WO1998008979A1 - Method and compounds for controlling capacitative calcium ion entry into mammalian cells - Google Patents

Abstract

A method for controlling capacitative calcium ion entry into a mammalian cell. The method is based on the discovery that mammalian transient receptor potential (trp) protein are essential for calcium ion entry. Two human trp proteins are disclosed. Htrp1 and Htrp3. The method involves treating cells with a trp-control agent to either raise or lower the amount of biologically active trp protein associated with the cell to thereby control capacitative calcium ion entry into the cell. Screening methods are also disclosed based upon using mammalian trp protein as a screening target.

Description

ETHOD AND COMPOUNDS FOR CONTROLLING CAPACITATIVE CALCIUM ION ENTRY INTO MAMMALIAN CELLS

This invention was made with government support under Grant No. HL-45198 from the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the capacitative entry of calcium ions (Ca^{2 +} ) into mammalian cells and the mechanisms by which such capacitative entry is accomplished. More particularly, the present invention is directed to the discovery of transient receptor potential (trp) proteins which are an essential part of the capacitative Ca^{2 +} entry (CCE) mechanism in mammalian cells. The invention further relates to methods for altering CCE in mammalian cells by controlling the expression of trp proteins or treating the cell with compounds which inhibit the biological activity of the trp protein. The invention also is directed to using the trp proteins as screening agents in methods for identifying c^rrϊpounds which may be useful in controlling CCE in mammalian cells.

2. Description of Related Art The publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and identified in the appended bibliography. The bibliography also includes a number of references which are not specifically referred to in the description. These references are listed as providing additional description of related art. Calcium regulation plays an important role in many cellular processes. In non-excitable mammalian cells, activation of phosphoinositide- specific phospholipase C (PLC) produces inositol 1 ,4,5-trisphosphate (IP₃), which in turn causes the release of intracelluiar calcium from its storage pools in the endoplasmic reticulum. This results in a transient elevation of cytosolic free

Ca^{2 +} , which is normally followed by a Ca^{2 +} influx from the extracellular space. By refilling the pools, Ca^{2 +} influx plays an important role in prolonging the Ca^{2 +} signal, allowing for localized signaling, and maintaining Ca^{2 +} oscillations [11.

Calcium influx in non-excitable cells is thought to occur through plasma membrane channels which, in contrast to the voltage-dependent Ca^{2 +} channels in excitable cells, are operated not by changes of membrane potentials but rather by how full the internal Ca^{2 +} stores are [2]. The Ca^{2 +} channels have variously been referred to as calcium release-activated calcium channels (CRACs), store- operated calcium channels (SOCs), and receptor-operated calcium channels (ROCs) (23, 24, 25 and 26). Because the entering Ca^{2 +} replenishes Ca^{2 +} stores that act like capacitors, it is also called capacitative Ca^{2 +} entry or CCE (27, 28).

Although studies using either fluorescent Ca^{2 +} indicators or electrophysiological techniques have suggested that multiple types of Ca ⁺ per meant channels may be involved in different cell types to fulfill the influx function, the molecular structure of the channels and the mechanism that regulates the influx have remained unclear and represent one of the major unanswered questions of cellular Ca^{2 +} homeostasis (3-5].

Candidates involved in voltage independent Ca^{2 +} entry into cells include a gene product missing in a Drosophila mutant, the transient receptor potential (trp), and its homologue, frp-like (trp ). The insect phototransduction pathway is mediated through the activation of PLC coupled by a G_q type protein [61. The consequent generation of IP₃ and the release of Ca^{2 +} from its intracelluiar storage pools is believed to lead to the opening of a light sensitive ion channel and generation of a depolarizing receptor potential. Similar to intracelluiar Ca^{2 +} changes in mammalian cells following stimulation by agonists acting via PLC, electroretinograms of Drosophila eyes are biphasic with an initial peak followed by a sustained phase of which the latter is dependent on extracellular Ca^{2 +} . This sustained phase is absent in the trp mutant which was therefore proposed to be caused by a defect in the Ca^{2 +} influx pathway [61. The trp gene was cloned [7,8]. Subsequently, molecular cloning of a Drosophila calmodulin binding protein showed it to be a homologue of the trp gene product and named ftp-like or trp\ [9]. A detailed analysis of the trpλ sequence showed that it shares moderate homology with voltage-dependent Ca^{2 +} and Na ⁺ channels at their putative transmembrane regions. However, in clear contrast with the voltage-dependent channels, it lacks the positively charged amino acid residues at the presumed S4 segment which are thought to act as voltage sensors that promote gating in response to changes in membrane potentials. The structural homology to Ca^{2 +} and Na ⁺ channels together with the absence of charged residues in trpλ and trp suggested that these proteins may form voltage independent ion channels. This was demonstrated recently by expression of the cDNAs for trp and trpλ in insect Sf9 cells using the baculovirus system. It was found that trp forms a Ca^{2 +} permeable cation channel which is activated by store depletion with thapsigargin

