane.

The existence of TREK-I and TREK-2 in human heart expands the understanding of human cardiac electrophysiology, in particular on the mechanisms of arrhythmias generated by abnormal mechanical stresses on the myocardium, or the physiological modulation of myocardial electrophysiology by ischemia and hormonal influences.

The expression of TREK-I and TREK-2 in the human heart further indicates that either or both of these ion channels may be used as a target for therapeutic applications involving diseases or conditions associated with altered cardiac rhythm, or for therapeutic applications involving diseases or conditions associated with altered cardiac K⁺ current activity or altered cardiac action potential.

Various terms that will be used throughout the specification have meanings that will be well understood by a skilled addressee. However, for ease of reference, some of these terms will now be defined.

The phrase "modulating the activity" or variants thereof as used throughout the specification in relation to the ion channels of the present invention is to be understood to include an alteration in the activity of the ion channels accomplished by any suitable means, including altering the function and/or expression of the ion channels. An alteration in function may be accomplished, for example, by contacting the ion channel with another molecule (eg a drug) or by modifying the ion-channel itself (eg by modifying the amino acid sequence of the ion channel). An alteration in expression may be accomplished, for example, by genetically altering the cell so as to alter the expression of an endogenous nucleic acid, genetically altering the cell to express an exogenous nucleic acid, exposing the mRNA for the ion channel to an agent such as an antisense RNA or a siRNA, and altering the stability of the mRNA and/or the protein.

The term "nucleic acid" as used throughout the specification is to be understood to mean to any oligonucleotide or polynucleotide. The nucleic acid may be DNA or RNA and may be single stranded or double stranded. The nucleic acid may be any type of nucleic acid, including a nucleic acid of genomic origin, cDNA origin (ie derived from a mRNA), derived from a virus, or of synthetic origin.

The term "polypeptide" as used throughout the specification is to be understood to mean two or more amino acids joined by peptide bonds. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring, recombinant, mutated or synthetic polypeptides.

The term "variant" as used throughout the specification is to be understood to mean an amino acid sequence of a polypeptide or protein that is altered by one or more amino acids.

A variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties to the replaced amino acid (e.g., replacement of leucine with isoleucine). A variant may also have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan) or a deletion and/or insertion of one or more amino acids.

A variant may also be a biologically active fragment of the full size protein, or be a polypeptide or protein having similar structural, regulatory, or biochemical functions as that of the full size polypeptide or protein.

In this regard, a biologically active fragment may be an amino or carboxy terminal deletion of a protein, an internal deletion of a protein, or any combination of such deletions. For example, a suitable amino terminal truncation is a truncation of the first 54 amino acid residues of TREK-I. A biologically active fragment will also include any such deletions fused to one or more additional amino acids.

The term "amplification" or variants thereof as used throughout the specification is to be understood to mean the production of additional copies of a nucleic acid sequence. For example, amplification may be achieved using polymerase chain reaction (PCR) technologies, essentially as described in Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.

The term "antibody" as used throughout the specification is to be understood to mean monoclonal or polyclonal antibodies, and fragments of antibody molecules, such as Fab, F(ab')₂, and Fv, which are capable of binding an epitopic determinant.

Brief Description of the Figures

Figure 1 shows cardiac action potential (A) and the schematic representation of the major ionic currents (B) contributing to its waveform. The amplitudes of the depolarizing (downward) and repolarizing (upward) currents are not on the same scales. In this figure it should be noted that the current expected to be carried through the TREK-I or TREK-2 ion channels is not depicted.

Figure 2 shows the structure of the human TREK-I gene (Genebank Number AF171068) located at chromosome Iq41; the full mRNA transcript contains 1252bp; the coding area is from bp 15 to 1250, which encodes 411 amino acids. Exons (El to E7) and introns of TREK-I are shown in the diagram. The gene consists of 7 exons and 6 introns, with a total length of approximately 493 Kbp.

Figure 3 shows PCR results for TREK-I expression in human atrial tissue. Panel A shows 1% agarose gel with DNA ladder on the left and a PCR product at 697 bp on the right. Panel B shows nested PCR results for TREK-I: 1% agarose gel, DNA ladder at left, PCR product at right with estimated size 372 bp. Figure 4 shows PCR results for TRAAK expression in human atrial tissue. Panel A shows 1% agarose gel with DNA ladder on the left and PCR product at 477 bp on the right. Panel B shows nested PCR results for TRAAK: 1% agarose gel, DNA ladder at left, PCR product at right with estimated size 250 bp.

Figure 5 shows reaction versus cycle of real time PCR results for amplification of TREK-I and TRAAK in human atrial and testicular tissue, compared to GAPDH as internal standard.

Figure 6 shows dissociation curve analysis for real time PCR experiments, confirming no primer-dimer formation.

Figure 7 shows the expression level of TREK-I and TRAAK in human atrial tissue and testicular tissue, quantified as a proportion of GAPDH expression as an internal standard. 7 human atrial samples (5 females and 2 males) were used in real-time PCR, all atrial samples had been stored at -80° C for at least 12 months. Human testicular tissue (from one patient) was used 3 hours after surgery. Error bars on atrial results show ± sem.

Figure 8 shows results of gene expression studies in fresh human atrial tissue (3 hours after surgery, 3 females) and testicular tissues by real-time PCR. Error bars on atrial results show ± sem.

Figure 9 shows results of gene expression studies in frozen human ventricular tissue (stored at -80 °C for 12 months) and testicular tissues by real-time PCR. Error bars on atrial results show ± sem. 5 tissue samples are used (3 male, 2 female). Testicle tissues were fresh (3 hours after surgery). Error bars on atrial results show ± sem.

Figure 10 shows effects of storage on expression level of TREK-I and TRAAK in human testicle tissue. A sample of fresh testicular tissue was divided into two equal- weight parts. One part was used in real-time PCR immediately, whereas the other part was stored at -80 °C for 3 months. Figure 11 shows PCR results for TREK-2a, TREK-2b and TREK-2c expression in three samples of human atrial tissue. The panels show 1% agarose gels with DNA ladder on the left and PCR products indicated.

Figure 12 shows nested PCR results for TREK-2a and TREK- 2c expression in human atrial tissue. Panel A panel shows the results of nested PCR for TREK-2a. Panel B shows the results of nested PCR for TREK- 2c.

Figure 13 shows Western analysis of human atrial tissue (six samples) using an anti- TREK-2 antibody.

Figure 14 shows PCR results for TREK-2a expression in human ventricle tissue. The panel shows a 1% agarose gel with DNA ladder on the left and PCR product at 647 bp on the right.

Figure 15 shows nested PCR results for TREK-2a expression in human ventricle tissue. The panel shows a 1% agarose gel with DNA ladder on the left and nested PCR products on the right.

Figure 16 shows PCR results for TREK- 2b and TREK-2c expression in human ventricle tissue. The panel shows a 1% agarose gel with DNA ladder on the left. No PCR product of approximately 946 bp was detected for amplification of TREK-2b (middle lane). A PCR product at approximately 952 bp was detected for amplification of TREK-2c (right lane).

Figure 17 shows nested PCR results for TREK-2c. A 1% agarose gel is shown with DNA ladder at left. Nested PCR of TREK- 2c with first set of primers gives a band of approximately 952 bp (middle lane), and nested PCR of TREK- 2c with second set of primers gives a product at right with estimated size 451 bp (right lane).

Figure 18 shows Western Blot of TREK-2a and 2c in human atrial tissue. 6 human atrial samples from patients undegoing coronary artery bypass were used for Western Blot experiments. Va (59KDa) and Vc (61KDa) could be readily detected in all the samples. Supernatant concentration of each sample was 25μg/30μL. [primary Ab] =1:500; [secondary Ab] =1:1000

Figure 19 shows Western Blot of TREK-2a and 2c in human ventricular tissue. Panel A showed the expression of Va and Vc in 4 human ventricular samples from donor heart with sample concentrations of 20μg/30μL. Panel B demonstrates that only Vc was found in 6 human ventricular samples under the sample concentration of 6μg/20μL. [primary Ab] =1:500; [secondary Ab] =1:1000.

Figure 20 shows Western Blot of TREK-2a and 2c in human disease ventricular tissue. Western blot result of variant A and variant C in 2 ventricular samples of ischemic cardiomyopathy (sample 1&2) and 5 ventricular samples of idiopathic dilated cardiomyopathy (samples 3 to 7) with identical sample concentrations of 20μg/30μL. [primary Ab] =1:500; [secondary Ab] =1:1000.

Figure 21 shows expression levels of Va and Vc in normal (panel A) and pathological human ventricular samples (panel B) as determined by Western Blot analysis.

Figure 22 shows immunohistochemistry of TREK-2a and TREK-2c in human atrial and ventricular tissues. Panel A: TREK-2 immunoreactivity in paraffin section of human atrial appendage. Within the muscle bundle the cardiac myocytes are different in diameter and some of them show an empty area without myofibrils in the nuclear region

(filled arrows). Punctate fine granules of TREK-2 immunoreactivity were located in most of cardiac myocytes. Some of TREK-2 positive granules were located along the membrane of the cardiac myocytes (hollow arrow). Connective tissue between the muscle bundles (arrow head) and centre of nuclear region in cardiac myocyte (filled arrows) were not labelled. No immunoreactivity was found in negative control preparation (Panel B). Calibration bar: 50 μm. Panel C: TREK-2 immunoreactivity in paraffin section of human cardiac ventricle. TREK-2 immunoreactivity was located in most of cardiac myocytes and their locations were similar to that in atrial myocytes, along the membrane (hollow arrow) and in the cytoplasm. Connective tissues between the muscle bundles (arrow head) were not immunoreactive. In an expanded view (Panel D) the immunoreactivity appeared to be spaced in striations. No immunoreactivity was found in negative control preparation (Panel E). Calibration bar: 50 μm.

Figure 23 shows expression level of TREK- 1/G APDH in human Ischemia Cardiomyopathy, Idopathic Dilated Cardiomyopathy and valvular diseases samples based on Table 6.

Figure 24 shows the level of TREK- 1/G APDH in human normal and diseased heart based on Table 7.

Figure 25 shows that TREK-I and GAPDH (positive control) were recognized by specific antibodies in human donor ventricle samples (n=4). [Protein supernatant]= 20μg/30μL; [primary Ab]= 1:1000; [secondary Ab]= 1:500.

Figure 26 shows that TREK-I was expressed in human IDC samples by Western blot analysis (n=7). GAPDH was used as a positive control. [Protein supernatant]= 20μg/30μL; [primary Ab]= 1:1000; [secondary Ab]= 1:500.

Figure 27 shows the expression level of TREK- 1/G APDH in IDC and donor samples based on Table 8 and 9.

Figure 28 shows immunohistochemistry of TREK-I in human atrial and ventricular tissues. A. Labeled TREK-I channels in longitudinal section of cardiac myocytes in atrial appendage. TREK-I immunoreactivity was observed as bright punctate granules in most of cardiac myocytes. Many of them were found along the membrane of the cardiac myocytes (unfilled arrows) and some in the cytoplasm. Connective tissue between the muscle bundles (arrow heads) and the center of nuclear region in cardiac myocytes (filled arrows) were not labeled. No immunoreactivity was found in negative control preparation after omission of primary antibody (B). C: Labeled TREK-I channels in cross-section of cardiac myocytes in ventricular tissue. Similar distribution of TREK-I immunoreactivity in atrial tissue was observed in ventricular tissue. Labelled TREK-I channels can also be found along membrane of cardiac myocytes and in the cytoplasm. No connective tissue and centre of nuclear region of cardiac myocytes were labelled. No positive labelling was observed in negative control preparation (D). Calibration bar: 25 μm for A and B and 50 μm for C and D.

Figure 29 shows immunohistochemistry of TREK-I expression in normal and diseased mycocytes of the human ventricle. Calibration bar: 50 μm.

General Description of the Invention

As mentioned above, in one form the present invention provides a method of modulating cardiac rhythm in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

This form of the present invention allows the cardiac rhythm in a human subject to be altered by an agent and/or treatment that modulates the activity of the TREK-I and/or TREK-2 potassium channels.

As discussed above, expression of TREK-I and TREK-2 has previously been shown in a number of human tissues (by reverse transcription-PCR analysis). However, despite the use of this sensitive technique, no conclusive expression of either of these genes has been detected in the human heart. This finding is contrary to the fact that these genes are expressed in rat heart. Taken together, these findings have led to the widespread belief that TREK-I and TREK-2 do not play a role in human cardiac physiology.

Contrary to expectation, the present study demonstrates that TREK-I and TREK-2 are indeed expressed in human heart. This indicates that these ion channels are potential targets for the treatment and/or prevention of diseases and conditions in humans associated with altered cardiac rhythm or altered cardiac action potential, and that modulation of the activity of these ion channels may lead to a modulation of cardiac rhythm in the human. This finding also indicates that TREK-I and/or TREK-2 may be used as targets to identify new compounds that may have therapeutic application. In addition, it has been found in the present study that the splice variants of TREK-2 are differentially expressed in the human heart, with expression of only the TREK-2a (Va) and TREK-2c (Vc) mRNAs being detected.

Thus, the ability to modulate the activity of TREK-I and/or TREK-2 in cardiac cells is likely to lead to an ability to modulate the ionic currents that contribute to the cardiac action potential. This has implications in the treatment and prevention of arrhythmias, in the control of ventricular rate in the presence of atrial flutter or fibrillation, and in the control of arrythmias arising from conditions which produce abnormal motion of the ventricular wall, such as cardiac ischemia and infarction.

In humans, TREK-I is located at chromosome Iq41. The genomic structure of the human gene is shown in Figure 2, consisting of 7 exons and six introns and giving a full mRNA of 1252 bp in length. The nucleotide and amino acid sequences are provided in Genbank Accession number AF171068. The coding region spans bp 15 to 1250, encoding a protein of 411 amino acids.

The genomic organisation of human TREK-2 consists of seven exons and six introns. The nucleotide and amino acid sequences are provided in GenBank Accession number AF279890.

