WO2000056887A1

WO2000056887A1 - Katp channel

Info

Publication number: WO2000056887A1
Application number: PCT/GB2000/001130
Authority: WO
Inventors: Frances Mary Ashcroft; Stefan Trapp; Sakura Hiroshi
Original assignee: Isis Innovation Limited
Priority date: 1999-03-24
Filing date: 2000-03-24
Publication date: 2000-09-28
Also published as: AU3444500A; GB9906843D0

Abstract

There is disclosed a nucleic acid molecule encoding a VMH KATP channel subunit designated SUR1bΔ33 comprising the amino acid sequence according to SEQ ID No 2 or the amino acid sequence of a functional equivalent, derivative or bioprecursor thereof. The subunit expressed also forms part of the invention. A VMH KATP channel comprising a Kir6.x subunit and a SUR1bΔ33 subunit is also disclosed.

Description

K_ATP CHANNEL

The present invention is concerned with an ATP sensitive K channel and, in particular, with such a channel which is found in hypothalamic neurones.

The ventromedial hypothalamus (VMH) plays an important role in glucose homeostasis by monitoring glucose status and regulating food intake. Glucose-receptive neurones within this nucleus are spontaneously active in the presence of glucose, but removal of the sugar produces membrane hyperpolarization and cessation of electrical activity (Ono et al., 1982). This effect requires glucose metabolism and is mediated by activation of ATP-sensitive potassium (K_ATP) channels, whose activity is inhibited by an increase in intracellular ATP (Ashford et al., 1990a). Leptin also mediates hyperpolarization of VMH neurones by activation of K_ATP channels (Spanswick et al., 1997).

K_ATP channels are found in a wide variety of tissues (Ashcroft, 1988), most notably the pancreatic β-cell where they play a key role in glucose-dependent insulin secretion (Ashcroft & Rorsman, 1989) . There are a number of differences between the properties reported for the K_ATP channel of VMH neurones and that of the β-cell K_AτP channel. First, the ATP-sensitivity of the VMH channel ( - 3mM; Ashford et al . , 1990a) is lower than that of the β-cell channel ( . ~ 10 μM, Ashcroft & Rorsman, 1989) . Secondly, whereas the β- cell K_ATP channel is blocked by the sulphonylurea tolbutamide in both cell-attached and excised membrane patches (Ashcroft & Ashcroft, 1992), the inhibitory effect of the drug on VMH K_AT? channels is lost following patch excision (Ashford et al., 1990b).

Thirdly, in contrast to the β-cell K_ATP channel (Ashcroft & Rorsman, 1989) , K-channel openers do not stimulate the activity of VMH K_AT? channels (Sellars et al., 1992) . The single-channel conductance of the β- cell K_ATP channel is 60-80 pS in symmetrical 140 mM k^~ (Ashcroft, 1988).

Molecular analysis of the β-cell K_ATP channel has revealed that it is an octameric (4:4) complex of two different types of subunit: Kir6.2 and SURl (Clement et al., 1997; Inagaki., 1997; Shyng et Al . , 1997c).

Kir6.2 is an inwardly-rectifying K⁺ (Kir) channel and SURl (the sulphonylurea receptor) is a member of the ATP-binding cassette (ABC) transporter superfamily (Inagaki et al., 1995a; Sakura et al., 1995; Aguilar- Bryan et al., 1995). It is now clear that Kir6.2 forms the channel pore and contains the site at which ATP mediates channel inhibition (Tucker et al., 1997; 1998). SURl serves as a regulatory subunit that endows the K_ATP channel with sensitivity to the stimulatory effects of MgADP and K⁺ channel openers and to the inhibitory effects of sulphonylureas (Nichols et al., 1996; Gribble et al . , 1997a, c; Shyng et al., 1997a; Tucker et al., 1997): in addition, it enhances the blocking action of ATP (the K_± shifts from 100 μM to 10 μM) and increases the channel open probability (Proks & Ashcroft, 1997; Tucker et al., 1997; Trapp et al., 1998).

To date, two Kir6.x genes have been described, Kirδ.l and Kir6.2 (Inagaki et al . , 1995a, b; Sakura et al . ,

1995) . Two closely related SURx genes, SURl AND SUR2, have also been cloned, the latter being alternatively spliced to give SUR2A and SUR2B (Aguilar-Bryan et al., 1995; Inagaki et al., 1996; Isomoto, 1996). Kir 6.2 serves as a common pore-forming subunit for K_ATP channels in a number of tissues, while the different SUP subunits account for the variable sensitivity to K channel openers and sulphonylureas . Thus, the β- cell K_A-._P channel is composed of Kιr6.2 and SURl, that in cardiac and skeletal muscle consists of Kιr6.2 and SUR2A (Inagaki et al . , 1996) and single-cell PCR studies have identified both Kιr6.2/SUR1 and Kιr6.2/SUR2B combinations in vagal and substantia nigra neurons (Liss et al . , 1997; Karschin et al., 1998) .

The identity of the K_ATP channels m ventro edial hypothalamic neurones is, however, far from clear since the show very different single-channel properties, ATP-sensitivity, and pharmacology from the "classical" K_ATP channel, exemplified by the β-cell channel. It has not been established whether the VMH K_ATo channel is related to those K_ATP channels cloned from other tissues or indeed if they represent a separate entity.

The present inventors have now found that the K_A-₃ channel of VMH neurones does consist of an SUR subunit, which associates with an, as yet unidentified pore-forming subunit. The latter may comprise Kιr6.2 but is more likely to be a novel type of K channel. Many of the different properties of the VMH channel result from the novel splice variant of tne SURl gene that the inventors have now identified. The novel SURl isoform that has been identified, when coexpressed with Kιr6.2, reproduces the pharmacological properties of the native K_^-,,. channel of ventromedial hypothalamic neurones. This isoform may also provide fresh insight into tne mechanism by which SURl regulates the properties of Kιr6.2. Therefore, according to a first aspect of the invention there is provided a nucleic acid molecule encoding a VMH K_AT. channel subunit designated SU^plbΔ33 comprising the amino acid sequence illustrated in Figure lb or the ammo acid sequence of a functional equivalent, derivative or bioprecursor thereof. The nucleic acid molecule may be a DNA, such as a genomic DNA or cDNA which comprises the sequence illustrated in Figure lc. The DNA may be double stranded or single stranded. If it is single stranded it may comprise the coding strand or non-coding (antisense) strand.

In accordance with the present invention a functional fragment, derivative or equivalent when referring to a polypeptide according to the present invention should be taken to mean a polypeptide which exhibits essentially the same biological function or activity as the polypeptide according to the invention.

Also provided by the present invention is a nucleic acid molecule, such as an antisense molecule, capable of hybridising to the molecule according to the invention under high stringency conditions. Stringency of hybridisation as used herein refers to conditions under which polynucleic acids are stable. The stability of hybrids is reflected in the melting temperature (T ) of the hybrids. Tm can be approximated by the formula:

! 1 . 5°C+ 16 . 6 ( log₁₀ [ Na⁺ ] +0 . 41 ( %G&C ) - 600 / 1

wherein "1" is the length of the hybrids in nucleotides. Tm decreases approximately 1-1.5°C with every 1% decrease in sequence homology. The term "stringency" refers to the hybridisation conditions wherein a single-stranded nucleic acid 301ns with a complementary strand when the puπne or pyrimidme bases therein pair with their corresponding base by hydrogen bonding. High stringency conditions favour homologous base pairing whereas low stringency conditions favour non-homologous base pairing.

"Low stringency" conditions comprise, for example, a temperature of about 37°C or less, a formamide concentration of less than about 50%, and a moderate to low salt (SSC) concentration; or, alternatively, a temperature of about 50°C or less, and a moderate to high salt (SSPE) concentration, for example 1M NaCl .