[10] whereas trpλ forms a Ca^{2 +} permeable non-selective cation channel which is not only constitutively active when over-expressed in S/9 cells but also can be up-regulated by receptor stimulation [1 1 -13]. However, it was also noticed that neither trp nor trpλ mimicked the endogenous Ca^{2 +} influx channel of the Sf9 cells, suggesting the existence of at least one other channel in insects involved in Ca^{2 +} entry [10].

SUMMARY OF THE INVENTION The present invention is based on our isolation of two trp proteins from human cells ( trpλ and Htrp2) and the discovery that the trp proteins are responsible for and essential to the capacitative calcium ion entry (CCE) mechanism found in mammalian cells. Among other things, this discovery allows one to provide methods which control calcium ion levels in cells by regulating the expression of biologically active trp proteins. In addition to being a target for controlling calcium ion entry, the trp proteins may also be used in screening procedures for determining whether or not certain compounds should be considered candidates for regulating calcium ion levels in mammalian cells. ln accordance with the present invention, a method is provided for controlling capacitative calcium ion entry into a mammalian cell where the cell naturally expresses a transient receptor potential (trp) protein that is required for capacitative calcium ion entry into the cell. The method includes the step of treating the ceil with a sufficient amount of a f/p-control agent to either raise or lower the amount of biologically active trp protein associated with the cell to thereby control capacitative calcium ion entry into said cell.

As a feature of the present invention, the f/p-control agent is a nucleotide sequence which codes for the expression of trp protein when said nucleotide sequence is introduced into said cell. The increase in expressed trp protein results in an increase in capacitative calcium entry into the cell. The frp-control agent may also be an anti-sense nucleotide sequence which is anti-sense to a nucleotide sequence which codes for the expression of trp protein. The anti-sense sequence can be used effectively to reduce the expression of trp protein and thereby reduces the influx of calcium ions into the cell. Inhibitors may also be used which bind to or otherwise inhibit the biological activity of the trp protein once it has been expressed by the cell.

As another feature of the present invention, methods are provided for screening compounds to determine their potential for use in controlling capacitative calcium ion entry into mammalian cells. The method involves providing a cell culture which expresses a transient receptor potential (trp) protein which is necessary for capacitative calcium ion entry into the cell. The cell expresses trp protein naturally in amounts which produces a naturally occurring level of biologically active trp protein associated with said cell. The cell culture is exposed to the compound of interest. A determination is then made to ascertain if the exposure of the cell culture to the compound produces an increase or decrease in the expression of the trp protein to thereby provide an indication of the compounds potential use in controlling capacitative calcium ion entry into mammalian cells. The above discussed and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 -3 are graphical representations of the results of screening tests using carbachol (FIGS. 1 and 2) and maitotoxin (FIG. 3) as exemplary compounds being screened.

DETAILED DESCRIPTION OF THE INVENTION The various aspects of the present invention are based upon the isolation and characterization of two human trp proteins. The invention is further based upon the discovery that these proteins, as well as other mammalian cell trp proteins, are essential components of the calcium ion entry mechanism. The following portion of this detailed description sets forth the procedures used to isolate, identify, clone and functionally characterize the trp proteins. Isolation and identification of trpλ

Expressed Sequence Tags (EST) are partial, "single-pass" cDNA sequences deposited in the Genbank database. Many of these sequences are homologous to proteins from other organisms and many of them may contain protein-coding regions that represent novel gene families [16]. We reasoned that such a cDNA sequence encoding a mammalian homologue for the trp gene might exist in the database. Therefore, we used the deduced amino acid sequence of the Drosophila trp as a query to search the Genbank database using 'tblastn', a program that allows comparison of a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. A human EST