It appears that three different mRNAs may be produced from the TREK-2 gene, presumably due to alterations in splicing, resulting in the production of protein with differing amino termini. The nucleotide sequence of TREK-2a (Va) is provided in GenBank Accession No. NM_021161, and encodes a protein of 538 amino acids. The nucleotide sequence of TREK-2b (Vb) is provided in GenBank Accession No. NM_138317 and encodes a protein of 543 amino acids. The nucleotide sequence of TREK-2c (Vc) is provided in GenBank Accession No. NM_138318, and encodes a protein of 543 amino acids. Preferably, the modulation of the activity of TREK-I and/or TREK-2 in the heart of the subject in this form of the present invention is decreased. Thus, inhibition of the activity of these ion channels in cardiac cells may be useful for treating diseases or conditions associated with altered cardiac rhythm.

Preferably, the decrease in activity of TREK-I and/or TREK-2 results in a reduction in outward potassium currents in cardiac cells of the subject and/or prolongs action potential duration in cardiac cells in the heart of the subject.

Preferably, the modulation of the activity of TREK-I and/or TREK-2 includes modulation of the activity of these ion channels in the ventricle of the heart of the subject.

Preferably, the activity of the TREK-I and/or TREK-2 ion channels in the various forms of the present invention is modulated in one or more cells in the heart of a human subject.

Preferably, the method of this form of the present invention is used to prevent and/or treat (ameliorate) an arrhythmia. Most preferably, the method is used to prevent and/or treat supraventricular tachycardia, including atrial flutter and atrial fibrillation, ventricular tachycardia and ventricular fibrillation.

The method of this form of the present invention is also useful in the control of ventricular rate in the presence of atrial flutter or fibrillation, and in the control of arrhythmias arising from conditions which produce abnormal motion of the ventricular wall, such as cardiac ischemia and infarction.

Accordingly, in another form the present invention provides a method of modulating ventricular rate in the presence of atrial flutter or fibrillation in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject. In a further form, the present invention provides a method of controlling an arrhythmia arising from abnormal motion of the ventricular wall in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

Preferably, the human subject is a subject susceptible to, or suffering from, a disease or condition associated with altered cardiac rhythm.

Examples of diseases or conditions associated with altered cardiac rhythm include disorders of the rate, rhythm or conduction of electrical impulses within the heart. The disorders include premature contractions (extrasystoles) originating in abnormal foci in atria or ventricles, paroxysmal supraventricular tachycardia, atrial flutter, atrial fibrillation, ventricular fibrillation and ventricular tachycardia, and arrhythmias arising from conditions which produce abnormal motion of the ventricular wall, such as cardiac ischemia and infarction. Cardiac arrhythmia is also often associated with coronary artery diseases, such as myocardial infarction and atherosclerotic heart disease.

The subject in the various forms of the present invention may also be suffering from, or susceptible to, cardiac hypertrophy (either concentric or dilated hypertrophy), cardiac myopathy, and cardiac ischemia, or be suffering from, or susceptible to, an arrhythmia arising from conditions which produce abnormal motion of the ventricular wall, such as cardiac ischemia and infarction.

In this regard, the present invention is also suitable for reducing the risk of a human subject suffering from a disease or condition associated with altered cardiac rhythm, by modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

Accordingly, in another form the present invention provides a method of reducing the risk of a human subject suffering from a disease or condition associated with altered cardiac rhythm, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject. In this case, the subject may also be predisposed to suffering from a disease or condition associated with altered cardiac rhythm, indicating an increased probability that a subject will suffer from a disease or condition associated with altered cardiac rhythm, as compared to the probability that another subject with similar other risk factors may suffer from a disease or condition associated with altered cardiac rhythm.

As discussed above, modulating the activity of TREK-I and/or TREK-2 may also be used to prevent, treat or ameliorate a disease or condition associated with altered cardiac rhythm in a human subject.

Accordingly, in another form the present invention provides a method of preventing and/or treating a disease or condition associated with altered cardiac rhythm in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

Preferably, the modulation of the activity TREK-I and/or TREK-2 includes administering to the human subject an effective amount of an agent that modulates the activity of TREK-I and/or TREK-2 in the heart of the subject.

The method of this form of the present invention may also be useful in the treatment and/or prevention of cardiac hypertrophy (either concentric or dilated hypertrophy), cardiac myopathy, and cardiac ischemia.

Accordingly, in another form the present invention provides a method of preventing and/or treating cardiac hypertrophy (including concentric or dilated hypertrophy), cardiac myopathy, and cardiac ischemia in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

In this regard, one of the primary events in cardiac ischemia is the release of phospholipids from the cell membrane by activation of phospholipases. TREK-I and TREK-2 are opened by arachidonic acid and by polyunsaturated fatty acids and as such these ion channels are likely to have a protective role in ischemia, since its activation would shorten the action potential in a way similar to activation of K_ATP channels. TREK-I is also activated by intracellular acidosis: lowering intracellular pH shifts the pressure / activation relationship so that, at lower pH levels, TREK-I is converted into a background channel. In ischemia, this effect would be synergistic with the activation due to polyunsaturated fatty acids acting on the membrane of cardiomyocytes.

In the case of this form of the present invention, preferably the activity of TREK-I and/or TREK-2 in the heart of the subject is increased.

In the case of TREK-2, preferably the modulation of TREK-2 activity includes modulation of the activity of TREK-2a and/or TREK-2c.

The step of modulating the activity of TREK-I and/or TREK-2 in the various forms of the present invention may be performed by exposing the subject to an effective amount of an agent, a combination of agents, and/or a treatment that results in an alteration of the activity of any one or more forms of the TREK-I and TREK-2 ion channels, including altering the activity of any one or more of splice variants of TREK2, such as TREK-2a, TREK- 2b and TREK-2c. A change in the activity of the ion channel may be accomplished, for example, by a change in the activity of the ion channel per se, by a change in the level of the expression of the ion channel, or by a change in the pattern of expression or pattern of activity of the ion channel.

Preferably, the activity of the ion channels in the various forms of the present invention is modulated in myocytes in the heart of the subject.

Confirmation that the cardiac rhythm has been modulated in the various forms of the present invention may be determined by a suitable method known in the art. For example, confirmation that the cardiac rhythm has been changed may be done by recording an electrocardiogram of the subject, recording of the cardiac action potential with intracardiac catheters, or recording the cardiac action potential with implanted electrical wires such as those attached to a pacemaker. All these methods will reveal electrophysiological changes produced as a consequence of modulation of TREK-I and/or TREK-2 activity. In this regard, it will be appreciated that a modulation in the activity of the TREK-I and/or TREK-2 ion channels in the various forms of the present invention includes any inhibition or augmentation in the activity of the ion channels, a change in the properties of the ion channels (for example an alteration in the current activity), a change in how the ion channels respond to a condition (for example, membrane stretch, cell swelling, shear stress or negative pressure), a change in the interaction of the ion channels with another molecule(s), or any other change that results in a physical change to the cell, a biochemical change to the cell, or a change in expression of one or more molecules within the cell.

For example, even though the TREK- 2b (Vb) form appears to be not expressed in the human heart, an increase in the activity of TREK-2 in the heart of a subject may be accomplished by expressing the TREK-2b channel in either or both of cells of the atrium or ventricle.

Preferably, the modulation of the activity TREK-I and/or TREK-2 in the various forms of the present invention includes administering to the human subject an effective amount of an agent that modulates the activity of TREK-I and/or TREK-2 in the heart of the subject.

Examples of agents that may modulate the activity of the ion channels in the various forms of the present invention include drugs, small molecules, nucleic acids, oligonucleotides, polypeptides, peptides, proteins, enzymes, polysaccharides, glycoproteins, hormones, receptors, ligands for receptors, co-factors, antisense oligonucleotides, ribozymes, small interfering RNAs, lipids, antibodies or a part thereof, aptamers, or viruses.

The agent in the various forms of the present invention may be administered to the subject in a suitable form to effect modulation of the activity of any one or more of the forms of the TREK-I and/or TREK-2 ion channels. The effective amount of agent to be administered is not particularly limited, so long as it is within such an amount and in such a form that generally exhibits a pharmacologically useful or therapeutic effect. In this regard, an effective amount of the agent may be appropriately chosen, depending upon, for example, the type and extent of cardiac rhythm to be altered, the age and body weight of the subject, the frequency of administration, and the presence of other active agents.

The administration of the agent as a pharmaceutical composition may be within any time suitable to produce the desired effect of modulating the activity of TREK-I and/or TREK-2. The agent may be administered orally, parenterally, topically or by any other suitable means, and therefore transit time of the agent must be taken into account.

The administration of the agent in the various forms of the present invention may also include the use of one or more pharmaceutically acceptable additives known in the art, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, taking into consideration the particular physical and chemical characteristics of the agent to be administered.

For example, the agent can be prepared into a variety of pharmaceutical compositions in the form of, e.g., an aqueous solution, an oily preparation, a fatty emulsion, an emulsion, a gel, etc., and these preparations can be administered as intramuscular or subcutaneous injection or as injection to an organ (including the heart), or as an embedded preparation or as a transmucosal preparation through nasal cavity, rectum, uterus, vagina, lung, etc. The composition may be administered in the form of oral preparations (for example solid preparations such as tablets, capsules, granules or powders; liquid preparations such as syrup, emulsions or suspensions). Compositions containing the agent may also contain a preservative, stabiliser, dispersing agent, pH controller or isotonic agent. Examples of suitable preservatives are glycerin, propylene glycol, phenol or benzyl alcohol. Examples of suitable stabilisers are dextran, gelatin, a-tocopherol acetate or alpha-thioglycerin. Examples of suitable dispersing agents include polyoxyethylene (20), sorbitan mono-oleate (Tween 80), sorbitan sesquioleate (Span 30), polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68) or polyoxyethylene hydrogenated castor oil 60. Examples of suitable pH controllers include hydrochloric acid, sodium hydroxide and the like. Examples of suitable isotonic agents are glucose, D-sorbitol or D-mannitol. The administration of the agent in the various forms of the present invention may also be in the form of a composition containing a pharmaceutically acceptable carrier, diluent, excipient, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavorant or sweetener, taking into account the physical and chemical properties of the agent being administered.

For these purposes, the composition may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically- acceptable carriers, or by any other convenient dosage form. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

When administered parenterally, the composition will normally be in a unit dosage, sterile injectable form (solution, suspension or emulsion) which is preferably isotonic with the blood of the recipient with a pharmaceutically acceptable carrier. Examples of such sterile injectable forms are sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable forms may also be sterile injectable solutions or suspensions in non-toxic parenterally- acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, saline, Ringer's solution, dextrose solution, isotonic sodium chloride solution, and Hanks' solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending mediums. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides, corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyl oleate, isopropyl myristate, and oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated versions, are useful in the preparation of injectables. These oil solutions or suspensions may also contain long- chain alcohol diluents or dispersants. The carrier may contain minor amounts of additives, such as substances that enhance solubility, isotonicity, and chemical stability, for example anti- oxidants, buffers and preservatives.

When administered orally, the agent will usually be formulated into unit dosage forms such as tablets, cachets, powder, granules, beads, chewable lozenges, capsules, liquids, aqueous suspensions or solutions, or similar dosage forms, using conventional equipment and techniques known in the art. Such formulations typically include a solid, semisolid, or liquid carrier. Exemplary carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and the like.

A tablet may be made by compressing or molding the agent optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered active ingredient and a suitable carrier moistened with an inert liquid diluent.

The administration of the agent in the various forms of the present invention may also utilize controlled release technology. The agent may also be administered as a sustained-release pharmaceutical. To further increase the sustained release effect, the agent may be formulated with additional components such as vegetable oil (for example soybean oil, sesame oil, camellia oil, castor oil, peanut oil, rape seed oil); middle fatty acid triglycerides; fatty acid esters such as ethyl oleate; polysiloxane derivatives; alternatively, water-soluble high molecular weight compounds such as hyaluronic acid or salts thereof (weight average molecular weight: ca. 80,000 to 2,000,000), carboxymethylcellulose sodium (weight average molecular weight: ca. 20,000 to 400,000), hydroxypropylcellulose (viscosity in 2% aqueous solution: 3 to 4,000 cps), atherocollagen (weight average molecular weight: ca. 300,000), polyethylene glycol (weight average molecular weight: ca. 400 to 20,000), polyethylene oxide (weight average molecular weight: ca. 100,000 to 9,000,000), hydroxypropylmethylcellulose (viscosity in 1% aqueous solution: 4 to 100,000 cSt), methylcellulose (viscosity in 2% aqueous solution: 15 to 8,000 cSt), polyvinyl alcohol (viscosity: 2 to 100 cSt), polyvinylpyrrolidone (weight average molecular weight: 25,000 to 1,200,000).

Alternatively, the agent may be incorporated into a hydrophobic polymer matrix for controlled release over a period of days. The agent may then be molded into a solid implant, or externally applied patch, suitable for providing efficacious concentrations of the agent over a prolonged period of time without the need for frequent re-dosing. Such controlled release films are well known to the art. Other examples of polymers commonly employed for this purpose that may be used include nondegradable ethylene- vinyl acetate copolymer a degradable lactic acid-glycolic acid copolymers which may be used externally or internally. Certain hydrogels such as poly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful, but for shorter release cycles than the other polymer release systems, such as those mentioned above.

The carrier may also be a solid biodegradable polymer or mixture of biodegradable polymers with appropriate time release characteristics and release kinetics. The agent may then be molded into a solid implant suitable for providing efficacious concentrations of the agent over a prolonged period of time without the need for frequent re-dosing. The agent can be incorporated into the biodegradable polymer or polymer mixture in any suitable manner known to one of ordinary skill in the art and may form a homogeneous matrix with the biodegradable polymer, or may be encapsulated in some way within the polymer, or may be molded into a solid implant.

It should also be appreciated that other methods of delivery of an agent to modulate the activity of TREK-I and/or TREK-2 are contemplated. For example, the agent may be delivered by way of a nucleic acid or vector that allows for expression of the agent in the appropriate target cells. For example, the agent may be delivered by way of a viral vector that causes expression of the agent in heart cells. Confirmation that an agent has the ability to modulate TREK-I and/or TREK-2 in the relevant forms of the present invention may be by, for example, use of cell based assay using TREK-I and/or TREK-2 transfected cells, such as COS cells, essentially as described in Lesage et al. (2000) J. Biol. Chem. 275(37): 28398-28405 or in Besana et al (2004) J. Biol. Chem. 279(32):33154-33160. In this case, transfected cells will display non-inactivating currents that are not present in control cells, and therefore the effect of agents on the activity of transfected cells can be determined.