"High stringency" conditions comprise, for example, a temperature of about 42°C or less, a formamide concentration of less than about 20%, and a low salt (SSC) concentration; or, alternatively, a temperature of about 65°C, or less, and a low salt (SSPE) concentration. For example, high stringency conditions comprise hybridization in 0.5 M NaHP0₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C (Ausubel, F.M. et al . Current Protocols in Molecular Biology, Vol. I, 1989; Green Inc. New York, at 2.10.3) .

"SSC" comprises a hybridization and wash solution. A stock 20X SSC solution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0.

"SSPE" comprises a hybridization and wash solution. A IX SSPE solution contains 180 mM NaCl, 9mM Na_;HPO and 1 mM EDTA, pH 7.4. The nucleic acid capable of nybridising to nucleic acid molecules according to the invention will generally be at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to tne nucleotide sequence illustrated in Figure lc.

The term "homologous" describes the relationship between different nucleic acid molecules or ammo acid sequences wherein said sequences or molecules are related by partial identity or similarity at one or more blocks or regions within said molecules or sequences .

According to a further aspect of the present invention, there is provided a VMH K_ATP channel subunit protein designated SURlbΔ33 or a functional equivalent, derivative or bioprecursor thereof, encoded by a nucleic acid molecule according to the invention. Preferably, the protein encoded by said nucleic acid molecule according to the invention comprises the ammo acid sequence illustrated in figure lb. The present invention also comprises within its scope not only proteins or polypeptides encoded by the nucleic acid molecules according to the invention, but functional equivalents, derivatives or bioprecursors thereof. Alternatively the polypeptide may be a recombinant polypeptide or a synthetic polypeptide prepared according to procedures known in the art.

The sequence of SURlb is identical to that which has been deposited in GenBank as Accession No. X97279. Reference to "SURlbΔ33" designated herein refers to a 131 bp deletion in the sequence of SURlb, which corresponds to exon 33. The nucleotide sequence for SUPlbΔ33 is identified in Figures lb. The 133 bp deletion introduces a frame shift which stops the open reading frame after 25 amino acids, truncating the protein at residue 1355.

There is also provided by the present invention a VMH K_ATP channel subunit designated SURlbΔ33 or a functional equivalent thereof, comprising an ammo acid sequence illustrated in Figure lb. Preferably, the SURlbΔ33 subunit is of human origin and also which may be encoded by a nucleic acid molecule as defined herein .

Also provided by the present invention is a VMH K_ATC channel comprising a Kirδ.x subunit and a SURlbΔ33 subunit according to the invention. According to the invention reference to "Kιr6.x" should be taken to mean the Kirδ.x family of subunits, which presently includes Kirβ.l and Kιr6.2 (Inagaki et al. 1995, 1996, Sakura et al. 1995) . Thus, the K_ATD channel may include either of the two previously identified Kιr6 subunits or isoforms thereof. Preferably, the Kirδ.x subunit comprises the sequence of the known subunit Kιr6.2.

The DNA molecules according to the invention may be included in a suitable expression vector to express said proteins m a suitable host. Incorporation of cloned DNA into a suitable expression vector for subsequent transformation of said cell and subsequent selection of transformed cells, is known to those skilled in the art as provided m Sambrook et al . , Molecular Cloning, A Laboratory Manual Cold Spring Harbour Laboratory Press. Thus, also provided by the present invention is a DMA expression vector, comprising a nucleic acid molecule according to the invention and which vector may, advantageously, be used to transform, transect or infect a host cell or organism to express the subunit according to the invention.

An expression vector according to the invention includes the nucleic acid sequences according to the invention operably linked to regulatory control sequences, such as promoter regions, that are capable of initiating transcription of said nucleic acid molecules. The term "operably linked" refers to a juxta position wherein the components described are in a relationship permitting them to function in their intended manner. Such vectors, as aforementioned, may be used to transform a suitable host cell to provide for expression of a subunit or channel according to the invention. Thus, in a further aspect, the invention provides a process for preparing subunits or channels according to the invention which comprises cultivating a host cell, transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the subunits or channels, and recovering the expressed proteins.

In this regard the nucleic acid molecule may encode a mature protein or a protein having a prosequence including encoding a leader sequence on the preprotein which is cleaved by the host cell to form a mature protein .

The vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for initiating transcription of said molecule and optionally a regulator of the promoter .

The vectors may include a coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the invention. In addition the vector may contain a sequence encoding a phenotypic trait for selection of transformed cells, such as, for example, ampicillin resistance.

Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector may include a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors may be obtained commercially or assembled from the sequences described by methods well known in the art.

In accordance with the present invention, a defined nucleic acid includes not only the identical nucleic acid but also any minor base variations including, in particular, substitutions in cases which result in a synonymous codon (a different codon specifying the same amino acid residue) due to the degenerate code in conservative amino acid substitutions. The term "nucleic acid sequence" also includes the complementary sequence to any single stranded sequence given regarding base variations.

Advantageously, human allelic variants or polymorphisms of the DNA molecule accorαing to the invention may be identified by, for example, probing cDNA or genomic libraries from a range of individuals, for example, from different populations. Furthermore, nucleic acids and probes according to the invention may be used to sequence genomic DNA from patients using techniques well known m the art, such as the Sanger Dideoxy chain termination method, which may, advantageously, ascertain any predisposition of a patient to certain disorders associated with a growth factor according to the invention.

The present invention also advantageously provides nucleic acid molecules of approximately from 5 to 50 contiguous nucleotides of a nucleic acid molecule according to the invention and preferably from 15 to 50 nucleotides. The sequences may, advantageously, be used as probes or primers to initiate replication, or the like. Such nucleic aciα sequences may be produced according to techniques well known m the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting for the presence of a nucleic acid molecule according to the invention. These tests generally comprise contracting the probe with the sample under hybridising conditions and detecting for the presence of any complex formation between the probe and any nucleic acid in the sample.

According to the present invention these probes may be anchored to a solid support. Preferably, they are present on an array so that multiple probes can simultaneously hybridize to a single biological sample. The probes can be spotted onto the array or syr.thesised in si tu on the array. (See Locknart et a l . , Nature Biotechnology, vol. 14, December 1996 "Expression monitoring by hybridisation to high density oligonucleotide arrays". A single array can contain more than 100, 500 or even 1,000 different probes in discrete locations.

The nucleic acid molecules according to the invention may be produced using recombinant or synthetic means, such as, for example, using PCR cloning mechanisms which generally involve making a pair of primers, which may be from approximately 15 to 50 nucleotides to a region of the gene which is desired to be cloned, bringing the primers into contact with mRNA, cDNA or genomic DNA from a human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the implicated region or fragment and recovering the amplified DNA. Generally, such techniques as defined herein, are well known in the art, such as described in Sambrook et al., (Molecular Cloning: A Laboratory Manual) .

The nucleic acid molecules according to the invention may carry a revealing label. Suitable labels include radioisotopes such as ³²p or ³⁹s enzyme labels or other protein labels may be added to the nucleic acid molecules and may be detected using known techniques.

Nucleic acid molecules according to the invention may be inserted into the vectors described an antisense orientation order to provide for the production of antisense RNA. Antisense RNA or other antisense nucleic acids may be produced by synthetic means.

Alternatively, the oligonucleotide described above car be delivered to cells by procedures m the art such that the anti-sense RNA and DNA may be expressed m vivo to inhibit production of a polypeptide of the invention in the manner described above.

Antisense constructs to the nucleotide sequence encoding the subunit or channel according to the invention, therefore, may inhibit the function or expression of the subunit or channel and may therefore be used to treat conditions associated with expression or overexpression of the subunit or channel according to the invention.