(EST05093) was found to encode an amino acid sequence that shares similarity with the Drosophila trp sequence from Glu33 to Asn80. The 297 nucleotide sequence of this EST was determined from a cDNA clone isolated from a fetal human brain cDNA library and was deposited in

GenBank by Adams et al. [16]. The deduced peptide sequence of EST05093 was then compared with the protein sequences of the Drosophila trpλ and a C. elegans trp homologue (ZC21 .2, Genbank accession # L16685). This revealed that the C-terminal region of the EST peptide is homologous to the N-terminal regions of all the ftp-type proteins. We thus synthesized an oligonucleotide according to the

3' region of the EST05093 and used it as a probe to screen a human kidney cDNA library. From 1.5 x 10^β recombinant phage, we isolated one positive clone, T23. An fcoRI digest of the purified Λgt10 phage DNA produced three fragments. Among them, a 470 bp fragment hybridized to the oligonucleotide probe used for screening. The sequence of this fragment was determined and found to contain the complete sequence of EST05093. The sequences of the other two fcoRI fragments were found to contain open-reading frames which encode amino acid sequences homologous to the ftp proteins down-stream from the region homologous to ETS05093. Thus, T23 was identified as a human trp homologue and has been named human ftp-1 or Hftp-1 (SEQ. ID. NO. 1 ). A 670 bp fcoRI fragment from T23 was then used as a probe to screen other human cDNA libraries, including a ΛZAP aorta, a ΛZAP cerebellum, a Λgt10 heart and a specifically primed Λgt10 library made from oiigo-dT-purified HEK 293 cell mRNA. From all isolated cDNA clones, 13 were sequenced completely. These cDNA clones cover an mRNA of about 5.5 Kb, with an open-reading frame of 2379 bases. Comparison of overlapping DNA sequences of clones obtained from kidney, aorta, cerebellum, and heart showed only two silent substitutions of nucleotides which may arise because of polymorphism. Therefore, all the cDNA clones should be the product of the same gene locus.

The open reading frame of the Hftp-1 encodes a protein of 793 amino acids. A stop codon is present at 366 bases upstream from the first methionine in the same reading frame. The codon for the second methionine in this sequence matches better than the first methionine codon the sequence characteristics for translation initiation as specified by Kozak [17]. Therefore, the translated open reading frame may contain only 792 instead of 793 codons. A more detailed analysis of the cDNA clones indicated that the primary transcript of Hftp-1 gene may be spliced in alternative ways. Many of the cDNA clones do not contain a stretch of 102 base pairs which encodes amino acids 109 to 143. This gives rise to a shorter form of Htrp-1 with only 759 amino acids.

Searching the Genbank database using 'blastp' and the trp-1 protein sequence as a query, we found that only Drosophila trp, Drosophila trpλ and C. elegans trp have probability scores higher than 300. The remainder of the matched sequences had scores lower than 70. The Hftp-1 is about 37% identical or 62% similar to each of the other three known trp proteins. Sequence alignment of all four trp proteins shows conserved clusters of short amino acid sequences distributed throughout the entire length of the polypeptides, except that Hftp-1 and C. elegans trp have much shorter C-termini. As seen with Drosophila trp, Drosophila trpλ and C. elegans trp, hydropathy analysis of the Hftp-1 protein suggests 8 hydrophobic regions. These could correspond to transmembrane segments.

The evolutionary distances between each pair of the four trp proteins determined by the Kimura method [19] are shown in Table 1.

TABLE 1 EVOLUTIONARY DISTANCES OF THE trp PROTEINS

Dtrp Dtrpλ Cftp

Hftp-1 124 122 128

Dtrp 78 130

Dtrpλ 124 Evolutionary distances were determined using the Kimura protein distance analysis method. The non-conserved regions at the N- and C-termini were not included for calculation of the distances.

A Northern analysis using a fragment of trp-1 as a probe shows that a transcript of about 5.5 Kb is abundant in human heart, brain, ovary, and testis.

Lower amounts of the transcript are also present in many other tissues including, kidney, lung, spleen, pancreas, thymus, skeletal and smooth muscle of the present invention. The Hftp-7 transcript is not detected in human liver mRNA by Northern blotting. However, a mouse trp-λ sequence which is 99% homologous to Hftp-1 is obtained from mouse liver mRNA by RT-PCR, indicating the presence of Hftp-1 in liver mRNA in low amounts.