In this case, methods for introducing nucleic acids expressing the ion channel into cells are known in the art. Preferably, the cells are transfected with nucleic acids encoding TREK-I, TREK-2a or TREK-2c ion channels, or a variant or functional fragment of these ion channels.

The identification that TREK-I and TREK-2 are expressed in the human heart also indicates that this ion channel may be used as a target for the identification of compounds that may have important therapeutic applications, as discussed previously.

Therefore, the present invention provides methods for identifying compounds that modulate cardiac rhythm, K⁺ current activity and action potential. The present invention further provides compounds identified by these methods, and therapeutic compositions including compounds identified by these methods.

Accordingly, in another form the present invention provides a method of identifying a compound that modulates cardiac rhythm in a human subject, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the heart of a human subject.

This form of the present invention provides a method of identifying a compound that modulates cardiac rhythm in a human subject. This screening procedure makes it possible to identify drugs that may be useful, for example, in the treatment and/or prevention of diseases or conditions associated with altered cardiac rhythm. Preferably, the method of identification involves identifying a compound that decreases the activity of TREK-I and/or TREK-2 in the heart of the subject.

The method of identification may further include the step of identifying the compound as a compound that reduces outward potassium currents in cardiac cells of the subject and/or prolongs action potential duration in the heart of the subject.

Preferably, the method of identification includes modulation of the activity of TREK-I and/or TREK-2 in cardiac myocytes.

Preferably, the method of identification includes the modulation of activity of TREK-I and/or TREK-2 in the ventricle of the heart of the subject.

In the case of TREK-2, preferably the modulation of TREK-2 activity includes modulation of the activity of TREK-2a and/or TREK- 2c.

The screening procedure also makes it possible to identify drugs that may be useful in controlling ventricular rate in the presence of atrial flutter or fibrillation, drugs useful in the control of arrhythmias arising from conditions which produce abnormal motion of the ventricular walls, such as cardiac ischemia and infarction, and drugs that may be useful in the treatment and/or prevention of cardiac hypertrophy, including concentric or dilated hypertrophy, cardiac myopathy and cardiac ischemia.

The present invention also provides a compound identified by this form of the present invention, and compositions including the compound for use in the various diseases and conditions of the present invention.

Accordingly, in another form the present invention provides a composition including a compound that modulates the activity of TREK-I and/or TREK-2 in the heart of a human subject. The formulation of such compositions is as previously hereinbefore described. Preferably, this method of the present invention includes the following steps: (a) identifying a compound that modulates the activity of TREK-I and/or TREK-2; (b) determining the ability of the compound so identified to modulate cardiac rhythm in a human subject; and (c) identifying the compound as a compound that modulates cardiac rhythm in a human subject.

Accordingly, in another form the present invention provides a method of identifying a compound that modulates cardiac rhythm in a human subject, the method including the steps of: (a) identifying a compound that modulates the activity of TREK-I and/or

TREK-2;

(b) determining the ability of the compound so identified to modulate cardiac rhythm in a human subject; and

(c) identifying the compound as a compound that modulates cardiac rhythm in a human subject.

The identification of compounds that modulate the activity of TREK-I and/or TREK-2 may be accomplished by a suitable cell-free or cell-based method. One method is to use expression of the channel in a heterologous system (eg COS, HEK cells) and patch clamp techniques to detect changes in the potassium current in the cells when exposed to an agent.

For example, a suitable method for identifying whether a candidate compound modulates the activity of TREK-I or TREK-2 is by way of electrophysiology experiments using a TREK-I or TREK-2 expression plasmid transfected into cells, such as CHO cells. In this case, the ability of a candidate compound to alter TREK-I or TREK-2 current in the transfected CHO cells may be determined by a patch clamp technique. A suitable method is as described essentially as in Besana et al (2004) /. Biol. Chem. 279(32):33154-33160.

In this regard, nucleic acids encoding TREK-I and TREK-2 ion channels in the various forms of the present invention may be cloned and introduced into a cell by a suitable method known in art. Methods for cloning nucleic acids are essentially as described in Sambrook, J, Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989).

As will be appreciated, expression of the relevant inserted DNA in plasmid or viral vectors will generally require various regulatory elements known in the art for the expression of inserted nucleic acids, for example promoters for driving the expression of an inserted nucleic acid in a particular cell, poly A signals for efficient polyadenylation of mRNA transcribed from inserted nucleic acids, or other regulatory elements to control translation, transcription or mRNA stability.

Depending upon the cell type to be modulated, the promoter driving the expression may be a constitutive promoter, an inducible promoter or a cell or tissue specific promoter.

Constitutive mammalian promoters include hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, and β-actin. Exemplary viral promoters which function constitutively in eukaryotic cells include promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus.

Inducible promoters include synthetic promoters regulated by the TetO/TetR system and inducible promoters such as metallothionein promoter, which may be used to induced transcription in the presence of certain metal ions. Other inducible promoters are known in the art.

The cell or tissue-specific promoter will depend upon the particular cell type of interest.

Nucleic acids may be introduced into a cell by methods such as transformation using calcium phosphate, viral infection, electroporation, lipofection, or particle bombardment. In this regard, transformed cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, or cells which transiently express the inserted DNA or RNA for limited periods of time. Methods for introducing exogenous DNAs into prokaryotic and eukaryotic cells are essentially as described in Sambrook, J, Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York. (1989).

Alternatively, to identify whether a candidate compound modulates the activity of TREK-I or TREK-2, conditions may be selected that minimise the contamination of TREK-I or TREK-2 currents by other K⁺ currents in isolated myocytes, and thus allow the effect of a candidate compound to modulate TREK-I or TREK-2 activity to be determined.

Preferably, the compounds identified do not result in undue prolongation of the action potential in normal myocardium. Most preferably, the compounds identified by this method do not result in undue prolongation of the action potential in the ventricles.

For example, in the case of a cell-based screening methodology, cells expressing the TREK-I and/or TREK-2 ion channel may be exposed to a candidate compound and the ability of the candidate compound to modulate the activity determined. In the case of TREK-2, preferably the cells express the TREK-2a and/or TREK-2c forms of the ion channel.

Other methods to identify whether a candidate compound modulates the activity of TREK-I or TREK-2 include fluorescence-based assays in which the channel is expressed in cells and the membrane potential monitored with a voltage sensitive fluorescent dye, or the channel can be expressed in a mutant yeast that lacks a potassium transporter and the compound screened by growth of the yeast on potassium deficient medium.

It will be appreciated that in the case of using a cell-based screening assay, the expression of the ion channel may be way of endogenous or exogenous expression of the ion channel. For example, isolated human cardiac cells expressing an endogenous form of the TREK-I and/or TREK-2 ion channels may be used to screen compounds that modulate the activity of the ion channels. Alternatively, isolated human non-cardiac cells endogenously expressing the ion channels may also be used.

Preferably, human cardiac cells, or cells derived from a human cardiac cell (such as the HCM cell line available from ScienCell Research Laboratories), are used to identify a compound that modulates the activity of TREK-I and/or TREK-2.

However, non-human cells expressing endogenous or exogenous TREK-I and/or TREK-2 may also be used to screen for compounds that modulate the activity of the ion channel. The cells may be cardiac cells or non-cardiac cells. Preferably the non-human cells are cardiac cells, or cells derived from a non-human cardiac cell. For example, mouse HL-I cells may be used.

An exogenous TREK-I or TREK-2 may be from a human or another species.

In this regard, TREK-I and TREK-2 ion channels may be identified by a method known in the art. For example, the BLAST algorithm may used to identify nucleic acids and proteins that encode TREK-I or TREK-2 ion channels. BLAST identifies local alignments between the sequences in the database and predicts the probability of the local alignment occurring by chance. The BLAST algorithm is as described in Altschul et ah, 1990, J. MoL Biol. 215:403-410.

Thus, it will be appreciated that this form of the present invention may utilise a non- human TREK-I or a TREK-2 ion channel for the step of determining whether a candidate compound modulates the activity of either or both of the ion channels.

For example, a suitable mouse or rat cardiac cell endogenously expressing the ion channel may be used to screen for compounds that modulate the activity of the ion channel. Alternatively, a suitable cell line engineered to express the ion channel may be used. For example, nucleic acids encoding the ion channel (or an active part or variant thereof) may be introduced into the cells, for example by transient or stable transfection procedures. As discussed previously herein, transcription of the nucleic acid will be driven from a suitable promoter known in the art which is active in the particular cell, and the nucleic acid will contain suitable post-transcriptional and translational regulatory signals known in the art to allow suitable expression of the encoded protein.

The cell may be transfected with a nucleic acid encoding a human or non-human form of the ion channel.

Preferably, the cell is transfected with a human TREK-I and/or a human TREK-2 ion channel, or a variant or functional fragment of these ion channels. In the case of TREK- 2, preferably the cell is transfected with TREK-2a and/or TREK-2c, or a variant or functional fragment thereof.

The cell will allow for expression of the ion channel, and will also allow the ability of a compound to modulate the activity of the ion channel to be determined. Preferably, the cell is a cardiac cell, or a cell derived from a cardiac cell.

In one form, a non-human cardiac cell may be transfected with human TREK-I and/or one or more of the forms of human TREK-2, so as to allow expression of the human ion channel in the non-human cell. Such cells may be useful for screening for candidate compounds that modulate the activity of human TREK-I and/or TREK-2.

Accordingly, in another form the present invention provides a non-human cardiac cell, the cell expressing human TREK-I and/or TREK-2, or a variant or functional fragment thereof.

The cells may be present in vitro, as part or whole of a tissue or organ, or in a non- human subject, such as a transgenic animal.

Preferably, the cells are mouse or rat cardiac cells. As will be appreciated, a cell-based method for screening for compounds that modulate the activity of TREK-2 may also include screening in whole animals. For example, the ability of a candidate compound to modulate the activity of TREK-I and/or TREK-2 may be performed in mice or rats.

In this regard, the animal may express one or more forms of human TREK-I and/or TREK-2 in one or more types of cells. For example, the animal may express human TREK-I and/or TREK-2 in the cardiac cells of the animal.

Accordingly, the present invention also provides a non-human animal including cardiac cells expressing human TREK-I and/or TREK-2, or a variant or functional fragment thereof.

Preferably, the animal is a transgenic animal expressing human TREK-I and/or TREK- 2 in cardiac cells of the animal. A suitable transgenic animal is a mouse or rat.

Methods for creating animals expressing an exogenous nucleic acid in one or more cells are known in the art. Confirmation that animals include cardiac cells that express a human TREK-I and/or TREK-2 (or a variant or functional fragment of these ion channels) may be performed by a suitable method known in the art, such as Southern analysis.

As discussed above, the cells expressing the TREK-I and/or TREK-2 potassium channels may be used for the screening of substances capable of modulating the activity of the potassium channels. This screening may be carried out by bringing into contact variable amounts of a substance to be tested with cells expressing the potassium channel and then determining the ability of the substance to alter the activity of the ion channel.

In the case of a whole animal, the test compounds may be administered to the animal in a suitable form. Methods for administration of compounds to a subject are as previously discussed. A further methodology for screening compounds that alter TREK-I and/or TREK-2 activity is the use of isolated tissues. For example, isolated perfused rat, rabbit or guinea pig heart preparations, or isolated tissues dissected from animal hearts can be used for screening.

Once a compound that modulates the activity of the ion channel has been identified, the compound may then be tested for its ability to modulate cardiac rhythm in a human subject.

For example, electrophysiology testing in human subjects with arrhythmia may be used to determine whether a particular compound has the ability to alter cardiac rhythm.

It will be appreciated that the screening methods of the various forms of the present invention may also include an additional step of using a suitable animal model to determine whether the compound that modulates the activity of TREK-I and/or TREK- 2 has the ability to modulate cardiac rhythm. In this regard, a number of methods for inducing arrhythmias in animals are known in the art.

As discussed previously herein, the present invention also provides compounds identified by the screening methods of the present invention, and a therapeutic composition including a compound identified by the methods. The present invention also provides the use of such compounds in the preparation of a medicament for the prevention and/or treatment of the aforementioned diseases and conditions.

Accordingly, in another form the present invention provides the use of an agent that modulates the activity of TREK-I and/or TREK-2 in the preparation of a medicament for preventing and/or treating a disease or condition associated altered cardiac rhythm in a human subject.

In another form the present invention provides a composition including a compound that modulates the activity of TREK-I and/or TREK-2 in the heart of a human subject. The formulation of such a composition may be achieved as previously herein described. Preferably, the compound modulates activity of TREK-I and/or TREK-2 in the ventricle.

Preferably, the compound decreases the activity of TREK-I and/or TREK-2 in the heart of the subject.

The compound preferably also reduces outward potassium currents in cardiac cells of the subject and/or prolongs action potential duration in the heart of the subject.

In the case of TREK-2, preferably the compound modulates the activity of TREK-2a and/or TREK-2c in the heart of a human subject.

Preferably, the composition modulates cardiac rhythm. Most preferably, the composition is useful for preventing and/or treating a disease or condition associated with altered cardiac rhythm, or for reducing the risk of a human subject suffering from a disease or condition associated with altered cardiac rhythm.

The composition may also be useful for preventing and/or treating cardiac hypertrophy (either concentric or dilated hypertrophy), cardiac myopathy or cardiac ischemia. The composition may also be useful in the control of ventricular rate in the presence of atrial flutter or fibrillation and in the control of arrhythmias arising from conditions that produce abnormal motion of the ventricular wall, such as cardiac ischemia and infarction.

A nucleic acid molecule used to express one or more forms of TREK-I and/or TREK-2 can also be used in genetic therapy strategies to compensate for a deficiency in, or to alter the activity of, the potassium channel in the heart of a patient. The invention thus also relates to a composition or medication containing nucleic acid molecules expressing TREK-I and/or TREK-2, or cells transformed by these nucleic acid molecules, for the treatment of disease or conditions of the heart. The present invention also provides a TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates cardiac rhythm in a human subject.

Preferably, the protein is a human TREK-I or TREK-2 protein, or a variant or fragment thereof.

Preferably, the TREK-I or TREK-2 is an isolated form of the ion channel, or a variant or a functional fragment thereof. In this regard, the term "isolated" is to be understood to mean a species (such as a nucleic acid, protein or cell) that is removed from its naturally occurring state. Generally, the isolated species will be substantially purified from other species of the same type.