The subunits or channels according to the invention include all possible ammo acid variants encoded by the nucleic acid molecule according to the invention, including a polypeptide encoded by said molecule and having conservative ammo acid changes. Conservative ammo acid substitution refers to a replacement of one or more ammo acids in a protein as identified m Table 1. Subunits or channels according to the invention include all variants of such ammo acid sequences, including naturally occurring allelic variants which are substantially homologous to said sequences. In this context, substantial homology is regarded as a sequence which has at least 70%, preferably 80 or 90% and even more preferably up to

95% ammo acid homology with the subunits or channels according to the invention.

As is well known in the art many proteins are produced in vivo with a (pre) signal at the N terminus of the protein and which may be required for transport of the protein across the cell membrane. Furthermore, such proteins may comprise a further pro sequence that represents a stable precursor to the mature protein. Such pre and pro sequences are not required for biological activity. Furthermore, in eukaryotic organisms many proteins are subjected to glycosylation so as to confer biological activity in vivo . References to a bioprecursor, in accordance with the present invention, refers to all such forms of the protein or polypeptide of the invention prior to any such post translational modification.

A further aspect of the invention provides a host cell or organism transformed or transfected with an expression vector according to the invention. The host cell or organism may advantageously be used in a method of producing a subunit or channel according to the invention, which comprises recovering any expressed protein from the host or organism transformed or transfected with the expression vector. Preferably, the cell is a eukaryotic cell such as an amphibian oocyte from, for example, Xenopus .

According to a further aspect of the invention there is also provided a transgenic cell, tissue or organism comprising a transgene capable of expressing a subunit or channel according to the invention. The term "transgene capable of expression" as used herein means a suitable nucleic acid sequence which leads to expression of a subunit or channel having the same function. The transgene, may include, for example, genomic nucleic acid isolated from human cells or synthetic nucleic acid, including DNA integrated into the genome or in an extrachromosomal state. Preferably, the transgene comprises the nucleic acid sequence encoding the subunits or channels according to the invention as described herein, or a functional fragment of said nucleic acid. A functional fragment of said nucleic acid should be taken to mean a fragment of the gene comprising said nucleic acid coding for the proteins according to the invention or a functional equivalent, derivative or a nonfunctional derivative such as a dominant negative mutant, or bioprecursor of said proteins. For example, it would be readily apparent to persons skilled m the art that nucleotide substitutions or deletions may be used using routine techniques, which do not affect the protein sequence encoded by said nucleic acid, or which encode a functional protein according to the invention.

Subunits or channels expressed by said transgenic cell, tissue or organism or a functional equivalent or bioprecursor of said protein also form part of the present invention.

Advantageously, the nucleic acid molecules or the subunits or channels encoded therefrom, may be used as a medicament, or m the preparation of a medicament, for treating diseases or conditions associated with lack of appetite regulation. Tne nucleic acid molecules or the subunits or channels according to the invention may be provided in a pharmaceutical composition together with a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent or excipient therefor.

Antibodies to the subunit or channel according to the invention may be prepared by techniques which are known in the art. For example, polyclonal antibodies may be prepared by inoculating a host animal, such as a mouse, with the subunit or channel according to tre invention or an epitope thereof and recovering immune serum. Monoclonal antibodies may be prepared according to known techniques, such as described by Kohler R. and Milstein C, Nature (1975) 256, 495- 497.

Such antibodies may also be used m a method of detecting for the presence of a subunit or channel according to the invention, which method comprises reacting the antibody with a sample and identifying any protein bound to said antibody. A kit may also be provided for performing said method which comprises an antibody according to the invention and means for reacting the antibody with said sample.

Proteins which interact with the polypeptide of the invention may be identified by investigating protein- protein interactions using the two-hybrid vector system first proposed by Chien et al (1991) . Proc. Natl. Acad. Sci. USA. 88 : 9578-9582.

This technique is based on a functional reconstitution in vivo of a transcription factor which activates a reporter gene. More particularly the technique comprises providing an appropriate host cell with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, expressing in the host cell a first hybrid DNA sequence encoding a first fusion of a fragment or all of a nucleic acid sequence according to the invention and either said DNA binding domain or said activating domain of the transcription factor, expressing in the host at least one second hybrid DNA sequence, such as a library or the like, encoding putative binding proteins to be investigated together with the DNA binding or activating domain of the transcription factor which is not incorporated in the first fusion; detecting any binding of the proteins to be investigated with a protein according to the invention by detecting for the presence of any reporter gene product in the host cell; optionally isolating second hybrid DNA sequences encoding the binding protein.

An example of such a technique utilises the GAL4 protein in yeast. GAL4 is a transcriptional activator of galactose metabolism in yeast and has a separate domain for binding to activators upstream of the galactose metabolising genes as well as a protein binding domain. Nucleotide vectors may be constructed, one of which comprises the nucleotide residues encoding the DNA binding domain of GAL4. The binding domain residues may be fused to a known protein encoding sequence, such as for example the nucleic acids according to the invention. The other vector comprises the residues encoding the protein binding domain in GAL4. These residues are fused to residues encoding a test protein. Any interaction between polypeptides encoded by the nucleic acid according to the invention and the protein to be tested leads to transcriptional activation of a reporter molecule in a GAL-4 transcription deficient yeast cell into which the vectors have been transformed. Preferably, a reporter molecule such as β-galactosidase is activated upon restoration of transcription of the yeast galactose metabolism genes. A further aspect of the present invention also provides a method of identifying a polypeptide of the invention in a sample, which method comprises contacting said sample with an antibody according to the invention and monitoring for any binding of any proteins to said antibody. A kit for identifying the presence of said protein in a sample is also provided comprising an antibody according to the invention and means for contacting said antibody with said sample.

The subunit or channel of the invention may be recovered and purified from recombinant cell cultures by methods known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography .

Compounds that modulate the activity of a VMH K_ATP channel according to the invention may be identified by a) contacting a cell expressing said channel with said compound in the presence of K⁺ ions, b) depolarising the cell membrane of said cell and detecting the current flowing into said cell, wherein the current that is detected is different from that produced by depolarising the same or a substantially identical cell containing said channel in the presence of said ions but in the absence of said compound.

Compounds which may be identified or are identifiable as modulators of a VMH K_ATP channel according to the invention may, advantageously, be used as a medicament, or in the preparation of a medicament to control appetite regulation, or diseases or conditions associated with malfunction of appetite regulation processes or diseases/conditions associated with over or underexpression of a VMH K_{P TΌ} channel of the invention. Such compounds may also be included in a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent or excipient therefor.

The therapeutic or pharmaceutical compositions of the present invention can be administered by any suitable route known in the art including for example intravenous, subcutaneous, intramuscular, transdermal, mtrathecal or mtracerebral or administration to cells in ex vivo treatment protocols. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation.

The protein of the invention can also be linked or con ugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. For example, it can be coupled to any substance known m the art to promote penetration or transport across the blood-brain barrier such as an antibody to the transferrm receptor, and administered by intravenous injection.

The antisense molecules or indeed the compounds identified as modulators of the channel or subunit according to the invention may be used in the form of a pharmaceutical composition, which may be prepared according to procedures well known in the art. Preferred compositions include a pharmaceutically acceptable vehicle or diluent or excipient, such as for example, a physiological saline solution. Other pharmaceutically acceptable carriers including other non-to/ic salts, sterile water or the like may also be used. A suitable buffer may also be present allowing the compositions to be lyophilized and stored in sterile conditions prior to reconstitution by the addition of sterile water for subsequent administration. Incorporation of the proteins or antisense molecules into a solid or semi-solid biologically compatible matrix may be carried out which can be implanted into tissues requiring treatment .

The carrier can also contain other pharmaceutically acceptable excipients for modifying other conditions such as pH, osmolaπty, viscosity, sterility, lipophilicity, solubility or the like.

Pharmaceutically acceptable excipients which permit sustained or delayed release following administration may also be included.