The materials and methods used to isolate and identify the Hftpl are as follows:

Isolation and sequencing of cDNA clones We used a synthetic 45 nucleotide long oligonucleotide sequence, 5'-

TTGAACATAAATTGCGTAG ATGTGCTTGGG AG AAATGCTGTTACC-3' (SEQ. ID. N0:3), labeled at the 5'-end with ³²P by incubating with l -³²P]ATP in the presence of T4 polynucleotide kinase to screen a Λgt10 human kidney cDNA library using standard protocols as described [14]. Hybridization was carried out in a shaking waterbath at 65 °C overnight. The filters were washed at 65 °C with 2 x SSC/0.1 % SDS (1 x SSC is 150 mM NaCI/15 mM sodium citrate, pH 7.0).

One positive clone was obtained from this library containing an insert of 1.5 Kb with multiple fcoRI sites. The EcoRI fragments were subcloned into plasmid Bluescript KS( + ) and sequenced. One 0.67 Kb EcoRI fragment was later used as a probe for subsequent screening of other human cDNA libraries after labeling with [αr-³²P]dCTP using the Klenow enzyme and random hexamers [15].

A primer specific library was constructed to facilitate the cloning of the N- terminal region of the Hftp-1gene. PolyA RNA was prepared from 2.5 x 10⁸ from human embryonic kidney cells, HEK 293, using an mRNA isolation kit from Collaborative Biomedical Products (Bedford, MA USA). Complementary DNA was synthesized, using a cDNA Synthesis module from Amersham, starting with 5 μg of the mRNA and a mixture of the following oligonucleotide primers: 5'- TCGCACGCCAGCAAGAAAAG-3' (SEQ. ID. N0:4), 5'- CGATGAGCAGCTAAAATGAC-3' (SEQ. ID. NO:5), and 5'- TGTCAGTCCAATTGTGAAAGA-3' (SEQ. ID. NO:6), each at the final concentration of 1 ,4pM. AΛgtl 0 library was constructed using Amersham cDNA cloning kits following manufacturer's protocols.

DNA inserts were sequenced by the dideoxynucleotide termination method usmg~[ZF³*S]dATP and Sequenase version 2.0 (United States Biochemical) as previously described [15]. The sequence was confirmed by-sequencing both strands using double-stranded plasmids as templates and either universal primers or Hftp-1 specific synthetic oligonucleotides as primers. Other standard nucleic acid and bacteriological manipulations were performed as described [14].

Database Searches and Sequence Analysis Protein and nucleic acid searches were performed using the BLAST network service of the National Center for Biotechnology Information via an e-mail server. DNA fragment assembly, restriction mapping, protein hydropathy analysis and alignment and all other sequence dependent analyses were performed using the Wisconsin Sequence Analysis Package from the Genetics Computer Group (GCG).

Northern Analysis

Human multiple tissue Northern blots (Clontβch) were prehybridized in a Rapid-hyb buffer (Amersham) at 60 °C for 2 hours and then hybridized in the same buffer with ³²P-labeled cDNA probe (4 x 10^β cpm/ml) at 60°C for 14 hours. After rinsing with 2 x SSC/0.05% SDS, the filters were washed twice in the same solution and then twice in 0.2 x SSC/0.1 % SDS at 60°C. The filters were exposed to X-ray film at -70 °C with intensifying screens for desired periods of time. The probe for Hftp was made from the 0.67 Kb EcoRI fragment of the Hftp- 1 cDNA and a control probe was a human cDNA for ?-actin. Both probes were labeled by random prime labeling with [σ-³²P]dCTP. Isolation and identification of Hfr--.3

The full length Hftp3 cDNA was cloned as follows: mRNA was prepared from human embryonic kidney cells (HEK 293 cells) \Zhu etal., 1995). A library for rapid amplification of cDNA ends through amplification by the polymerase chain reaction (RACE-PCR) was prepared using 1 μg HEK mRNA, adaptors, reagents and protocols provided by Clontech in the Marathon cDNA Amplification k i t . S p e c i f i c o l i g o n u c l e o t i d e p r i m e r s S 1 ( 5 ' -

TGACTTCCGTTGTGCTCAAATATGATCACAAATTCATAG-3') (SEQ. ID. NO:7), S2 (5'-ATGGAATATACAATGTAACTATGGTGGTCG-3') (SEQ. ID. NO:8), A1 (5'- GGACTAGGAACTAGACTGAAAGGTGGAGGTAATGTTTTTCCATCATCA-3')(SEQ. ID. NO:9), and A2 (5'-CGAGCAAACTTCCATTCTACATCACTGTC-3') (SEQ. ID.