In one form, the TREK-I and/or TREK-2 proteins used as a target for identifying a compound that modulates cardiac rhythm in a human subject are present in a cell-free system. In this case, molecules that bind or interact with the target may be first identified using affinity chromatography techniques known in the art. Molecules may then be screened to identify compounds that modulate cardiac rhythm in a human subject, as previously herein described.

In another form, the TREK-I and/or TREK-2 protein may be expressed in a cell, and used to identify a compound that modulates the activity of the ion channel, as herein described. The cells may be present in vitro, as part or whole of a tissue or organ, or in a non-human animal subject. Molecules may then screened to identify compounds that modulate cardiac rhythm in a human subject, as previously herein described.

The present invention also allows the identification of human subjects suffering from, or susceptible to, a disease or condition associated with altered cardiac rhythm, by identifying an altered activity of TREK-I and/or TREK-2 in the subject. A subject that is susceptible to a disease or condition associated with altered cardiac rhythm may be predisposed to such a disease or condition and/or have a higher risk of developing the disease or condition. Accordingly, in another form the present invention provides a method of identifying a human subject suffering from, or susceptible to, a disease or condition associated with altered cardiac rhythm, the method including the step of identifying an altered activity of TREK-I and/or TREK-2 in the subject.

Preferably, the disease or condition associated with altered cardiac rhythm is an arrhythmia. Most preferably, the disease or condition is supraventricular tachycardia, including atrial flutter and atrial fibrillation, ventricular tachycardia and ventricular fibrillation.

Preferably, the altered activity of TREK-I and/or TREK-2 is an altered activity of either or both of these ion channels in the heart of the subject. More preferably, the activity of TREK-I and/or TREK-2 is increased in the heart of the subject suffering from, or susceptible to, a disease or condition associated with altered cardiac rhythm.

In the case of TREK-2, preferably the altered activity of TREK-2 is an altered activity of TREK-2a and/or TREK-2b.

An altered activity of TREK-I or TREK-2 may be identified in the subject by a suitable method. For example, an altered activity may be identified by administration of a suitable diagnostic reagent to the human subject.

It will be appreciated that an alteration in the activity of TREK-I and/or TREK-2 may be due to a variety of causes. For example, the altered activity may be due to other diseases or conditions in the subject that affect the level of these and other ion channels, such as chronic atrial fibrillation and cardiac hypertrophy.

Alternatively, the alteration in the activity of TREK-I or TREK-2 may be due to a mutation in one or more genes in the subject. Therefore, in some subjects an altered activity may also be identified by a suitable genetic test performed on the subject.

For example, the presence of a mutation in the gene coding for the ion channel that alters the activity may be determined by a suitable method known in the art. It will be appreciated that such mutations may be located in the gene for TREK-I or TREK-2, or may be located in other genes or in an extragenic region.

Preferably, the mutation is located in the TREK-I gene or the TREK-2 gene.

Accordingly, in another the present invention provides a method of identifying a human subject suffering from, or susceptible to, a disease or condition associated with altered cardiac rhythm, the method including the step of identifying a mutation in the TREK-I and/or TREK-2 gene in the subject.

The mutation may be present in one or both alleles of a particular gene.

In this regard, the ability of a specific mutation to alter the activity of the ion channels may be determined by a suitable method, as discussed previously. For example, the effect of a specific mutation may be determined by the activity of the ion channels introduced into a heterologous expression system (eg COS cells, HEK cells). Such systems have been discussed previously herein.

The identification of a mutation in the subject that alters the activity of TREK-I or TREK-2 may be determined by a suitable method known in the art.

DNA may be isolated from the subject by a suitable method known in the art. A suitable method for isolating genomic DNA from a subject is from whole venous blood as described in Miller et al. (1988) Nucleic Acids Research 16(3):1215.

Various methods may be used to identify the mutation. DNA sequencing (either manual sequencing or automated fluorescent sequencing) can be used to detect the mutation. In this case, identification of the mutation will usually involve amplification of the region containing the mutation from nucleic acid isolated from the subject (generally genomic DNA), although it is also possible to identify the mutation by sequencing a clone of the region derived from a particular subject, with or without amplification. Another approach for identifying mutations is the single-stranded conformation polymorphism assay (SSCA) (as described in Orita et al. (1989) Genomics 5(4): 874- 879. This method does not detect all sequence changes, especially if the DNA fragment size is greater than 200 bp, but can be optimized to detect most DNA sequence variation. Fragments which have shifted mobility on SSCA gels are then sequenced to determine the exact nature of the DNA sequence variation.

Another approach is based on the detection of mismatches between two complementary DNA strands, including clamped denaturing gel electrophoresis (as described in Sheffield et al. (1991) Am. J. Hum. Genet. 49:699-706), heteroduplex analysis (as described in White et al. (1992) Genomics 12:301-303) and chemical mismatch cleavage (as described in Grompe et al. (1989) Proc. Natl. Acad. ScL USA 86:5855- 5892). Once a specific mutation is identified, an allele specific detection approach such as allele specific oligonucleotide hybridization can be utilized.

If DNA sequence analysis of genomic DNA is used to identify a mutation, the presence of a mutation in one allele (ie the subject is heterozygous for the mutation) will be by the presence of two nucleotides at the relevant position in the DNA sequence. Sequence of the DNA from a subject homozygous for the normal allele or homozygous for the mutation will yield only the presence of the appropriate nucleotide at the relevant position of the DNA sequence.

To provide a suitable template for sequencing, a region of the genomic DNA isolated from the subject may be amplified using appropriately designed primers. Sequencing reactions with an appropriate primer and the analysis of the DNA sequence may be performed by a suitable method known in the art.

Alternatively, the presence of a mutation may be determined using sequence specific primers that will only amplify either the wild type allele or the allele with the mutation from the DNA isolated from the subject. If sequence specific primers are used to amplify the DNA, a consensus primer and one of two alternative primers will be used. Each of the alternative primers will have a 3' terminal nucleotide that either corresponds to the wild type sequence (a WT primer) or the polymorphic sequence (a SNP primer). In this case, amplification will only occur from the template having the correct complementary nucleotide.

Other methods to identify mutations involve hybridization of nucleic acid containing the mutation with other nucleic acids (ie a reporter nucleic acid) that allows discrimination between differences in nucleic acid sequences. For example, Southern analysis with an oligonucleotide may be used to detect mutations. Alternatively, methods are known in the art in which the oligonucleotide is attached to a solid substrate, such as chip, and the binding of a nucleic acid containing a mutation detected by binding (or lack thereof) to the oligonucleotide. In these cases, the identification of a mutation in a subject also includes detection of the mutation by hybridisation of nucleic acid isolated or derived from the subject to a reporter nucleic acid.

Preferably, the identification of a mutation includes amplification of a region containing the mutation from nucleic acid isolated or derived from the subject.

Preferably, the identification of a mutation includes detection of the mutation by hybridisation of nucleic acid isolated or derived from the subject to a reporter nucleic acid.

As will be appreciated, identifying an altered activity of TREK-I and/or TREK-2 also allows for the identification of subjects in need of (or suitable for) medical intervention to prevent and/or treat cardiac arrhythmia, or for the identification of a subject susceptible to, predisposed to, or at risk of suffering from a disease or condition associated with altered cardiac rhythm.

For example, in one form the present invention provides a method of identifying a human subject suitable for intervention to prevent and/or treat cardiac arrhythmia, the method including the step of identifying an altered activity of TREK-I and/or TREK-2 in the subject.

The present invention also allows for the modulation of the K⁺ current activity in a human cardiac cell, by modulating the activity of TREK-I and/or TREK-2. Accordingly, in another form the present invention provides a method of modulating K⁺ current activity in a human cardiac cell, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the cell.

This form of the present invention allows the K⁺ current activity in a human cardiac cell to be altered by an agent and/or treatment that modulates the activity of the TREK-I and/or TREK-2 potassium channels.

Preferably, the modulation of the activity of TREK-I and/or TREK-2 in the heart of the subject in this form of the present invention is decreased. Thus, inhibition of the activity of these ion channels in cardiac cells may be useful for treating diseases or conditions associated with altered cardiac rhythm or altered K⁺ current activity.

Preferably, the decrease in activity of TREK-I and/or TREK-2 results in a reduction in outward potassium currents in cardiac cells of the subject and/or prolongs action potential duration in cardiac cells in the heart of the subject.

Preferably, the modulation of the activity of TREK-I and/or TREK-2 includes modulation of the activity of these ion channels in the ventricle of the heart of the subject.

In the case of TREK-2, preferably the activity of the TREK-2a and/or TREK- 2c ion channel is modulated in the human cardiac cell.

The cells may be present in vitro, as part or whole of a tissue or organ, or in an animal human subject.

Preferably, the activity of the TREK-I and/or TREK-2 ion channels is modulated in one or more cells in the heart of a human subject. Accordingly, in another form the present invention provides a method of modulating cardiac K⁺ current activity in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of a human subject.

Preferably, the method of this form of the present invention is used to prevent and/or treat (ameliorate) a disease or condition associated with altered K⁺ current activity, including an arrhythmia. Examples of diseases or conditions associated with altered K⁺ current activity include disorders of the rate, rhythm or conduction of electrical impulses within the heart. The disorders include premature contractions (extrasystoles) originating in abnormal foci in atria or ventricles, paroxysmal supraventricular tachycardia, atrial flutter, atrial fibrillation, ventricular fibrillation and ventricular tachycardia, and arrhythmias arising from conditions which produce abnormal motion of the ventricular wall, such as cardiac ischemia and infarction. Cardiac arrhythmia is also often associated with coronary artery diseases, such as myocardial infarction and atherosclerotic heart disease.

Preferably, the method of this form of the present invention is used to prevent and/or ameliorate an arrhythmia in a human subject. Most preferably, the method may be used to prevent or ameliorate supraventricular tachycardia, including atrial flutter and atrial fibrillation, ventricular tachycardia and ventricular fibrillation.

The method of this form of the present invention is also useful in the control of ventricular rate in the presence of atrial flutter or fibrillation, and in the control of arrhythmias arising from conditions that produce abnormal motion of the ventricular wall, such as cardiac ischemia and infarction.

In addition, the method of this form of the present invention is also useful in the treatment in human subjects of cardiac hypertrophy (either concentric or dilated hypertrophy), cardiac myopathy, and cardiac ischemia.

Preferably, the human subject is a subject susceptible to, or suffering from, a disease or condition associated with altered K⁺ current activity. Most preferably, the human subject is a subject susceptible to, or suffering from, supraventricular tachycardia, including atrial flutter and atrial fibrillation, ventricular tachycardia and ventricular fibrillation.

The subject may also be suffering from, or susceptible to, cardiac hypertrophy (either concentric or dilated hypertrophy), cardiac myopathy, and cardiac ischemia, or be suffering from, or susceptible to, an arrhythmia arising from conditions which produce abnormal motion of the ventricular wall, such as cardiac ischemia and infarction.

As discussed, it will be appreciated that modulating the activity of TREK-I and/or TREK-2 may also be used to prevent and/or treat a disease or condition associated with altered K⁺ current activity in the heart of a human subject.

Accordingly, in another form the present invention provides a method of preventing and/or treating a disease or condition associated with altered K⁺ current activity in the heart of a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

As discussed previously, the step of modulating the activity of the TREK-I and/or TREK-2 potassium channels may be performed by exposing the subject to an agent, a combination of agents, and/or a treatment that results in an alteration of the activity of the ion channels. Confirmation that the activity has been altered may be by a suitable method known in the art. Modulation of the K⁺ current activity in the heart of a human subject may be determined by a suitable method known in the art.

The present invention also allows the identification of compounds that modulate cardiac K⁺ current activity in cardiac cells in a human subject, by screening compounds that modulate the activity of TREK-I and/or TREK-2.

Accordingly, in one form the present invention provides a TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates K⁺ current activity in a human cardiac cell. In another form, the present invention provides a TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates cardiac K⁺ current activity in a human subject.

Preferably, the protein is a human TREK-I or TREK-2 protein, or a variant or fragment thereof.

Preferably, the TREK-I or TREK-2 is an isolated form of the ion channel, or a variant or a functional fragment thereof. In this regard, the term "isolated" is to be understood to mean a species (such as a nucleic acid, protein or cell) that is removed from its naturally occurring state. Generally, the isolated species will be substantially purified from other species of the same type.

In one form, the TREK-I and/or TREK-2 proteins used as a target for identifying a compound that modulates K⁺ current activity in a cell-free system. In this case, molecules that bind or interact with the target may be first identified using affinity chromatography techniques known in the art. Molecules may then be screened to identify compounds that modulate K⁺ current activity, as previously herein described.

In another form, the TREK-I and/or TREK-2 protein may be expressed in a cell, and used to identify a compound that modulates the activity of the ion channel, as herein described. The cells may be present in vitro, as part or whole of a tissue or organ, or in a non-human animal subject. Molecules may then screened to identify compounds that modulate K⁺ current activity, as previously herein described.

In another form, the present invention provides a method of identifying a compound that modulates cardiac K⁺ current activity in a human subject. This screening procedure makes it possible to identify drugs that may be useful, for example, in the treatment and/or prevention of diseases or conditions associated with altered cardiac rhythm.

Accordingly, in another form the present invention provides a method of identifying a compound that modulates cardiac K⁺ current activity in a human subject, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the heart of a human subject.

Preferably, the method of identification involves identifying a compound that decreases the activity of TREK-I and/or TREK-2 in the heart of the subject.

The method of identification may further include the step of identifying the compound as a compound that reduces outward potassium currents in cardiac cells of the subject and/or prolongs action potential duration in the heart of the subject.

Preferably, the method of identification includes modulation of the activity of TREK-I and/or TREK-2 in cardiac myocytes.

Preferably, the method of identification includes the modulation of activity of TREK-I and/or TREK-2 in the ventricle of the heart of the subject.

In the case of TREK-2, preferably the modulation of TREK-2 activity includes modulation of the activity of TREK-2a and/or TREK- 2c.

The screening procedure also makes it possible to identify drugs that may be useful in controlling ventricular rate in the presence of atrial flutter or fibrillation, drugs useful in the control of arrhythmias arising from conditions which produce abnormal motion of the ventricular walls, such as cardiac ischemia and infarction, and drugs that may be useful in the treatment and/or prevention of cardiac hypertrophy, including concentric or dilated hypertrophy, cardiac myopathy and cardiac ischemia.