The protein or the nucleic acid molecules or compounds according to the invention may be administered orally. In this embodiment they may be encapsulated and combined with suitable carriers m solid dosage forms which would be well known to those skilled in the art.

As would be well known to those of skill in the art, the specific dosage regime may be calculated according to the body surface area of the patient or the volume of body space to be occupied, dependent upon the particular route of administration to be used. The amount of the composition actually administered will, however, be determined by a medical practitioner, based on the circumstances pertaining to the disorder to be treated, such as the severity of the symptoms, the composition to be administered, the age, weight, ana response of the individual patient and the cnosen route of administration.

TABLE 1

The present invention may be more clearly understood from the following example, which is purely exemplary, with reference to the accompanying drawings wherein;

Fig. la is an illustration of putative membrane topology of SURlb (after Tusnady et al., 1997) with the position of the amino acids tr.at are altered in SURlb marked (•) . "NBD" refers to a nucleotide binding domain.

Fig. lb & lc are illustrations of the truncation that occurs in the SURlbΔ33 as indicated in the expanded view of NBD2 (insert, right). B, C Differences in the amino acid sequence (B) and nucleotide sequence (C) of SURlb and SURlbΔ33.

Fig. 2a to 2c are illustrations of effects of ATP and diazoxide on Kir 6.2/SURlb and

Kir6.2/SURlbΔ33 currents.

a. Macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from -110 mV to

+100 mV from oocytes injected with mRNAs encoding Kir 6.2 and either SURlb (left) or SURlbΔ33 (right) . ATP, diazoxide (DZ) or MgADP were added to the internal solution as indicated by the bars .

b. Mean ATP dose-response relationships for Kir6.2/SURlb currents (•, n=5) and SURlbΔ33/Kirδ .2 currents

(o, n=5) . Test solutions were alternated with control solutions and the slope conductance (G) is expressed as a percentage of the mean (G_c) of that obtained in control solution before and after exposure to ATP. Tre lines are the nest fit of the data tc the Hill equa ior: for SURlb, K_L = 22 ± 3μM, h = 1.30± 0.16 and for SURlbΔ33, i. = 86± 4 μM, h = 1.20 ± 0.07.

c. Mean macroscopic slope conductance recorded in the presence of MgADP, ATP OR ATP plus diazoxide (DZ) , expressed as a percentage of the slope conductance control solution (no additions), for Kιrδ.2/SURlb and Kιr6.2/SURlbΔ33 currents. The dashed line indicates the current level in control solution. The number of oocytes is given above the bars.

Figs. 3a & 3b are illustrations of the effect of sulphonylureas on Kιrδ.2/SURlb and Kιr6.2/SURlbΔ33 currents.

a. Macroscopic currents recorded from inside-out patches m response to a series of voltage ramps from -110 mV to +100 mV from oocytes injected with mRNAs encoding Kιr6.2 and either SURlb (above) or SURlbΔ33 (below) . Tolbutamide (TB) , glibenclamide (Glib, or ATP were added to the internal solution as indicated by the bars.

b. Mean relationship between tolbutamide concentration and the macroscopic K.,-.., conductance, expressed as a fraction of its amplitude in the absence of the drug. •, solid line: Kir6.2/SURlb currents (n=4) o, dashed line: Kirδ .2/3UR1DΔ33 currents (n=7). The lines are the best fit of the data to eqn. 2 of the text. Kir6.2/SURlb currents: K_lλ = 2.1 μM, hi = 0.83, K_l2 = 1.2 mM, h2 = 1.0, L = 26%. Kir6.2/SURlbΔ33 currents: k_Ll = 1.2 μM, hi = 1.5, K_l2 = 1.4 mM, h2 = 1.1, L = 72%.

Figs. 4a & 4b are illustrations of the interactions between MgADP and tolbutamide.

a. Macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from -110 mV to +100 mV from oocytes injected with mRNAs encoding Kirδ.2 and either SURlb (left), SURlbΔ33 (middle) and a C- terminally truncated form of SURlb, SURlbΔC (right) . Tolbutamide (TB) and MgADP were added to the internal solution as indicated by the bars.

b. Mean macroscopic slope conductance recorded in the presence of ADP, tolbutamide (TB) or ADP plus tolbutamide, expressed as a percentage of the slope conductance in control solution (no additions), for Kir6.2/SURlb, Kirδ .2/SURlbΔ33 and Kirδ .2/SURlbΔC currents. Experiments were carried out in the presence of Mg²⁺ except where indicated. The dashed line indicates the current level in control solution. The number of oocytes is given above the bars. " p<0.05, ** p<0.01 as compared with Kir6.2/SURlb. ++ p,0.01 as compared with data obtained for the same type of channel in the presence of Mg²⁺-

Figs. 5a & 5c are illustrations of Single-channel currents.

a. illustrates single-channel currents recorded at -60 mV from inside-out patches excised from oocytes injected with mRNAs encoding Kirδ.2 plus SURl,

SURlb, SURlbΔ33, or SURlbΔC. The dashed lines indicate the zero current level .

b. illustrates single-channel currents recorded at the indicated membrane potentials from an inside-out patch excised from an oocyte injected with mRNAs encoding Kirδ.2 and SURlbΔ33.

c. is a mean single-channel current- voltage relations recorded for Kirδ.2/SURlbΔ33 currents (n=3).

Figs .6a to 6c are illustrations of effects of metabolic inhibition.

a. illustrates mean whole-cell current amplitudes recorded at -100 mV in control solution (white bars), 15 min after exposure to 3 mM azide (hatcned bars), and the continued presence of azide plus eitner 0.01 mM tolbutamide (TB, grey bars or 0.1 mM tolbutamide 5 (TB, black bars) . Oocytes were comjected with mRNA encoding Kirδ.2 and either SURl, SURlb, SURlbΔ33 or SURlbΔC, as indicated; or were injected with mRNA enclosing Kιrδ.2ΔC26. The 10 number of oocytes is given above the bars .

b. illustrates mean whole-cell current amplitudes recorded at -100 mV in

15 control solution, m the presence of 3 mM azide, in the presence of 3 mM azide plus 0.01 mM tolbutamide (TB) , 0.1 mM tolbutamide or 0.1 μM glibenclamide (Glib) , as indicated. Oocytes were

20 comjected with mRNA encoding Kιr6.2 and SURlbΔ33. The number of oocytes is given above tne bars .

c. illustrates mean current amplitudes 25 recorded at -100 mV in the excised patch or whole-cell (TEVC) configuration, expressed as a fraction of the mean current recorded for SURIA/Kirδ .2. Whole-cell currents were

30 recorded in the presence of 3 mM azide.

Oocytes were comjected with mRNA encoding Kirδ.2 and either SURl, SURlb, SURlbΔ33 or SURlbΔC as indicated; or were injected with mRNA encoding

35 Kιrδ.2ΔC26. Data indicated by * is taken from Tucker et al . , (1997).

Example

The present inventors have isolated mRNA from the rat hypothalamus and carried out RT-PCR using redundant primers based on regions of Kιr6.x and SURx that are highly conserved throughout the inwardly-rectifying K⁺ channel and sulphonylurea receptor families. Six clones of each family were analysed. For the Kir 6.x family, all six clones were Kirδ.2, while for the SURx family, 5 were SURl and 1 was SUR2. A rat hypothalamic cDNA library (5 x 10⁵ clones) was screened with probes for either the Kirδ.x or SURx family. Only two Kirδ.x clones were isolated, both of which were identified as Kirδ.2. For SUR, however, more than 40 positive clones were obtained. Analysis of 24 of these clones revealed that 15 were similar to SURl while 9 were identical to SUR2b. All of the clones homologous to SURl differed from the rat SURl sequence originally reported by five ammo acids: these included four substitutions (T487S, P835Q, G836R and G1313R) and the insertion of an additional serine residue after S741. For the purposes of the present application this variant is referred to as SURlb for clarity: as we show, however, its functional properties are identical with those of SURl, and thus it does not constitute a true variant and should be referred to as SURl elsewhere. The sequence of SURlb is identical to that deposited in GenBank as Accession No. X97279. One clone of the SURlb type had a 131 bp deletion efore the second nucleotide-bindmg domain (NBD2) : since the missing 131 base pairs correspond to exon 33 of human SURl, this clone was called SURlbΔ33. As shown m Fig 1, this deletion introduced a frame shift at residue 1330, and the open reading frame stopped after a further 25 amino acids, truncating the protein at residue 1355. This resulted in the presence of 25 different residues and the deletion of the whole of NBD2.