NO:10) were synthesized according to the sequence of EST R34716 from the GenBank dbEST database. Primary RACE-PCR amplifications were performed using AP1 (adaptor-ligated primer provided by the manufacturer) in combination with primer S1 for 3' amplification or AP1 with primer A1 for 5' amplification of Hftp3. Nested-PCR amplifications were performed using internal primers AP2

(Clontech) plus S2 for the 3' RACE or AP2 plus A2 for the 5' RACE. Polymerase chain reactions were carried out in a thermal cycle controller (MJ Research) using the Takara ExTaq polymerase for 30 cycles each consisting of a denaturing step at 94°C for 40 sec and an annealing plus extension step at 70°C for 5 min. PCR products were extracted from agarosegel following electrophoresis and subcloned into a T/A cloning plasmid, pCRII (Invitrogen). Positive clones were identified using end-labeled oligonucleotides A1 and S1 for the 3' and 5' RACE, respectively, following a standard colony screening protocol {Sambrook et al. (14)]. DNA was sequenced by the dideoxy-chaintermination method of Sanger et al. (49) using double stranded DNA as template as described by Levy et al. (15). The sequence was confirmed by isolating overlapping partial cDNAs made directly from HEK 293 cell mRNA by RT-PCR with multiple sets of specific primers derived from the Hftp3 sequence. The nucleotide sequence of the Hftp3 cDNA has been deposited in GenBank (see below) and is set forth in SEQ. ID. NO. 2.

Partial cDNA fragments of murine trp homologues were cloned by reverse transcribing polyA ⁺ RNA from liver, brain and kidney and subjecting the transcripts to amplification by the polymerase chain reaction (RT-PCR). The primers used for amplification of reverse transcripts were: 5'- GCNGA(G/A)GGNCTCTT(T/C)GC (SEQ. ID. NO: 1 1 ) (sense)/5'- CGNGC(G/A)AA(C T)TGCA(A/G)(A/G)T (SEQ. ID. NO: 12) (antisense) for Mftp2(a); 5'-TGGGNCCN(C T)TGCA(A/G)(A/G)T (SEQ. ID. NO:13) (sense)/5'- CGNGC(G/A)AA(C T)TTCCA(C T)TC (SEQ. ID. NO: 14) (antisense) Mtrpλ and

Mftp2(b); 5'-ACCTCTCAGGCCTAAGGGAG (SEQ. ID. NO:15) (sense)/ 5'- CCTTCTGAAGTCTTCTCCTTCTGC (SEQ. ID. NO: 16) (antisense) for Mftp3; 5'- TCTGCAGATATCTCTGGGAAGGATGC (SEQ. ID. NO: 17) (sense)/5'- AAGCTTTGTTCGAGCAAATTTCCATTC (SEQ. ID. NO:18) (antisense) for Mftp4 and Mftpδ; and 5'-A(C/A)(G/A)CCNTT(C T)ATGAA(G/A)TT (SEQ. ID. NO: 19)

(sense)/5'-CCACTCCACGTCCGCATCATCC (SEQ. ID. NO:20) (antisense) for Mftp6.

The primers used for amplification of murine genomic DNA isolated from the 129Sv embryonic stem cell AB2.2 as described by Rudolph et al. (50) were: 5'-GGTTTAGCTATGGGGAAGAGC (SEQ. ID. NO :21 ) (sense)/5'-

TTTCCA(T/C)TCTTTATCCTCATG (SEQ. ID. NO:22) (antisense) for Mftpl ; 5'- TGGACATG CCTCAGTTCCTGG (SEQ. I D . NO : 23) (sense)/5 '- TTTCCA(T/C)TCCACATCAGCATC (SEQ. ID. N0:24) (antisense) for Mftp2; 5'- GGCTATGTTCTTTATGGGATAT (SEQ. ID. NO: 25) (sense)/5 '- CCATCATCAAAGTAGGAGAGCC (SEQ. ID. NO:26) (antisense) for Mftp3; 5'- ATGTCAAAGCCCAGCACGAGT (SEQ. ID . NO : 27 ) (sense ) /5 '- AAGCTTTGTTCGAGCAAATTTCCATTC (SEQ. ID. NO:28) (antisense) for Mftp4;