Preferably, this method of the present invention includes the following steps: (a) identifying a compound that modulates the activity of TREK-I and/or TREK-2; (b) determining the ability of the compound so identified to modulate cardiac K⁺ current activity in a human subject; and (c) identifying the compound as a compound that modulates cardiac K⁺ current activity in a human subject. Accordingly, in another form the present invention provides a method of identifying a compound that modulates cardiac K⁺ current activity in a human subject, the method including the steps of:

(a) identifying a compound that modulates the activity of TREK-I and/or TREK-2;

(b) determining the ability of the compound so identified to modulate cardiac K⁺ current activity in a human subject; and

(c) identifying the compound as a compound that modulates cardiac K⁺ current activity in a human subject.

Preferably, the compounds identified by this method do not result in undue prolongation of the action potential in normal myocardium. Most preferably, the compounds identified by this method do not result in undue prolongation of the action potential in the ventricles.

The identification of compounds that modulate the activity of TREK-I and/or TREK-2 may be accomplished as previously described, including the use of cell-free, cell-based or in vivo methods as previously described.

Once a compound that modulates the activity of the ion channel has been identified, the compound may then be tested for its ability to modulate K⁺ current activity in a human subject by a suitable method known in the art. Methods for administering compounds to a subject are as previously discussed.

However, it will also be appreciated that the method of this form of the present invention may include the additional step of using a suitable animal model to determine whether the compound that modulates the activity of TREK-I and/or TREK-2 has the ability to modulate cardiac K⁺ current activity.

The present invention also provides compounds identified by this method and a therapeutic composition including a compound identified by the method. The present invention also provides the use of such compounds in the preparation of a medicament for the prevention and/or treatment of the aforementioned diseases and conditions. In a similar fashion, the present invention also allows the identification of compounds that K⁺ current activity in a human cardiac cell.

Accordingly, in another form the present invention provides a method of identifying a compound that modulates K⁺ current activity in a human cardiac cell, the method including the steps of identifying a compound that modulates the activity of TREK-I and/or TREK-2.

In this case, the method of this form of the present invention may further include identifying a compound that modulates the activity of TREK-I and/or TREK-2, determining the ability of the compound so identified to modulate K⁺ current activity in a human cardiac cell, and identifying the compound as a compound that modulates K⁺ current activity in a human cardiac cell.

Accordingly, in another form the present invention provides a method of identifying a compound that modulates K⁺ current activity in a human cardiac cell, the method including the steps of:

(a) identifying a compound that modulates the activity of TREK-I and/or TREK-2;

(b) determining the ability of the compound so identified to modulate K⁺ current activity in a human cardiac cell; and

(c) identifying the compound as a compound that modulates K⁺ current activity in a human cardiac cell.

The identification of compounds that modulate the activity of TREK-I and/or TREK-2 may be accomplished as previously described, including the use of cell-free, cell-based or in vivo methods as previously described.

Methods for determining that a compound modulates the activity of TREK-I and/or TREK-2 are as previously discussed. Methods for determining K⁺ current activity are known in the art. The present invention also provides compounds identified by this method and a therapeutic composition including a compound identified by the method. The present invention also provides the use of such compounds in the preparation of a medicament for the prevention and/or treatment of the aforementioned diseases and conditions.

It will also be appreciated that the present invention allows for the identification of a subject suffering from, or susceptible to, a disease or condition associate with altered cardiac K⁺ current activity, by identifying an altered activity of TREK-I and/or TREK-2 in the subject.

Accordingly, in another form the present invention provides a method of identifying a human subject suffering from, or susceptible to, a disease or condition associated with altered cardiac K⁺ current activity, the method including the step of identifying an altered activity of TREK-I and/or TREK-2 in the subject.

Preferably, the disease or condition associated with altered cardiac rhythm is an arrhythmia. Most preferably, the disease or condition is supraventricular tachycardia, including atrial flutter and atrial fibrillation, ventricular tachycardia and ventricular fibrillation.

The subject may also be suffering from, or susceptible to, cardiac hypertrophy (either concentric or dilated hypertrophy), cardiac myopathy, and cardiac ischemia, or be suffering from, or susceptible to, an arrhythmia arising from conditions which produce abnormal motion of the ventricular wall, such as cardiac ischemia and infarction.

Preferably, the altered activity of TREK-I and/or TREK-2 is an altered activity of these ion channels in the heart of the subject.

As discussed previously, an altered activity of TREK-I or TREK-2 may be identified in the subject by a suitable method. For example, an altered activity of TREK-I or TREK- 2 may be identified by administration of a suitable diagnostic reagent to the human subject. Alternatively, an altered activity of TREK-I or TREK-2 may be identified by a suitable genetic test performed on the subject, as previously discussed. This form of the present invention is also useful for identifying subjects in need of medical intervention to prevent and/or treat a disease or condition associated with an altered cardiac K⁺ current activity.

Accordingly, in another form the present invention also provides a method of identifying a human subject suitable for intervention to prevent and/or treat a disease or condition associated with an altered cardiac K⁺ current activity, the method including the step of identifying an altered activity of TREK-I and/or TREK-2 in the subject.

Modulation of the activity of TREK-I and/or TREK-2 will also allow modulation of the cardiac action potential in a human subject.

Accordingly, in another form the present invention provides a method of modulating cardiac action potential in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

This form of the present invention allows the cardiac action potential in a human subject to be altered by an agent or treatment that modulates the activity of either or both of the TREK-I and TREK-2 potassium channels.

Preferably, the modulation of the activity of TREK-I and/or TREK-2 in the heart of the subject in this form of the present invention is decreased. Thus, inhibition of the activity of these ion channels in cardiac cells may be useful for treating diseases or conditions associated with altered cardiac action potential.

Preferably, the decrease in activity of TREK-I and/or TREK-2 results in a reduction in outward potassium currents in cardiac cells of the subject and/or prolongs action potential duration in cardiac cells in the heart of the subject.

Preferably, the modulation of the activity of TREK-I and/or TREK-2 includes modulation of the activity of these ion channels in the ventricle of the heart of the subject. Preferably, the activity of the TREK-I and/or TREK-2 ion channels in the various forms of the present invention is modulated in one or more cells in the heart of a human subject.

In the case of TREK-2, preferably the method of this form of the present invention involves modulating the activity of the TREK-2a and/or TREK- 2c ion channels in the heart of the subject.

Preferably, the method of this form of the present invention may be used to prevent or ameliorate an arrhythmia. Most preferably, the method may be used to prevent or ameliorate supraventricular tachycardia, including atrial flutter and atrial fibrillation, ventricular tachycardia and ventricular fibrillation.

The method of this form of the present invention may also be useful in the control of ventricular rate in the presence of atrial flutter or fibrillation.

In addition, the method of this form of the present invention may also be useful in the treatment of cardiac hypertrophy (either concentric or dilated hypertrophy), cardiac myopathy, and cardiac ischemia, and in the control of arrhythmias arising from conditions which produce abnormal motion of the ventricular wall, such as cardiac ischemia and infarction.

Preferably, the human subject is a subject susceptible to, or suffering from, a disease or condition associated with altered cardiac action potential. Most preferably, the human subject is a subject susceptible to, or suffering from, supraventricular tachycardia, including atrial flutter and atrial fibrillation, ventricular tachycardia and ventricular fibrillation.

The subject may also be suffering from, or susceptible to, cardiac hypertrophy (either concentric or dilated hypertrophy), cardiac myopathy, and cardiac ischemia, or be suffering from, or susceptible to, an arrhythmia arising from conditions which produce abnormal motion of the ventricular wall, such as cardiac ischemia and infarction. In this regard, it will be appreciated that modulating the activity of TREK-I and/or TREK-2 may also be used to prevent and/or treat a disease or condition associated with altered cardiac action potential in the human subject.

Accordingly, in another form the present invention also provides a method of preventing and/or treating a disease or condition associated with altered cardiac action potential in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

Examples of diseases or conditions associated with altered cardiac action potential include disorders of the rate, rhythm or conduction of electrical impulses within the heart. The disorders include premature contractions (extrasystoles) originating in abnormal foci in atria or ventricles, paroxysmal supraventricular tachycardia, atrial flutter, atrial fibrillation, ventricular fibrillation and ventricular tachycardia, and arrhythmias arising from conditions which produce abnormal motion of the ventricular wall, such as cardiac ischemia and infarction. Cardiac arrhythmia is also often associated with coronary artery diseases, such as myocardial infarction and atherosclerotic heart disease.

As discussed previously, the step of modulating the activity of the TREK-I and/or TREK-2 potassium channels may be performed by exposing the subject to an agent, a combination of agents, or a treatment that results in an alteration of the activity of TREK-I and/or TREK-2. Confirmation that the activity of TREK-I and/or TREK-2 has been altered may be by a suitable method known in the art. Modulation of the cardiac action potential in a human subject may be determined by a suitable method known in the art.

The present invention also allows modulation of the action potential of a human cardiac cell. Accordingly, in another form the present invention provides a method of modulating the action potential of a human cardiac cell, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the cell.

Preferably, the modulation of the activity of TREK-I and/or TREK-2 in this form of the present invention is decreased. Thus, inhibition of the activity of these ion channels in cardiac cells may be useful for treating diseases or conditions associated with altered action potential.

Preferably, the decrease in activity of TREK-I and/or TREK-2 results in a reduction in outward potassium currents in cardiac cells of the subject and/or prolongs action potential duration in cardiac cells in the heart of the subject.

Preferably, the modulation of the activity of TREK-I and/or TREK-2 includes modulation of the activity of these ion channels in the ventricle of the heart of the subject.

Preferably, the activity of the TREK-I and/or TREK-2 ion channels in the various forms of the present invention is modulated in one or more cells in the heart of a human subject.

In the case of TREK-2, preferably the method of this form of the present invention involves modulating the activity of the TREK-2a and/or TREK- 2c ion channels in the cell.

It will also be appreciated that the present invention may be used to identify compounds that modulate cardiac action potential, by identifying compounds that modulate the activity of TREK-I and/or TREK-2.

Accordingly, in another form the present invention provides a method of identifying a compound that modulates cardiac action potential in a human, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the heart of a human subject. In this case, the method of this form of the present invention may further include identifying a compound that modulates the activity of TREK-I and/or TREK-2, determining the ability of the compound so identified to modulate cardiac action potential in a human subject, and identifying the compound as a compound that modulates cardiac action potential in a human subject.

Accordingly, in another form the present invention provides a method of identifying a compound that modulates cardiac action potential in a human subject, the method including the steps of:

(a) identifying a compound that modulates the activity of TREK-I and/or TREK-2;

(b) determining the ability of the compound so identified to modulate cardiac action potential in a human subject; and (c) identifying the compound as a compound that modulates cardiac action potential in a human subject.

This form of the present invention provides a method of identifying a compound that modulates cardiac action potential in a human subject. This screening procedure makes it possible to identify drugs that may be useful in the treatment or prevention of diseases or conditions associated with altered cardiac action potential.

The screening procedure also makes it possible to identify drugs that may be useful in controlling ventricular rate in the presence of atrial flutter or fibrillation, drugs useful in the control of arrhythmias arising from conditions which produce abnormal motion of the ventricular walls, such as cardiac ischemia and infarction, and drugs that may be useful in the treatment and/or prevention of cardiac hypertrophy, including concentric or dilated hypertrophy, cardiac myopathy and cardiac ischemia.

The method includes the steps of identifying a candidate compound that modulates the activity of TREK-I and/or TREK-2, and determining whether a compound so identified is capable of modulating cardiac action potential in a human subject. Preferably, the compounds identified by this method do not result in undue prolongation of the action potential in normal myocardium. Most preferably, the compounds identified by this method do not result in undue prolongation of the action potential in the ventricles.

The identification of compounds that modulate the activity of TREK-2 may be accomplished as previously described.

Once a compound that modulates the activity of the ion channel has been identified, the compound may then be tested for its ability to modulate cardiac action potential in a human subject, by a suitable method known in the art.

However, it will be appreciated that the method of this form of the present invention may include the additional step of using a suitable animal model to determine whether the compound that modulates the activity of TREK-a and/or TREK-2 has the ability to modulate cardiac action potential.

The present invention also contemplates compounds identified by this method, and a therapeutic composition including a compound identified by the method. The present invention also provides the use of such compounds in the preparation of a medicament for the prevention and/or treatment of the aforementioned diseases and conditions.

In a similar fashion, the present invention also allows the identification of compounds that modulate the action potential of a human cardiac cell, by identifying compounds that alter the activity of TREK-I and/or TREK-2.

Accordingly, in another form the present invention provides a method of identifying a compound that modulates K⁺ current activity in a human cardiac cell, the method including the steps of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in a human cardiac cell.

In this case, the method of this form of the present invention may further include identifying a compound that modulates the activity of TREK-I and/or TREK-2, determining the ability of the compound so identified to modulate action potential in a human cardiac cell, and identifying the compound as a compound that modulates action potential in a human cardiac cell.

Accordingly, in another form the present invention provides a method of identifying a compound that modulates the action potential of a human cardiac cell, the method including the steps of:

(a) identifying a compound that modulates the activity of TREK-I and/or TREK-2; (b) determining the ability of the compound so identified to modulate the action potential of a human cardiac cell; and

(c) identifying the compound as a compound that modulates the action potential of a human cardiac cell.

The present invention also contemplates compounds identified by this method, and a therapeutic composition including a compound identified by the method. The present invention also provides the use of such compounds in the preparation of a medicament for the prevention and/or treatment of the aforementioned diseases and conditions.

The identification of compounds that modulate the activity of TREK-I and/or TREK-2 in the heart of a human subject may be accomplished by a suitable method, as previously discussed.

Once a compound that modulates the activity of the ion channel has been identified, the compound may then be tested for its ability to modulate cardiac action potential in a human subject or in human cardiac cells.

For example, electrophysiology testing in human subjects may be used to determine whether a particular compound has the ability to alter cardiac action potential.

The present invention also contemplates compounds identified by this method, and a therapeutic composition including a compound identified by the method. The present invention also provides the use of such compounds in the preparation of a medicament for the prevention and/or treatment of the aforementioned diseases and conditions.

The present invention also allows the identification of a subject suffering from, or susceptible to, a disease or condition associated with altered cardiac action potential, by identifying an altered activity of TREK-I and/or TREK-2.