Inside-out patches

To examine whether the functional properties of K_ATP channels containing SURlb or SURlbΔ33 differ from those containing SURl, the different sulphonylurea receptor variants were co-expressed with Kirδ.2 in Xenopus oocytes. In all cases, no significant current was detected in the cell-attached configuration but large currents developed following patch excision into nucleotide-free solution. The mean current amplitude at -100 mV was 10.5 ± 1.8 nA (n=19) for Kirδ.2/SUR1, 7.1 ± 1.3 nA (n=21) for Kirδ .2/SURlb, and 0.9 ± 0.2 nA (n=25) for Kirδ .2/SURlbΔ33. Therefore, both SURlb and SURlbΔ33 are able to support the expression of full- length Kirδ.2, although Kirδ .2/SURlbΔ33 is less effective than SURlb. Thus NBD2 does not appear to be essential for the interaction of SURl with Kirδ.2 that enables its functional expression.

The effect of the different SURl variants on the channel ATP-sensitivity was then examined (Fig. 2) . Kirδ.2/SURlb channels were blocked by ATP with a k_. of 22 ± 3 μM and a Hill coefficient ( ) of 1.30 ± 0.16 (n=6) , values which are similar to those reported for Kirδ.2/SUR1 channels (Sakura et al., 1995; Gribble et al., 1997b). However, Kirδ .2/SURlbΔ33 currents were significantly less ATP sensitive: . = 86 ± 4 μM, h = 1.20 ± 0.07 (n=5) . As is me case for Kιr6.2/3UP1 channels, both MgADP ano. di zo/icle are capable of enhancing the activity of Kirδ.2/SURlb currents (Fig 2A,C), By contrast, diazoxide did not activate Kirδ .2/SURlbΔ33 currents and 100 μM MgADP was actually inhibitory (Fig. 2A,C). Diazoxide was tested in the presence of MgATP because its stimulatory action is dependent upon the presence of mtracellular hydrolysable nucleotides (Kozlowski et al., 1989) . As discussed in more detail below, the inability of MgADP and diazoxide to stimulate

Kirδ .2/SURlbΔ33 currents is consistent with the fact that this SUR variant lacks NBD2.

The ability of sulphonylureas to block K_ATC channels which contain different SURl variants was then compared. Fig. 3A shows that both tolbutamide and glibenclamide were significantly less effective at blocking Kirδ .2/SURlbΔ33 than Kirδ.2/SURlb currents. Indeed, the effect of glibenclamide was so slow that it was difficult to distinguish from rundown. The mean dose-response relationships are given m Fig 3B and, like that for Kιrδ.2/SUR1 (Gπbble et al., 1997c) , were best fit by assuming the drug interacts with both a high-affmity and a low-affmity site (eqn. 1). The low-affmity site lies on Kιr6.2 (Gπbble et al, 1997c) and it is therefore not surprising that the K_x for this site was independent of the SURl variant, being 1.2 + 0.6 mM (n=4) and 1.4 ± 0.1 mM (n=7) for Kir 6.2/SURlb and Kirδ .2/SURlbΔ33, respectively. The K_λ for the high-aff ity site - which lies on SURl- was also comparable: 2.1 ± 0.8 μM for Kirδ.2/SURlb and 1.2 ± 0.4 uM for Kirδ .2/SURlb_Δ33 Similar values of . were also found for both the high (2.0 μM) and low (1.8 mM) affinity sites of Kιrδ.2/SUR1 channels (Gπbble et al., 1997c). Although tne affinity for tolbutamide was unaffected by deletion of NBD2, there was a marked difference lr the ratio of high to low-affinity inhibition. Tl^~e percentage of tolbutamide olock attributable to the high-affmity site was 57% for Kιrδ.2/SUR1 (Gribble et al., 1997c) and 72 ± 4% (n=4) for Kirδ .2/SURlb, but only 26 ± 6% (n=7) for Kirδ .2/SURlbΔ33.

Effect of NBD2 deletion

In addition to lacking NBD2, SURlbΔ33 possesses an additional 25 ammo acids at its C-termmus . The present inventors explored the contribution of these ammo acids to the difference in the functional properties of SURlbΔ33 and SURlb, by truncating SURlb at residue 1330 (SURlbΔC) . To their surprise, channels comprising SURlbΔC and Kirδ.2 had a similar ATP-sensitivity ( . = 24 ± 4 μM, n=7 ) to Kirδ.2/SURlb channels. Like Kirδ .2/SURlbΔ33, however, tolbutamide produced less inhibition and MgADP inhibited (rather than activated) the channel (Fig. 4). This indicates that the additional 25 ammo acids, rather than the deletion of NBD2, is responsible for modifying the ATP-sensitivity of Kirδ .2/SURlbΔ33. The second NBD is required, however, for normal MgADP, diazoxide and tolbutamide sensitivity.

Fig. 4 also shows that tolbutamide blocks

Kirδ.2/SURlb channels less efficiently m the absence of Mg^2τ. A similar effect has been reported for native β-cell K^_? channels (Lee et al., 1994). In contrast, tolbutamide block of both Kirδ .2/SURlbΔ33 and Kirδ.2/SURlbΔC channels is unaffected by Mg^9* Furthermore, in the absence of Mg , the extent of tolbutamide block of Kirδ .2/SURlbΔ33 and Kirδ.2/SURlb channels was not significantly different. This raises the possibility that the aoility of Mg to enhance tolbutamide inhibition m/olves NBD2 of SURl or requires the functional integrity of both NBDs (as is the case for MgADP; Gribble et al., 1997a).

Single-channel recordings

Fig. 5A illustrates single-channel currents recorded from K_ATP channels containing different SURl variants. It is evident that the open probability (P₀) of Kirδ .2/SURlbΔ33 channels is greater than that of either Kιrδ.2/SUR1 or Kirβ.2/SURlb channels. The mean P₀ at -60 mV was 0.27 ± 0.05 (n=5) for Kιr6.2/SUR1, 0.29 ± 0.05 (n=4) for Kirδ .2/SURlb, 0.35 ± 0.08 (n=4) for Kιrδ.2/SURlbΔC currents and 0.63 ± 0.05 (n=5) for Kιrδ.2/SURlbΔ33. Thus deletion of NBD2 has only a small effect on P₀, suggesting that it is the presence of the additional 25 ammo acids in SURlbΔ33 that is responsible for the higher P_^ of K_ATP channels containing this variant.

The single-channel current-voltage relation of Kirδ .2/SURlbΔ33 currents shows slight inward rectification at potentials positive to +20 mV (Fig. 5B,C), as is observed for Kιrδ.2/SUR1. The single- channel conductance, measured between -20 mV and -80 mV, was 63 ± 1 pS (n=3) for Kirδ .2/SURlbΔ33 channels, a value which is not significantly different from that observed for Kιrδ.2/SUR1 or Kιr6.2ΔC26 (67-76 pS - Sakura et al., 1995; Tucker et al., 1997).

Whole-cell studies

To explore the effect of metabolic inhibition on Kirδ.2/SURlb, Kirδ .2/SURlbΔ33 and Kirδ .2/SURlbΔC channels, the present inventors recorded whole-cell K,,_τ; currents from intact oocytes and used 3 mM azide as a metabolic poison. In control solution, oocytes expressing these three types of channel exhibited current amplitudes similar to those of water-injected oocytes. As shown in Fig. 6A, however, metabolic poisoning produced a large increase in Kirδ.2/SUR1 and Kirδ.2/SURlb currents but a very much smaller increase in Kirδ.2/SURlbΔ33 or Kirδ .2SURlbΔC currents.

The lower activation of Kirδ .2/SURlbΔC currents cannot be attributed to a reduced level of expression, since this channel shows large currents in excised patches (10.4 ± 2.3 nA, n=16) , comparable to those observed for Kirδ.2/SURlb: nor is the ATP-sensitivity different from that of Kirδ .2/SURlb. Thus the reduced activation of Kirδ .2/SURlbΔC channels on metabolic poisoning indicates that some metabolic intermediate interacts with NBD2 to enhance channel activity. One possible candidate would be MgADP, which has previously been suggested to play a role in metabolic activation (Nichols et al., 1996); Gribble et al., 1997a; Shyng et al . , 1997). As shown in Fig.4, unlike Kirδ.2/SURlb channels, Kirδ .2/SURlbΔC channels are not activated by MgADP.

Expression of Kirδ .2/SURlbΔ33 channels is only about one tenth that of Kirδ .2/SURlb, as assessed from the macroscopic current amplitude in excised patches.

When this difference is taken into account, metabolic inhibition produces a similar increase in the whole- cell currents (Fig.δC). Since Kir6.2/SURlbΔ33 lacks NBD2 and is insensitive to MgADP, it seems likely that azide-induced current activation reflects the fall in mtracellular ATP. To test this idea ιr6.2 containing a 26 amino acid truncation ( Kirδ .2 C26) v/as used, which has an ATP-sensitivity similar to that of Kιr6.2/SURlbΔ33 and is not activated by MgADP (when expressed in the absence of SURl; Tucker et al.,

1997). Fig. 6 shows that the extent of Kir6.2ΔC26 current activation in response to azide was comparable to that found for Kirδ .2/SURlbΔ33, as was the current amplitude in excised patches (0-7 ± 0.2 nA, n=17) The lower activation of Kirδ .2/SURlbΔC produced by metabolic inhibition, as compared to Kirδ .2/SURlbΔ33 is therefore likely to be a consequence of the greater ATP-sensitivity (K , -20 μM) of this channel. In contrast to what was observed for excised patches, in mtact oocytes both tolbutamide and glibenclamide blocked Kirδ .2/SURlbΔ33 currents as potently as Kirδ.2/SUR1 currents (Fig. 6A,B).

Effects of MgADP

Comparison of the data given in Figs 3 and 6 reveals that sulphonylureas are more effective inhibitors of both Kirδ.2/SURlb and Kirδ.2 SURlbΔ33 currents in the mtact cell than m the mside-out patch. A similar difference has been noted for native K_ΛT-, channels m pancreatic β-cells, and for Kιrδ.2/SUR1 channels, where it has been attributed to the ability of mtracellular MgADP to enhance the apparent efficacy of tolbutamide block (Zunckler et al . , 1988; Gribble et al., 1997c). The effect of MgADP on the inhibitory potency of tolbutamide on Kirδ .2/SURlb, Kιrδ.2/SURlbΔ33 and Kirδ .2/SURlbΔC channels was therefore examined (Fig. 6) . In all cases, the extent of tolbutamide inhibition was greater in the presence of 100 μM MgADP and was not significantly different between the different types of channel. Tne ability of mtracellular MgADP to enhance the apparent efficacy of tolbutamide bloc-- probably accounts for the greater inhibition produced by sulphonylureas in the mtact oocyte.

MgADP has two effects on the K_ATP channel: it inhibits channel activity by interaction with Kιr6.2 and it stimulates the channel via the NBDs of SURl (Tucker et al., 1997). The latter effect requires the presence of Mg²⁺ (Fig. 4B) and is abolished by mutations in a single NBD (Gribble 1997a) . It is therefore not unexpected that deletion of NBD2, as m Kirδ.2 SURlbΔ33 and Kirδ .2/SURlbΔC, prevents the potentiatory action of MgADP (Fig. 4B) . It is noteworthy that

MgADP blocked Kirδ .2/SURlbΔ33 to the same extent m both the presence and absence of Mg²⁺ (Fig. 4B) . This supports the view that the ADP (like ATP) is actually less potent at inhibiting Kirδ .2/SURlbΔ33 channels than Kirδ .2/SURlbΔC channels, rather than the alternative idea that Kirδ .2/SURlbΔ33 retains some ability to be stimulated by MgADP which partially masks the inhibitory effect of the nucleotide. Indeed the extent of block of Kirδ .2/SURlbΔ33 by 100 μM MgADP (and by ATP) is similar to that observed for Kιr6.2ΔC36 (Tucker et al . , 1998).

Many of the properties of the rat VMH K..- channel differ from those of the archetypal K_A-_D channel. Compared with Kιrδ.2/SUR1, tne VMH channel has a reduced ATP-sensitivity ( K , l-3mM) and a different single-channel conductance (46pS or 140pS) : it is also not activated by K-channel openers, and is blocked by tolbutamide in mtact cells but not in excised patches. Since the s gle-cnannel conductance and ATP-inhibition of archetypal K_{A P} channels are intrinsic to Kirδ.2, while the sulphonylurea and K- channel opener sensitivity are conferred by SUR, these results suggest that both the pore-forming and regulatory subunits of the VMH K_ATP channel may differ from those of archetypal K_ATP channels.

Channels formed by coexpression of SURlbΔ33 with Kirδ.2 share many properties with those of the native K_ATP channel of VMH neurones. In particular, the cloned channels are not activated by diazoxide or MgADP, and are blocked less by tolbutamide in excised patches than in intact oocytes. It is therefore possible that the clone the present inventors have isolated may encode the SUR subunit of the VMH K_ATP channel. One argument against this idea is the fact that only a single SURlbΔ33 clone was detected in the hypothalamic cDNA library, compared with 15 SURlb clones. This suggests that the expression level of SURlbΔ33 in the hypothalamus is low. It remains possible, however, that SURlbΔ33 is confined to a specific neuronal population.

Identification of the pore-forming subunit of the K_{A P} channel from VMH neurones is complicated by the fact that single-channel conductances of 46pS and ~150pS have been reported for rat (Routh et al., 1997; Ashford et al . , 1990a) and 70ps for mouse (Rowe et al . , 1998). The latter value is close to that found for Kir6.2 (Sakura et al., 1995); Tucker et al., 1997), but the former values suggest a different protein is involved. The reduced ATP-sensitivity of the VMH K_ATP channel also supports this idea. Tolbutamide sensitivity

In excised patches, Kirδ .2/SUR1DΔ33 channels exhibit a lower tolbutamide sensitivity than wild-type K_ATP channels. The results presented support that this effect is not a consequence of a reduced binding affinity for the drug, since the . for tolbutamide block is unaffected and Kιr6.2/SURlbΔ33 currents exhibit a normal tolbutamide sensitivity m intact oocytes. Rather they suggest that the reduced drug sensitivity results from a partial uncoupling of SURlbΔ33 from Kirδ.2, since the fraction of block associated with the high-aff ity site is lower for Kιrδ.2/SURlbΔ33 channels than for Kirδ.2/SURlb channels. There are at least three possible explanations for this finding. First, residues within the C-termmus of SURl, not present in SURlbΔ33, may be directly involved m coupling SUR to Kirδ.2. Secondly, the reduced drug potency may be a consequence of the higher open probability (P₀) of Kirδ .2/SURlbΔ33 channels, if tolbutamide stabilizes the long closed state. The inventors have shown elsewhere that an increased P₀, resulting from a mutation in Kιr6.2, is associated with a reduced block by tolbutamide (Trapp et al., 1998). Thirdly, the fact that Mg²" is unable to enhance tolbutamide block of Kirδ .2/SURlbΔ33 currents may contribute to the lower efficacy of the drug. The third explanation is favoured since 100 μM tolbutamide blocked Kιrδ.2/SURlbΔ33 and Kirδ.2/SURlb channels to a similar extent in the absence of Mg^2~