Accordingly, in another form the present invention provides a method of identifying a human subject suffering from, or susceptible to, a disease or condition associated with altered cardiac action potential, the method including the step of identifying an altered activity of TREK-I and/or TREK-2 in the subject.

Preferably, the disease or condition associated with altered cardiac potential is an arrhythmia. Most preferably, the disease or condition is supraventricular tachycardia, including atrial flutter and atrial fibrillation, ventricular tachycardia and ventricular fibrillation.

Preferably, the altered activity of TREK-I and/or TREK-2 is an altered activity of these ion channels in the heart of the subject.

In the case of TREK-2, preferably the altered activity is an altered activity of TREK-2a and/or TREK-2c in the heart of the subject.

As discussed previously, an altered activity of TREK-I and/or TREK-2 may be identified in the subject by a suitable method. For example, an altered activity of TREK-I and/or TREK-2 may be identified by administration of a suitable diagnostic reagent to the human subject.

Alternatively, an altered activity of TREK-I and/or TREK-2 may be identified by a suitable genetic test performed on the subject, as previously discussed. This form of the present invention is also useful for identifying subjects in need of medical intervention to prevent and/or treat a disease or condition associated with an altered cardiac action potential.

Accordingly, in another form the present invention provides a method of identifying a human subject suitable for intervention to prevent and/or treat a disease or condition associated with an altered cardiac action potential, the method including the step of altering the activity of TREK-I and/or TREK-2 in the subject.

Description of the Preferred Embodiments

Reference will now be made to experiments that embody the above general principles of the present invention. However, it is to be understood that the following description is not to limit the generality of the above description.

Example 1

Primers

Primers for use in PCR experiments were designed with the use of Primer Express software (PE Applied Biosystems, Foster city, CA, USA). The primers used were as follows:

(i) TREK-I Primers

TREK-I primers for PCR amplification:

Sense: 5' GAGGACCACCATTGTGATCCA 3' (SEQ ID NO.l)

Antisense: 5' TCCCACCTCTTCTTTTGTCTTTTT 3' (SEQ ID NO.2)

TREK-I primers for nested PCR amplification:

Sense: 5' TTTGGAAAAGGAATTGCCAAA 3' (SEQ ID NO.3) Antisense: 5' CCCGCTGGAACTTGTC ATAAA 3' (SEQ ID NO.4) Sense: 5' TTTGGAAAAGGAATTGCCAAA 3' (SEQ ID NO.5) Antisense: 5' TCCCACCTCTTCTTTTGTCTTTTT 3' (SEQ ID NO.6)

TREK-I primers for real-time PCR: Sense: 5' GCTGTCCTGAGC ATGATTGGA 3' (SEQ ID NO.7) Antisense: 5' CCCGCTGGAACTTGTCATAAA 3' (SEQ ID NO.8)

(ii) TREK-2 Primers

Note that the specificity of the primers for each of the variants relies on the sequence differences in the first exon.

TREK-2a primers for PCR amplification in ventricle tissue: Sense: 5' TTGGAGC AGCCCTTTGAGA 3' (SEQ ID NO. 9) Antisense: 5' AGGCC AAC AAGGATCCAAAA 3' (SEQ ID NO. 10)

TREK-2a primers for PCR amplification in atrial tissue:

Sense: 5' AGGAACGCTAGGCAGTCTCTTTC 3' (SEQ ID NO. 11)

Antisense: 5' AGGCCAACAAGGATCCAAAA 3' (SEQ ID NO. 12)

Primers for nested PCR amplification of TREK-2a: Sense: 5' TTGGAGCAGCCCTTTGAGA 3' (SEQ ID NO. 13) Antisense 1: 5' CAGTG CTCGGAGCAATATTCC 3' (SEQ ID NO. 14) Antisense 2: 5' TGGACTGACTCCCGCATTG 3' (SEQ ID NO. 15)

TREK-2b primers:

Sense: 5' TTGGTATATGGAGGATGGATTTAAGG 3' (SEQ ID NO. 16)

Antisense: 5' AGGCCAACAAGGATCCAAAA 3' (SEQ ID NO. 17)

TREK-2c primers:

Sense: 5' GGGC AACG AAGCAATGAAAT 3' (SEQ ID NO. 18) Antisense: 5' AGGCCAACAAGGATCCAAAA 3' (SEQ ID NO. 19) Primers for nested PCR amplification of TREK- 2c: Sense: 5' GGGC AACG AAGCAATGAAAT 3' (SEQ ID NO. 20) Antisense 1: 5' TGGACTGACTCCCGCATTG 3' (SEQ ID NO. 21) Antisense 2: 5' CAGTG CTCGGAGCAATATTCC 3' (SEQ ID NO. 22)

(iii) TRAAK Primers

TRAAK primers for PCR amplification:

Sense: 5' ACCATCGGCTATGGCAATGT 3' (SEQ ID NO.23) Antisense: 5' GGACACTACTCGC AGCC AGTT 3' (SEQ ID NO.24)

TRAAK primers for nested PCR amplification: Sense: 5' CGGCTGCCTGCTCTTTGT 3'(SEQ ID NO.25) Antisense: 5' GGACACTACTCGC AGCC AGTT 3' (SEQ ID NO.26)

TRAAK primers for real-time PCR amplification:

Sense: 5' CGGCTGCCTGCTCTTTGT 3' (SEQ ID NO.27)

Antisense: 5' AAAGCCC ACGGTGGTAAGC 3' (SEQ ID NO.28)

(iv) GAPDH Primers

GAPDH (housekeeping gene) for real-time PCR amplification: Sense: 5' ATGGAAATCCC ATC ACC ATCTT 3' (SEQ ID NO.29) Antisense: 5' GGTGC AGGAGGC ATTGCT 3' (SEQ ID NO.30)

Example 2

Preparation ofmRNA, cDNA, PCR and real-time PCR

Fresh human right atrial appendages were obtained from patients undergoing coronary bypass surgery. These patients had no cardiac disease other than coronary artery disease. Frozen human ventricular tissues were obtained from either transplant donor hearts or from explanted hearts with diagnoses of ischaemic cardiomyopathy (IC) or idiopathic dilated cardiomyopathy (IDC). Positive control tissue was human testicle, obtained from a patient undergoing orchidectomy to control prostate cancer. All procedures were overseen by Human Research Ethics Committees of the RAH and the University of Adelaide.

Fresh tissues were frozen in liquid nitrogen and then stored at -8O⁰C until RNA extraction. Total RNA isolation from human atrial, ventricular and testicle tissues was done with TRI-Reagent (SIGMA-ALDRICH, Australia) and RNA kit (BIO-RAD, Sydney, Australia) according to the manufacturer's instructions. TRI-Reagent-treated RNA isolation was used to prepare cDNA for normal PCR, whereas BIO-RAD-treated RNA extraction was used to prepare cDNA for real-time PCR. Two-step RT-PCR method was used. Reverse transcriptase was from Invitrogen, SYBR Green real-time PCR reagent from BIO-RAD, and Applied Biosystems (US). RT-PCR was performed by heating to 65°C for 5 min and quick chilling on ice for at least 1 min, after which the tube was centrifuged and RTase mixer added. Mix was then incubated at 25°C for 10 min, followed by 42°C for 50 min then 70°C for 15min. The PCR products were detected in 1.2% agarose gel and visualised by a Gel documentation system (BIO-RAD, Sydney, Australia). Selected bands were cleaned by UltraClean Kit (MO BIO Laboratories, CA, US) and used for nested PCR, with the nested PCR product being purified by ExoSAP-IT (USB corporation, Cleveland, Ohio, US), and sequenced.

The parameters for normal PCR were 1 cycle of 94⁰C for 3 min, 35 cycles of 94⁰C for 45 s, 5O⁰C for 1 min and 72⁰C for 1 min, one cycle of 72⁰C for 10 min. The PCR products were detected on a 1.2% agarose gel and visualised by Gel documentation system (BIO-RAD, Sydney, Australia). Bands were cut and cleaned by UltraClean Kit (MO BIO Laboratories, CA, US). Gel-clean PCR products were used in nested PCR. When a single, correct size band occurred in nested PCR, the product was purified by ExoSAP-IT (USB corporation, Clevenland, Ohio, US), and then sequenced (Institute of Medical and Veterinary Science).

For real-time PCR, 25 μL reaction solution included 12.5 μL SYBR Green PCR Master Mix, 5.5 μL cDNA, 1 μL of sense primer (10 μM), 1 μL of antisense primer (10 μM), and 5 μL of molecular-level water. Real-time PCR assay was performed in 96-well optical plates on an ABI Prism 5700 Sequence Detection System (PE Applied Biosystems, Forster city, CA, US). The process of real-time PCR was 1 cycle of 95⁰C for 10 min, and then 40 cycles of 6O⁰C for 1 min and 95⁰C for 15 s. The data from realtime PCR was analysed in Sigmaplot (12.01 Version, SPSS, Richmond, CA, US), as described in: Liu W, Saint DA. (2002) "Validation of a quantitative method for real time PCR kinetics" Biochem Biophys Res Commun. 294(2):347-353 and Liu W, Saint DA. (2002) "A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics" Anal Biochem. 302(l):52-59.

Example 3

Western Analysis

Human atrial and ventricular tissues stored below -80⁰C were homogenized by general methods for protein extraction. Protein content was quantified spectrophotometrically. The same concentration of protein from each sample was denatured and run on a SDS- PAGE mini-gel. After fractionation, the samples were transferred on nitrocellulose membrane. The membrane was blocked with non-fat milk solution and probed with anti-TREK antibody (Alomone, Israel) and HRP- Anti-Rabbit antibody. Visualization of immunoreactivity was done with ECL reagents (Sigma- Aldrich, Australia). Images on X-ray Film (Amersham) were analysed by Gel documentation system (Bio-Rad, Australia).

(i) Protein Extraction Protocol

1. Retrieve tissue from -8O⁰C freezer and keep on dry ice. Use the tissue hammer to break up stock tissue while in the bag. Place sample tube on balance and zero the balance. Use the scalpel to cut tissue on the chopping board, and weigh approx. lOOmg of tissue into sample tube. Record exact weight, and store on dry ice. Stock tissues should be returned to the minus 8O⁰C freezer ASAP. 2. To each sample tube, add 1 niL of homogenising buffer, and homogenise each sample with rotor to full speed.

3. Pour the samples from the white-capped tubes into the centrifuge tubes and spin in Beckman J6 Centrifuge for 30 minutes at 1800rpm (Pre-cool centrifuge to 4°C).

4. Remove tubes from centrifuge and place on ice again. Slowly pour off the supernatant layer from the sample and place into a labelled centrifuge tube. The pellet can be discarded. The extracted protein can be stored at minus 2O⁰C if being used in the short term, or -8O⁰C for long-term storage.

(ii) SDS-Polyacrylamide Gel Electrophoresis

SDS-PAGE Preparation: Prepare the stacking gel and resolving gel in 10ml tubes. Mix thoroughly after adding each component.

Prepare 300-500ml 10% APS. Add 10ml TEMED to the resolving gel and immediately add 100ml 10% APS solution. Mix quickly by inverting to ensure no bubbles are formed. Slowly pour the resolving gel in between the glass plates and carefully overlay the resolving gel with iso-propanol (or iso-butanol).

For resolving gel, add 10ml TEMED and 100ml 10% APS solution to the stacking gel, mix quickly by inverting to ensure no bubbles are formed. Use a glass pasteur pipette, slowly add the stacking gel in between the glass plates slowly, allow air bubbles to escape from the gel as you fill up the apparatus. Once the apparatus is filled, slowly and evenly push the comb the rest of the way in. Allow the gel layer to set fully (about 30 minutes).

Sample buffer (SB) containing 5% β-mercaptoethanol is added to protein samples in a 1:1 ratio. Denature at 95°C for 5 minutes and cool on ice. Kaleidoscope molecular weight markers require no denaturation Place gel into staining fixative (if Coomassie staining the gel) or transfer buffer (if transferring).

(iii) Western Transfer Protocol 1. Remove the gel and pour on 1 x Western Buffer + 20% methanol (WBM) to cover the gel and wash for -10 min to remove the electrode buffer and equilibrate the gel

2. Cut nitrocellulose membrane and filter paper and soak in IXWBM along with filter paper.

3. On the transfer cassette, place one of the pre-wetted filter pads and place a piece of filter paper on this. Place the gel on the filter paper being careful not to tear the gel, and roll the glass rod over the gel to remove air bubbles. Place the nitrocellulose membrane on the gel, roll with the glass rod, place the other piece of chromatography paper on top of the membrane, roll with the glass rod, and place the filter pad on top of this.

4. Place the transfer cassette into the transfer tank, positioning the 'Bio-ice' cooling unit next to it. Place a magnetic stir flea in the base of the tank, and place the tank on a magnetic stirrer pad. Fill the tank with the IXWNM and place the lid on the tank.

5. Transfer for a minimum ofl hr. β.When transfer is complete, disassemble the transfer cassette, discard the gel and chromatography paper and proceed to the immunodetection protocol. (iv) Western Immunodetection Protocol

1. Wash membrane 3 X 5 minutes in TBS (IX).

2. Block the membrane in TBS-T + 5% MILK (powder form) blocking solution for lhr at room temperature.

3. Wash 3 X 5 minutes in TBS-T. 4. Dilute Primary Antibody

5. Incubate overnight at 4⁰C on the shaker in the cold room.

6. Wash membrane 3 X 5 minutes in TBS-T.

7. Dilute the 2° Ab. Pour over membrane.

8. Incubate for 1 hr at room temperature. 9. Wash membrane 3 X 5 minutes in TBS-T. 10. Prepare ECL reagents 11. Mix solutions together and incubate membrane for 1 min with agitation. Place membrane in plastic wrap and into film cassette ready for exposure of x-ray film (usually 30s, 1 min, 2.5 m and/or 5 min).

12. If membrane is to be used again, strip immediately. 13. Incubate for 30 min at 50⁰C.

14. Wash membrane 3 X 5 minutes in TBS.

15. Repeat step 2 with appropriate blocking solution.

16. Wash membrane 3 X 5 minutes in TBS.

17. Block the membrane in TBST (TBS + 0.1% Tween-20) + 5% milk (powder form) blocking solution for lhr at room temperature

18. Wash 3 X 5 minutes in TBST

19. Dilute Primary Antibody (i.e. STAT5)

STAT5 - dilute 1:250 (manufacturers recommendation) in TBST+2% milk

20. Incubate overnight at 4⁰C on the gyrating shaker in the cold room. 21. Wsh membrane 3 X 5 minutes in TBST

22. Dilute the 2° AB (HRP conjugated Donkey a-mouse IgG) eg for STAT5 -dilute 1:1000 in bocking buffer (TBST+5% milk)

23. Pour over membrane in the small tray. Make sure there are no air bubbles under membrane. Cover tray with parafilm. 24. Incubate for lhr at room temperature.