As is the case for native VMH K^_. channels, the sulphonylurea sensitivity of Kirδ .2/SURlbΔ33 channels varies markedly between the mtact cell and the excised patch. Glibenclamide, for example, produces no clear inhibition in the excised patch, yet causes complete block of the whole-cell current. A possible explanation for this difference is afforded by the fact that the ability of sulphonylureas to inhibit both the mtracellular MgADP. In mtact cells, Mg- nucleotides are likely to be present and will therefore contribute to the enhanced block by sulphonylureas. Indeed, both Kirδ.2/SURlb channels and Kirδ.2/SURlbΔ33 channels show greater inhibition by tolbutamide m intact cells than m excised patches (as is also the case for Kιr6.2/SUR1 channels; cf. Gribble et al., 1997b, c).

ATP-sensitivity

The ATP-sensitivity of Kirδ .2/SURlbΔ33 channels is approximately 4-fold lower than that of Kιrδ.2/SUR1 channels. One might expect the ATP-sensitivity of Kιr6.2/SURlbΔ33 channels to oe higher than that of wild-type K_ATP channels, since mutations within the Walker A and Walker B motifs of NBD2 enhance channel inhibition by ATP (Gribble et al., 1997b; 1998). This is because MgATP (like MgADP) enhances K_A-.- channel activity by interaction with the NBDs of SURl, and thereby produces an apparent reduction m ATP- sensitivity (Gribble et al . , 1998). One possible reason for the paradoxical reduction in ATP- sensitivity of Kirδ .2/SURlbΔ33 is that the loss of NBD2 might prevent the ability of SURl to "sensitize" Kirδ.2 to ATP: Tucker et al. (1997) have shown that when a truncated form of Kirδ.2 is expressed by itself the K for ATP-mhibition is -100 μM, but this is shifted to 10-30μM in the presence of SURl. This idea seems unlikely, however, because Kirδ .2/SURlbΔC channels exhibit normal ATP-sensitivity, yet they lack NBD2. An alternative explanation for the reduced ATP- sensitivity of Kirδ .2/SURlbΔ33 currents is that it results from the higher channel open probability. This idea is supported by the fact that mutations within Kirδ.2 which enhance P₀ produce a concomitant reduction in ATP-sensitivity (Shyng et al., 1997b; Trapp et al., 1998). Furthermore, the open probability of Kirδ .2/SURlbΔ33, like the ATP- sensitivity, is similar to that of Kir 6.2/SURlb channels .

Metabolic activation

In contrast to Kirδ.2/SUR1 or Kirδ .2/SURlb,

Kirδ.2/SURlbΔ33 shows only a small activation by metabolic inhibition when expressed in Xenopus oocytes. Comparison with the level of expression seen in inside-out patches excised from the same batch of oocytes, suggests that this difference principally reflects the reduced expression of Kirδ .2/SURlbΔ33. The data further argue that deletion of NBD2 removes the contribution of SURl to metabolic stimulation of channel activity, and that azide-induced activation of Kirδ .2/SURlbΔ33 currents may be simply a consequence of the fall in mtracellular ATP, which is sensed by the Kirδ.2 subunit. The effect of metabolism on K_ATP channels which contain SUR1PΔ33 will be determined by the properties of the pore-forming subunit. Thus if the ATP-sensitivity of the pore-forming subunit of the VMH channel differs from that of Kirδ.2, the regulation of the channel by metabolism may also vary from that described here (we predict that the lower the ATP-sensitivity, the faster the activation by metabolic inhibition) . SURlb differs from SURl at only five amino acids. When SURlb was coexpressed v/ith Kirδ.2, the properties of the K_Λ1T, currents were not different from those of Kir6.2/SUR1 channels. Thus, the five different amino acids may simply be polymorphisms and in future SURlb should therefore be referred to as SURl.

The results herein presented indicate that the C- terminus of SURl influences K_ATP channel gating, that it plays an important role in the regulation of K_ATP channel activity by nucleotides and drugs, but that it is not needed for functional channel expression.

Materials and Methods

Molecular biology

Isola tion of mRNA from VMH

The region containing the VMH was isolated from the brains of 11 Wistar rats (-5 mg per rat) . These fragments inevitably also contained the arcuate nucleus, but we attempted to exclude the lateral nucleus of the hypothalamus. mRNA was purified using an mRNA purification kit (Stratagene) and was used for RT-PCR (100 ng) or for making a cDNA library (600 ng) .

RT-PCR

mRNA was reverse transcribed and the polymerase chain reaction (PCR) was carried out on the resulting cDNA using an Access RT-PCR system (Promega) and primers based on conserved regions of the Kir6.x and SURx families. The primers used for Kirδ.x were designed to amplify the pore region: 5' -TTCTCCATCGAGGT (CT) CA (AG) GT (AG) AC (CT) AT (CT ) GG-3 '

(sense) and

5' -AATGATCATGCT(CT)TT (CT)C(GT) (CG) AGGTC ( C ) CC (CT / AC-3 '

(antisense) . Primers used for the SURx family, which were designed to amplify NBD2, were:

5' -ATCTG (CT ) GG (CT ) CGCAC (AC) GG (CT ) AGTGG ( G) AA (AG) TCCTC- 3' (sense) and

5' -GCCATGTCGATGGA (GA) GC (AC) GT (GT) GC (CT) TC (AG) TCCA-3' (antisense) . The PCR protocol consisted of 40 cycles of 94 °C for 30 sec, 60 °C for 1 min and 68 °C for 2 min. Amplified cDNAs were purified, subcloned and sequenced.

Screening of cDNA Library

A hypothalamic cDNA library was constructed using a ZAP Express cDNA Synthesis Kit (Stratagene) . This library (~1 x 10⁶ independent clones) was screened under low stringency conditions (hybridised with 30% formamide, 5 x Denhardt's, 5 x SSPE, 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA at 37 °C and washed with 2 x SSC, 0.2% SDS at room temperature) with probes for Kirδ.x and SURx. A mixed probe containing full length Kir 6.1 and Kir 6.2 was used for isolating Kirδ.x genes: to detect SURx, the probe consisted of the PCR products containing NBDl and NBD2 of SURl and SUR2. PBK-CMV phagemids containing cDNA inserts of positive clones were excised using ExAssist helper phage (Stratagene) and sequenced.

Expression studies

Full length SUR1A, SURlb or SURlbΔ33 was coexpressed with mouse Kirδ.2 (Genbank D50581, Inagaki et al. 1995a, b; Sakura et al., 1995). Synthesis of capped mRNA was carried out using the miMessage mMachine large scale in vitro transcription kit (A bion, Austin, TX, USA) . Oocyte collection

Female Xenopus laevis were anaesthetised with MS222 (2g/l added to the water) . One ovary was removed via a mini-laparotomy, the incision sutured and the animal allowed to recover. Once the wound had completely healed, the second ovary was removed in a similar operation and the animal was then sacrificed by decapitation whilst under anaesthesia. Immature stage V-VI Xenopus oocytes were incubated for 60 min with 1.0 mg/ml collagenase (Sigma, type V) and manually defolliculated. Ooctyes were coinjected with -0.1 ng Kirδ.2 and ~2 ng of SUR (wild-type or variant), giving a 1:20 ratio. The final injection volume was -50 nl/oocyte. Control oocytes were injection with water. Isolated oocytes were maintained in Barth' s solution and studied 1-4 days after injection (Gribble et al . , 1997b) .

Eletrophysiology

Patch pipettes were pulled from thick-walled glass and had resistances of 250-500 kΩ when filled with pipette solution. Macroscopic currents were recorded from giant excised inside-out patches at a holding potential of 0 mV and at 20-24 °C (Gribble et al . , 1997b) . Currents were evoked by repetitive 3 s voltage ramps from -110 mV to +100 mV and recorded using an EPC7 patch-clamp amplifier (List Electronik, Darmstadt, Germany) . They were filtered at 5 kHz and stored on digital audio tape. Currents were subsequently filtered at 0.5 kHz, digitised at 1kHz using a Digidata 1200 Interface and analysed using Pclamp software (Axon Instruments, Burlingame, USA). Single-channel currents were recorded from small inside-out patches, filtered at 1 kHz and sampled at 3 kHz.

The pipette (external) solution contained (mM) : 140 KC1, 1.2 MgCl₂, 2.6 CaCl₂, 10 HEPES (pH 7.4 with KOH) . The standard mtracellular (bath) solution contained (mM) : 107 KC1, 2 MgCl₂, 1 CaCl₂, 10 EGTA, 10 HEPES (pH 7.2 with KOH; final [K⁺] -140 mM) and nucleotides as indicated. Mg-free solution contained (mM) : 107 KC1, 2.6 CaCl₂, 10 EDTA, 10 HEPES (pH 7.2 with KOH; final [K⁺] -140 mM) . Tolbutamide was made up as a 0.05 M stock solution in 0.1 M KOH and diazoxide as a 0.02 M stock solution in 0.1 M KOH. Solutions containing nucleotides were made up fresh each day. The pH of all solutions was checked and readjusted, if required, after drug and nucleotide addition. Rapid exchange of solutions was achieved by positioning the patch in the mouth of one of a series of adjacent inflow pipes placed in the bath.

Data analysis

The slope conductance was measured by fitting a straight line to the current-voltage relation between -20 mV and -100 mV: the average of 5 consecutive ramps was calculated in each solution.

ATP dose-response relationships were measured by- alternating the control solution with a test ATP concentration. The conductance (G) was then expressed as a fraction of the mean of the value obtained in the control solution before and after application of the nucleotide (G,__,. Dose-response curves v/ere fitted to the Hill equation (eqn.l) G/G. = 1 / (1 + ( [ATP] / )^h) where [ATP] is the ATP concentration, . is the concentration at which inhibition is half maximal and h is the slope factor (Hill coefficient) .

Tolbutamide dose-response curves were fit to the following equation (Gribble et al., 1997c):

G = x *y eqn.2

G_c

Where x is a term describing the high affinity site and Y a term describing the low affinity site.

x = L + (1-L) eqn.3

(l+( [Tolb] /K,)"

y = 1 eqn.4 l+( [Tolb]/ _l2)^r-²

where [Tolb] is the tolbutamide concentration, K_{ll r} K ₂ are the tolbutamide concentrations at which inhibition is half maximal at the high and low-affinity sites, respectively; hi , h2 are the Hill coefficients (slope factors) for the high and low-affinity sites, respectively; and L is the fractional conductance remaining when all of the high-affinity inhibitory sites are occupied.

Data are given as mean ± one S.E.M, and the symbols in the figures indicate the mean and the vertical bars indicate one S.E.M. (where this is larger than the symbol) . Statistical significance was tested using an unpaired Student's t-test or by ANOVA, as appropriate. P values of -O.05 (*) or O.Q1 ( * ^k ) were taken to indicate that the data were significantly different.

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Sequence Listing

SEQ ID No 1 is a nucleotide sequence encoding the

SURlbΔ33 subunit of a VMH K^* channel according to the invention, illustrated in Figure lc.

SEQ ID No 2 is an amino acid sequence of a SURlbΔ33 subunit of a VMH K⁺ channel, illustrated in Figure lb.

Claims

CLAIMS :

1. A nucleic acid molecule encoding a VMH K channel subunit designated SURlbΔ33 comprising the ammo acid sequence according to SEQ ID No 2 or the ammo acid sequence of a functional equivalent, derivative or bioprecursor thereof.

2. A nucleic acid molecule according to claim 1 which is a cDNA sequence.

3. A nucleic acid molecule according to claim 1 or 2 which comprises the sequence illustrated in SEQ ID No 1.

4. A nucleic acid molecule capable of hybridising to a nucleic acid molecule according to any of claims 1 to 3 under high stringency conditions.

5. An expression vector comprising a nucleic acid molecule according to any of claims 1 to 4.

6. A VMH K_Aτ_D channel subunit designated SURlbΔ33 or a functional equivalent, derivative or bioprecursor thereof comprising an ammo acid sequence according to SEQ ID No 2.

7. A VMH K_ATP channel subunit designated SURlbΔ33 or a functional equivalent, derivative or bioprecursor thereof comprising an ammo acid sequence encoded by the nucleic acid molecule according to any of claims 1 to 3.

8. A subunit according to claim 7 comprising the ammo acid sequence illustrated m SEQ ID No 2.

9. A subunit according to any of claims 6 to 8 which is of human origin.

10. A VMH K_ATP channel comprising a Kirδ.x subunit and a SURlbΔ33 subunit according to any of claims 6 to 9.

11. A VMH K_ATP channel according to claim 10 wherein said Kirδ.x subunit comprises a Kirδ.2 subunit.

12. A host cell transformed or transfected with a vector according to claim 5.

13. A host cell according to claim 12 which is a eukaryotic cell and preferably an amphibian oocyte, which is preferably a Xenopus oocyte.

14. A host cell according to claims 12 or 13 which comprises a functional VMH K_ATP channel that contains at least one subunit encoded by the nucleic acid sequence according to any of claims 1 to 3.

15. A transgenic cell, tissue or organism comprising a transgene capable of expressing a subunit or channel according to any of claims 6 to 9 or 10 and 11 respectively.

16. A nucleic acid molecule according to any of claims 1 to 4 or a functional equivalent or fragment thereof, for use a medicament.

17. Use of a nucleic acid molecule according to any of claims 1 to 4, or a functional fragment thereof, in the preparation of a medicament for controlling appetite regulation.

18. A pharmaceutical composition comprising a nucleic acid molecule according to any of claims 1 to 5 together with a pharmaceutically acceptable carrier, diluent or excipient therefor.

19. A subunit or channel according to any of claims 6 to 9 or 10 and 11 respectively, for use as a medicament .

20. Use of a subunit or channel according to any of claims 6 to 9 or 10 and 11 respectively in the preparation of a medicament for controlling appetite regulation.

21. An antibody which is capable of binding to a VMH K_ATP channel subunit according to any of claims 6 to 9 or an epitope thereof.

22. A pharmaceutical composition comprising an antibody according to claim 21 together with a pharmaceutically acceptable carrier, diluent or excipient therefor.

23. An antibody according to claim 21 for use in a method of treatment of the human or animal body.

24. Use of an antibody according to claim 21 in the manufacture of a medicament for controlling appetite regulation.

25. A method for identifying a compound that modulates the activity of a VMH K_ATP channel, which method comprises,

a) contacting a cell expressing said channel with said compound in the presence of K' ions, b) depolarising the cell membrane of said cell and detecting the current flowing into said cell, wherein the current that is detected is different from that produced by depolarising the same or a substantially identical cell containing said channel in the presence of said ions but in the absence of said compound.

26. A compound identifiable as a modulator of a VMH K_ATP channel according to the method of claim 25.

27. A compound according to claim 26 for use as a medicament.

28. Use of a compound according to claim 26 in the preparation of a medicament for controlling appetite.

29. A pharmaceutical composition comprising a compound according to claim 26 together with a pharmaceutically acceptable carrier, diluent or excipient therefor.