25. Wash membrane 3 X 5 minutes in TBST

26. ECL reagents

SOLUTION A: 5 ml 10OmM Tris HCL pH 8.5

22 μl Courmaric Acid (stored at -2O⁰C) 50 μl Luminol (stored at -2O⁰C)

SOLUTION B: 5 ml 10OmM Tris HCL pH 8.5

3 μl H₂O₂

27. Mix solutions A + B together and incubate membrane for 1 min with agitation.

28. If membrane is to be used again, it must be stripped immediately. Add 350 μl of β- mercaptoethanol to 50 ml of stripping solution.

29. Incubate for 30 min at 65°C.

30. Wash membrane 3 X 5 minutes in TBST

31. Repeat step 2 with appropriate blocking solution. Sample Buffer (SDS reducing buffer) 9.5ml 4.55ml MiIIiQ H2O

1.25ml 0.5M Tris-HCl; pH 6.8

2.50ml glycerol (lab N434) 1.00ml 20% SDS

0.20ml 0.5% (w/v) Bromophenol Blue dye

Store at room temperature.

Just prior to use, aliquot out the desired amount, and add 5% D- mercaptoethanol - ie 200ml SB + 10ml β-ME.

1OX Western Buffer (WB), pH 8.3 1000ml 30.3g Tris-Base

144.0g Glycine

Dissolve and bring total volume to 1000ml with MiIIiQ H₂O. pH will be 8.3, do not adjust.Store at 4⁰C, and warm to room temperature before use if it precipitates. Use at IX concentration.

Stripping Buffer 500ml

50 ml 20% SDS

31.25ml lM Tris pH 6.8

Make up to 500 ml. For working solution, add 350μl D -mercaptoethanol per 50 ml of solution

Example 4

TREK-I and TRAAK are expressed in human heart

Normal and nested amplification of TREK-I in human heart tissue was performed using TREK-I sense and antisense primers, as discussed above. The results are shown in Figure 3. Normal PCR of TREK-I produced a near 700-bp band (theoretical size 697bp), on a 1.2% Agarose gel, as shown in panel A. The 700 bp fragment was exised from the gel and used in nested PCR (panel B). This resulted in a band of approximately 400 bp as visualized on a 1.2% agarose gel (theoretical size 372bp). Sequencing of the nested PCR product deomstrated that it was identical to TREK-I in human brain as published in Genebank.

Normal and nested PCR amplification of TRAAK by specific primers in human heart tissue is shown in Figure 4. Panel A shows that normal PCR of TRAAK produced a band of approximately 480 bp (theoretical size 477bp) on a 1.2% Agarose gel. Panel B shows that cutting the nearly 480 bp band for nested PCR produced a single band of 250 bp as visualized on a 1.2% Agarose gel. Sequencing of 132 bp within this product demonstrated that the sequence is 100% homologous with that published for human TRAAK.

Example 4

Quantification of TREK-I in human atrial, ventricular and testicle tissues by real-time PCR

Mathematical models and statistical methods have been published in Liu and Saint (2002) Biochem Biophys Res Commun. 2002 294(2):347-53 and Liu and Saint (2004) Clin Exp Pharmacol Physiol. 31(3):174-8. Relative-method of real-time PCR was applied to quantify TREK-I in human atrial, ventricular and testicular tissues, in which quantification of TREK-I is based on the ratio between fluorescence value of TREK-I and GAPDH. All data from real-time PCR are analyzed by Sigmaplot (version 8.0).

Figure 5 shows real time PCR results for amplification of TREK-I and TRAAK in human atrial and testicular tissue, compared to GAPDH as internal standard.

Figure 6 shows dissociation curve analysis for real time PCR experiments, confirming no primer-dimer formation. The results for quantification of TREK-I, TRAAK and GAPDH gene expression obtained in 7 samples of human atrial tissue frozen for 12 months are shown in Table 1.

Table 1

The results for quantification of TREK-I, TRAAK and GAPDH gene expression obtained in 3 samples of fresh human atrial tissue are shown in Table 2.

Table 2

The results for quantification of TREK-I, TRAAK and GAPDH gene expression obtained in 5 samples of human ventricular tissue frozen for 12 months are shown in Table 3. Table 3

The results for quantification of TREK-I, TRAAK and GAPDH gene expression obtained in 1 sample of fresh human ventricular tissue and one sampled frozen for 3 months are shown in Table 5.

Table 5

Figure 7 shows the expression level of TREK-I and TRAAK in human atrial tissue and testicular tissue, quantified as a proportion of GAPDH expression as an internal standard. In this case, 7 human atrial samples (5 females and 2 males) that had been stored at -80° C for at least 12 months, and human testicular tissue (from one patient) used 3 hours after surgery, were analysed by real-time PCR.

Figure 8 shows the expression level of TREK-I and TRAAK in human atrial tissue and testicular tissue, quantified as a proportion of GAPDH expression as an internal standard. In this case, fresh human atrial tissue (3 hours after surgery, 3 females) and human testicular tissue (from one patient) used 3 hours after surgery, were analysed by real-time PCR.

Figure 9 shows the expression level of TREK-I and TRAAK in human frozen ventricular tissue and testicular tissue, quantified as a proportion of GAPDH expression as an internal standard. In this case, in frozen human ventricular tissue (-80 ⁰C for 12 months) and human testicular tissue (from one patient) used 3 hours after surgery, were analysed by real-time PCR.

Figure 10 shows effects of storage on expression level of TREK-I and TRAAK in human testicle tissue. A sample of fresh testicular tissue was divided into two equal- weight parts. One part was used in real-time PCR immediately, whereas the other part was stored at -80 ⁰C for 3 months before being used for real-time PCR.

The gene expression level of TREK-I in human atrial and ventricular tissue is similar in magnitude to that of GAPDH. Expression levels are higher in ventricle than in atria. TRAAK expression levels are much lower than TREK-I.

In conclusion, the results demonstrate that the gene expression level of TREK-I in human atrial and ventricular tissue is similar in magnitude to that of GAPDH. Expression levels are higher in ventricle than in atria. TRAAK expression levels are generally much lower than TREK-I.

Example 5

TREK-2a and TREK-2c are expressed in human atrial tissue

PCR amplification of cDNAs from human atrial tissue (there samples) was performed using TREK-2a, TREK- 2b and TREK-2C specific primers. The date is shown in Figure 11.

As can be seen, PCR amplification with the TREK-2a primers resulted in the detection of a 1065 bp band in each of the three samples tested. The size of the band detected was consistent with the exprected size of the amplification product using the TREK-2a primers.

PCR amplification with the TREK- 2c primers resulted in the detection of a 961 bp band in each of the three samples tested. The size of the band detected was consistent with the exprected size of the amplification product using the TREK-2c primers. However, PCR amplification with the TREK-2b primers did not result in the detection of an appropriate band (945 bp) in each of the three samples tested.

To determine whether TREK-2a is expressed in human ventricle tissue, PCR amplification of cDNAs from human ventricle tissue was also performed.

Nested PCR was applied to the 1065 bp band detected for TREK-2a and the 961 bp band detected for TREK-2c. The data is shown in Figure 12A and 12B. Nested PCR for TREK-2a (Fig. 12A) and nested PCR for TREK-2c (Fig. 12B) resulted in the amplification of bands of sizes consistent with the 147 bp and 647 bp as expected.

Figure 13 shows the results of Western analysis of 6 atrial samples using an anti TREK- 2 antibody (from Alomone labs, Antibody AntiOK2plO.l, Cat # APC-055). Bands of 61 kDa and 59 kDa were detected, consistent with the sizes of the TREK-2a (59 kDa) and TREK-2c (61 kDa) forms.

The above data confirms that TREK-2a and TREK- 2c are expressed in human atrial tissue, but that TREK-2b is not expressed in this tissue.

Example 6

TREK-2a is expressed in human venticle

To determine whether TREK-2a is expressed in human ventricle tissue, PCR amplification of cDNAs from human ventricle tissue was performed.

Figure 14 shows the results of PCR amplification with TREK-2a primers on cDNAs isolated from human ventricle tissue. The size of the product expected to to be amplified for TREK-2a was 647 bp. As shown in Figure 14, a PCR product of this size was produced, demonstrating that TREK-2a is exressed in human ventricle tissue. Nested PCR method was applied to the 647 bp TREK-2a PCR product. The theoretical sizes of products produced for TREK-2a are 196bp and 255bp. Figure 15 shows that bands of this size were produced upon nested PCR, confirming expression of the TREK-2a mRNa in ventricle tissue.

Sequencing of the nested PCR product identified the product sequence as human TREK-2a, by BLAST comparison with the human sequence published in Genebank.

These data confirm that TREK-2a is expressed in the human ventricle.

Example 7

TREK-2b is not expressed in human heart ventricle tissue

PCR amplification of cDNAs from human ventricle tissue was also used to assess whether TREK-2b is expressed in human ventricle tissue. PCR amplification with TREK-2ab primers on cDNAs isolated from human ventricle tissue is shown in Figure 16. The size of the product expected to be amplified for TREK-2b was 946 bp, but as can be seen from the Figure, no band of this size was produced.

This data indicates that TREK-2b is also not expressed in human ventricle tissue.

Example 8

TREK-2c is expressed in human ventricle tissue

Figure 16 shows the results of PCR amplification with TREK- 2c primers on cDNAs isolated from human ventricle tissue. The size of the product expected to to be amplified for TREK- 2c was 952 bp. As shown in Figure 16, a PCR product of this size was produced, demonstrating that TREK-2c is exressed in human ventricle tissue.

Nested PCR method was applied to the 952 bp TREK-2c PCR product. The theoretical sizes of products produced for TREK- 2c are 196bp and 255bp. Figure 17 shows that bands of this sixe were produced upon nested PCR, confirming expression of the TREK-2c mRNa in ventricle tissue.

Sequencing of the nested PCR product identified the product sequence as human TREK-2c, by BLAST comparison with the human sequence published in Genebank.

These data confirm that TREK-2c is expressed in the human ventricle.

Example 9

Quantification of TREK-2a and 2c in human atrial and ventricular tissue by Western Blot experiments

Real-time PCR could not be used to quantify the expression level of the TREK-2 splice variants since specific primers suitable for real-time PCR could not be designed to the different sequences. However, since the variants have different molecular weights, it was possible to quantify them at the protein level using Western blot. 6 human right atrial samples were used. Non-mammalian tissue was used as negative control. The anti-TREK-2 primary antibody recognized Va (59KDa) and Vc (61KDa) (Fig 18). OD values of Va and Vc in 6 samples demonstrated that the protein expression of Vc was 1.5 times more than that of Va in human atrial tissue (1.85 ± 3% compared to 1.19 ± 5% respectively). For human ventricular samples, different sample concentrations were applied to detect the quantity of Va and Vc, (25μg/30μL, 20μg/30μL and 6μg/20μL). With decreasing gel loading, the level of Va fell below the level of detection before that of Vc (Figure 19), indicating that the expression level of Vc was higher than that of Va in these tissues. With a sample concentration of 6μg/20μL, Vc was detected on the nitrocellulose membrane whereas Va was not detected. Hence we conclude that Vc is expressed at a higher level in both atrial and ventricular tissue from patients with no obvious cardiac disease. Example 10

Expression levels of TREK-2a and TREK-2c in normal and pathological human ventricular samples

Figure 20 shows Western blot analysis of Va and Vc in 2 ventricular samples of ischemic cardiomyopathy (sample 1&2) and 5 ventricular samples of idiopathic dilated cardiomyopathy (samples 3 to 7) with identical sample concentrations of 20μg/30μL. [primary Ab] =1:500; [secondary Ab] =1:1000.

Figure 21 shows a comparison of expression level of variant A and C between normal human ventricular samples (panel A) and pathological ventricular samples (panel B) from Western blot experiments.

The results indicate that unlike the situation in normal ventricle, TREK-2c in pathological samples is elevated with respect to TREK-2a.

Example 11

lmmunohisto chemistry

Four fresh cardiac atrial appendages and four frozen ventricle tissues were used for immunohistochemsitry. Preparations were washed with phosphate buffered saline (PBS: 0.15 M NaCl in 0.01 M sodium phosphate, pH 7.2) and fixed (15 % saturated picric acid and 2% formaldehyde in phosphate buffer 0.1 M) overnight at 4⁰C. They were then cleared in DMSO (3 x 10 minute) and washed three times in PBS. Preparations were then excised into 2-5 mm thick blocks, dehydrated in a series of concentrations (70-100%) of ethanol, 100% safsolvent and embedded in wax. Paraffin sections (5 μm thick) were cut with a microtome (Leitz, Germany) and placed on chromium potassium sulphate- gelatin coated glass slides. After removal of paraffin wax with 100% safsolvent and washing in a series of concentrations (100-70%) of ethanol and PBS, the sections were incubated with 20% normal horse serum for at least 60 minutes. The preparations were then incubated with antisera against TREK-2 (raised in rabbit, dilution: 1:1000, Alomone AN-Ol) at 4 ⁰C for 72 hours. After three washes in PBS, sections were incubated with donkey anti-rabbit CY3 (1:50, Jackson 63578) secondary antisera at room temperature for two hours. After several washes in PBS, preparations were then mounted in 100% buffered glycerol and viewed on an Olympus AX70 epifluorescence microscope with appropriate filter blocks. Digital images were taken with Precision Digital Imaging System (V++, digital optics limited, Auckland, New Zealand). Negative controls were carried out by omission of primary antibody during the incubation.

Example 12

lmmunohisto chemistry of TREK-2a and TREK-2c in human atrial and ventricular tissues

In order to confirm the tissue localisation of the variants of TREK-2 in atrial and ventricular tissue, immunohistochemistry was performed. In human atrial and ventricular tissue, cardiac myocytes are formed in bundles separated by connective tissue and small blood vessels (Fig 22A). In paraffin sections of human atrial tissue, TREK-2 immunohistochemical reactivity was observed as bright punctate granules in the cardiac myocytes, with some of this immunoreactivity along the membrane of the cardiac myocytes. No connective tissue was labelled. No immunoreactivity was found in negative control preparation after omission of primary antibody (Fig 22B).

In human ventrical tissue there was much less connective tissue between the muscle bundles. TREK-2 immunohistochemical reactivity was also observed as bright punctate granules in cardiac myocytes in ventricular tissue, with no connective tissue labelling

(Fig 22C). TREK-2 immunoreactivity appeared to be distributed in the myocytes with the same spacing as the t- tubule system (magnified image in fig 22D). No immunoreactivity was found in negative control preparation after omission of primary antibody (Fig 22E).

Figure 22 shows immunohistochemistry of TREK-2a and TREK-2c in human atrial and ventricular tissues. Panel A: TREK-2 immunoreactivity in paraffin section of human atrial appendage. Within the muscle bundle the cardiac myocytes are different in diameter and some of them show an empty area without myofibrils in the nuclear region (filled arrows). Punctate fine granules of TREK-2 immunoreactivity were located in most of cardiac myocytes. Some of TREK-2 positive granules were located along the membrane of the cardiac myocytes (hollow arrow). Connective tissue between the muscle bundles (arrow head) and centre of nuclear region in cardiac myocyte (filled arrows) were not labelled. No immunoreactivity was found in negative control preparation (Panel B). Calibration bar: 50 μm. Panel C: TREK-2 immunoreactivity in paraffin section of human cardiac ventricle. TREK-2 immunoreactivity was located in most of cardiac myocytes and their locations were similar to that in atrial myocytes, along the membrane (hollow arrow) and in the cytoplasm. Connective tissues between the muscle bundles (arrow head) were not immunoreactive. In an expanded view (Panel D) the immunoreactivity appeared to be spaced in striations. No immunoreactivity was found in negative control preparation (Panel E). Calibration bar: 50 μm.

By way of summary, the results demonstrate that the splice variants of TREK-2 Va and Vc, but not Vb, are expressed in human atrial and ventricular tissues and that TREK-2 channels are located immunohistochemically in the myocytes in both tissues. The proportion of expression of each of the splice variants was altered in cardiac disease (ischaemic cardiomyopathy or idiopathic dilated cardiomyopathy) compared to donor hearts.

Example 13

Expression of TREK-I in human pathological conditions

For Western Blot experiments of all the samples, primary antibodies of GAPDH and TREK-I were diluted into 1:1000 and secondary antibodies of HRP-Donkey- anti- Rabbit and HRP-Donkey-anti-Mouse were diluted into 1:500. cDNA used in real-time PCR was decreased to lμL and the total volume of real-time PCR reaction was lOμL. Data of Western Blot experiments were calculated by Gel-Dock system (Bio-Rad, Australia). The results for expression of TREK-I in human pathological samples (40yr - 60yr) are shown in Table 6.

Table 6

The expression level of TREK-1/GAPDH in human Ischemia Cardiomyopathy, Idopathic Dilated Cardiomyopathy and valvular diseases is also shown in Figure 23, based on Table 6. TREK-I was expressed more highly in the groups of IDC and IC than valvular disease, in which 0.44±11.47% in IDC (n=7), 0.35± 6.83% in IC (n=4) and 0.01 in Valvular disease (n=l).

Table 7 shows a comparison of expression of TREK-I in normal and pathological human ventricular samples (40yr - 60yr). Table 7

The data is also shown in Figure 24. There were remarkable differences in the expression level of TREK-1/GAPDH in human donor and pathological groups, all of which were in the range of 40 yr to 60 yr. Quantity of TREK-I in IDC (0.44+11.47%; n=7) was 5 times more than that of Donor group (0.08+1.56%; n=5) whereas TREK-I in IC (0.23+7.96%; n=3) was 3 times more than that of Donor group (0.08+1.56%; n=5). The expression level of TREK-I in IDC group was notably higher than human donor group.

Figure 25 shows that TREK-I and GAPDH (positive control) were recognized by specific antibodies in human donor ventricle samples (n=4). [Protein supernatant]= 20μg/30μL; [primary Ab]= 1:1000; [secondary Ab]= 1:500. The data is also provided in Table 8. Table 8

Figure 26 shows that TREK-I was expressed in human IDC samples by Western blot analysis (n=7). GAPDH was used as a positive control. [Protein supernatant]= 20μg/30μL; [primary Ab]= 1:1000; [secondary Ab]= 1:500. The data is also shown in Table 9.

Table 9

Figure 27 shows the expression level of TREK-1/GAPDH in IDC and donor samples based on Table 8 and 9.

Protein level of TREK-I was parallel with real-time PCR results of TREK-I in IDC and donor samples. Intensity of TREK-I protein in IDC (1.80±14.26%; n=7) was 2.13 times more than that of TREK-I in Donor group (0.84±7.46%; n=7) (P<0.00028) whereas 5 times more than TREK-I in Donor group at mRNA level (P≤O.001).

The location of TREK-I channel in atrial appendage was revealed by immunohistochemical labelling with specific antibody for TREK-I channels. In paraffin section, human atrial cardiac myocytes are formed in bundles, which are separated by connective tissue (Fig 28A). TREK-I immunoreactivity was observed as bright punctate granules in most of cardiac myocytes. Many of them were found along the membrane of the cardiac myocytes (Fig 28A). No connective tissue was labelled. No immunoreactivity was found in negative control preparation after omission of primary antibody (Fig 28B).

In human ventricular tissue, cardiac myocytes are also formed in bundles but of larger size. There was much less connective tissue between the muscle bundles. TREK-I immunoreactivity was also observed as bright punctate granules in ventricular cardiac myocytes, with no connective tissue labelling (Fig 28C). No immunoreactivity was found in negative control preparation after omission of primary antibody (Fig 28D).

Figure 29 shoes immunohistochemistry for TREK-I in normal and disease cardiac mycocytes of the human ventricle. Significant overexpression of TREK-I is seen in diseased cardiac mycocytes.

Example 14

Electrophysiology in Transfected COS Cells

COS cells may be seeded at a density of 20,000 cells/35 mm dish 24 h before transfection. Cells may be transiently transfected by the DEAE-dextran method with a plasmid for expression of TREK-I or TREK-2, or with a suitable control plasmid.

For whole-cell experiments, the patch electrode solution (INT) will contain 150 mM KCl, 3 mM MgCl₂, 5 mM EGTA, and 10 mM HEPES, adjusted to pH 7.3 with KOH; the external solution (EXT) will contain 150 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 1 niM CaCl₂, and 10 mM HEPES, adjusted to pH 7.4 with NaOH. For outside-out patch recordings, the pipette solution will be the INT solution, and the external solution the EXT solution (5 mM K+) or a K⁺-rich EXT solution that contains 150 mM KCl instead of 150 mM NaCl. For inside-out patch recordings, pipettes may be filled with the EXT solution, and the bathing solution the INT solution buffered either at pH 7.3 or at pH 5.6 in the internal acidosis experiments. Cells will be continuously superfused with a microperfusion system during the experiment (0.2 ml/min) to be performed at room temperature. A patch-clamp amplifier may be used for whole-cell and single-channel recordings.

Finally, it will be appreciated that various modifications and variations of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the present invention.

Claims

Claims:

1. A method of modulating cardiac rhythm in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

2. A method according to claim 1, wherein the activity of TREK-I and/or TREK- 2 in the heart of the subject is decreased.

3. A method according to claim 2, wherein the decrease in activity of TREK-I and/or TREK-2 results in a reduction in outward potassium currents in cardiac cells of the subject and/or prolongs action potential duration in the heart of the subject.

4. A method according to any one of claims 1 to 3, wherein the modulation of activity of TREK-I and/or TREK-2 includes modulation of the activity in the ventricle of the heart of the subject.

5. A method according to any one of claims 1 to 4, wherein the modulation of TREK-2 activity includes modulation of the activity of TREK-2a and/or TREK-2c.

6. A method according to any one of claims 1 to 5, wherein the method is used to prevent and/or treat an arrhythmia.

7. A method according to any one of claims 1 to 5, wherein the method is used to prevent and/or treat supraventricular tachycardia, including atrial flutter and atrial fibrillation, ventricular tachycardia and ventricular fibrillation.

8. A method according to any one of claims 1 to 5, wherein the method is used to control ventricular rate in the presence of atrial flutter or fibrillation.

9. A method according to any one of claims 1 to 5, wherein the method is used to control an arrhythmia arising from one or more of the group consisting of cardiac hypertrophy, cardiac myopathy, cardiac ischemia and a condition which produces abnormal motion of the ventricular wall, including an infarction.

10. A method according to any one of claims 1 to 9, wherein the modulation of activity of TREK-I and/or TREK-2 includes administering to the subject an effective amount of an agent that modulates the activity of TREK-I and/or TREK-2 in the heart of the subject.

11. A method of preventing and/or treating a disease or condition associated with altered cardiac rhythm in a human subject, the method including the step of administering to the human subject an effective amount of an agent that modulates the activity of TREK-I and/or TREK-2 in the heart of the subject.

12. Use of an agent that modulates the activity of TREK-I and/or TREK-2 in the preparation of a medicament for preventing and/or treating a disease or condition associated altered cardiac rhythm in a human subject.

13. A method of reducing the risk of a human subject suffering from a disease or condition associated with altered cardiac rhythm, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

14. A method of identifying a compound that modulates cardiac rhythm in a human subject, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the heart of a human subject.

15. A method according to claim 14, wherein the method of identification involves identifying a compound that decreases the activity of TREK-I and/or TREK-2 in the heart of the subject.

16. A method according to claims 14 or 15, wherein the method of identification includes the further step of identifying the compound as a compound that reduces outward potassium currents in cardiac cells of the subject and/or prolongs action potential duration in the heart of the subject.

17. A method according to any one of claims 14 to 16, wherein the method of identification includes modulation of the activity of TREK-I and/or TREK-2 in the ventricle of the heart of the subject.

18. A method according to any one of claims 14 to 17, wherein the method of identification includes modulation of the activity of TREK-2a and/or TREK- 2c.

19. A compound identified according to any one of claims 14 to 18.

20. A method of identifying a compound that modulates cardiac rhythm in a human subject, the method including the steps of:

(a) identifying a compound that modulates the activity of TREK-I and/or TREK-2;

(b) determining the ability of the compound so identified to modulate cardiac rhythm in a human subject; and

(c) identifying the compound as a compound that modulates cardiac rhythm in a human subject.

21. A compound identified according to the method of claim 21.

22. A TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates cardiac rhythm in a human subject.

23. A protein according to claim 22, wherein the protein is a human TREK-I or TREK-2 protein, or a variant or fragment thereof.

24. A protein according to claims 22 or 23, wherein the protein is expressed in a cell.

25. A non-human cardiac cell, the cell expressing human TREK-I and/or TREK-2, or a variant or fragment thereof.

26. A non-human animal including cardiac cells expressing human TREK-I and/or TREK-2, or a variant or a fragment thereof.

27. A method of identifying a human subject suffering from, or susceptible to, a disease or condition associated with altered cardiac rhythm, the method including the step of identifying an altered activity of TREK-I and/or TREK-2 in the subject.

28. A method according to claim 27, wherein the method of identifying an altered activity of TREK-I and/or TREK-2 in the subject includes the step of identifying a mutation in the subject that alters the activity of TREK-I and/or TREK-2.

29. A method according to claim 28, wherein the mutation is in the TREK-I gene or the TREK-2 gene.

30. A method of preventing and/or treating a disease or condition selected from the group consisting of cardiac hypertrophy, cardiac myopathy and cardiac ischemia in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

31. A method according to claim 30, wherein the activity of TREK-I and/or TREK-2 in the heart of the subject is increased.

32. A method according to claims 30 or 31, wherein the modulation of TREK-2 activity includes modulation of the activity of TREK-2a and/or TREK-2c.

33. A method according to any one of claims 30 to 32, wherein the modulation of activity of TREK-I and/or TREK-2 includes administering to the subject an effective amount of an agent that modulates the activity of TREK-I and/or TREK-2 in the heart of the subject.

34. A method of modulating K⁺ current activity in a human cardiac cell, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the cell.

35. A method of identifying a compound that modulates K⁺ current activity in a human cardiac cell, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the cell.

36. A compound identified according to the method of claim 35.

37. A TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates K⁺ current activity in a human cardiac cell.

38. A method of modulating cardiac K⁺ current activity in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of a human subject.

39. A method of identifying a compound that modulates cardiac K⁺ current activity in a human subject, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the heart of a human subject.

40. A compound identified according to the method of claim 39.

41. A TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates cardiac K⁺ current activity in a human subject.

42. A method of preventing and/or treating a disease or condition associated with altered cardiac K⁺ current activity in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

43. A method of reducing the risk of a human subject suffering from a disease or condition associated with altered cardiac K⁺ current activity in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

44. A method of identifying a human subject suffering from, or susceptible to, a disease or condition associated with altered cardiac K⁺ current activity, the method including the step of identifying an altered activity of TREK-I and/or TREK-2 in the subject.

45. A method of modulating cardiac action potential in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

46. A method of identifying a compound that modulates cardiac action potential in a human subject, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the heart of a human subject.

47. A compound identified according to the method of claim 46.

48. A TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates cardiac action potential in a human subject.

49. A method of preventing and/or treating a disease or condition associated with altered cardiac action potential in a human subject, the method including the step of modulating the activity of TREK-2 in the heart of the subject.

50. A method of reducing the risk of a human subject suffering from a disease or condition associated with altered cardiac action potential in a human subject, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the heart of the subject.

51. A method of identifying a human subject suffering from, or susceptible to, a disease or condition associated with altered cardiac action potential, the method including the step of identifying an altered activity of TREK-I and/or TREK-2 in the subject.

52. A method of modulating the action potential of a human cardiac cell, the method including the step of modulating the activity of TREK-I and/or TREK-2 in the cell.

53. A method of identifying a compound that modulates the action potential of a human cardiac cell, the method including the step of identifying a compound that modulates the activity of TREK-I and/or TREK-2 in the cell.

54. A compound identified according to the method of claim 53.

55. A TREK-I or a TREK-2 protein, or a variant or fragment thereof, when used as a target for identifying a compound that modulates cardiac action potential in a human cardiac cell.