5'-ATGTGAAGGCCCGACATGAGT (SEQ. ID. NO:29) (sense)/5'- TTTCCATTCAATATCAGCATG (SEQ. ID. NO:30) (antisense) for Mftpδ; and 5'- ATCGGCTACGTTCTGTATGGTGTC (SEQ. ID. NO:31 ) (sense)/5'- GGAAAACCACAATTTGGCCCTTGC (SEQ. ID. NO:32) (antisense) for Mftp6. PolyA⁺ RNA was prepared from mouse tissues using an mRNA isolation kit from Collaborative Biomedical Products (Bedford, MA, USA). The first strand cDNAs were synthesized using Moloney Murine Leukemia Virus Reverse Transcriptase (Gibco BRL) with either random hexamers or oligo-dT as primers following established protocols (14). The PCR reaction mixture was composed of the cDNA, 0.2 mM dNTP, 0.2 or 1 pM of each primer, 1.5 mM of MgCI₂, and

25 unit/ml of Taq polymerase (Perkin Elmer). PCR reactions using reverse transcripts were carried out in a Thermal Controller (MJ Research Inc.). For amplification of reverse transcripts the cycles were: 1 min at 94°C, 1 min at the annealing temperature listed next to the primers, and 1 min at 72 °C for 30 to 35 cycles. For genomic DNA (from 129Sv mouse embryonic stem cells), the cycles were 30 sec at 94°C, 60 sec at 55°C and 3.5 min at 72°C, ending with 10 min at 72°C.

The PCR products were separated on a 1 % agarose gel by electrophoresis. Appropriate DNA fragments were extracted with Qiagen Gel Extraction kit and subcloned into a TA cloning vector, pCRII (Invitrogen). These and all other cDNA fragments used in this work were sequenced as described above. The DNA sequences were confirmed by sequence analysis of produces obtained from at least one additional independent PCR reaction for each specific ftp-related gene fragment. Expression Piasmids

The Mtrpλ (470 bp), Mftp2 (470 bp), Mftp3 (1 ,200 bp), Mftp4 (1 ,200 bp), Mftpδ (450 bp), and Mftp6 (270 bp) cDNA fragments obtained by RT-PCR were subcloned in negative orientation downstream of the CMV promoter of expression vector pGW1 H (British Biotech Pharmaceuticals, Oxford, UK).

The full length cDNAs encoding the M5 muscarinic receptor (32), Hftpl (29), Hftp3 and murine luteinizing hormone receptor, mLHR were subcloned downstream of the CMV promoter of the expression plasmid pcDNA3 (Invitrogen).

Transfection of COS-M6 and Ltk^" Cells

COS-M6 cells were transfected by the DEAE-dextran/chloroquine shock method (14) as described (30) with changes. Sixteen hours prior to transfection, COS-M6 cells that had been kept subconfluent were plated at a density of 2 x 10^δ cells/well onto 25 mm glass coverslips placed at the bottom of the wells of

6-WΘII plates. Cells in the individual wells were then transfected with 160 pi of transfection mixture (30) containing 0.1 pg pcDNA3 with the M5 receptor cDNA, a three fold molar excess of pcDNA3 vector carrying either the Hftp3, Hftpl or mLHR cDNA to bring the final concentration of DNA to 4 pg/ml. Cell were used 40 to 48 hours after transfection.

Mouse fibroblast Ltk^' cells (3 x 10° cells/100 mm dish) were transfected by the calcium phosphate/glycerol shock method with 5 pg each of the plasmids with the antisense cDNAs and 0.5 pg of the pcDNA3 carrying the M5 receptor. The control cells received only the M5 muscarinic receptor cDNA in pcDNA3. One day after transfection, the cells were trypsinized and diluted with Minimum

Essential Medium -a medium containing 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, 50 pg/ml streptomycin, and 400 pg/ml G418 (GIBCO). Serial 1 :4 dilutions of the cells were transferred into 96- well plates and G418 resistant clones were allowed to develop for two weeks in the G418-containing medium. Single colonies were then expanded and the cells used for Fur a 2 fluorescence measurement of muscarinic receptor induced [Ca^{2 +} ]_j transients. Of 17 control cell lines, 5 responded to CCh, and increased [Ca^{2 +} ]_j through the capacitative influx path by 96 ± 5 nM (difference between [Ca^{2 +} ]_j at time of Ca^{2 +} addition and [Ca^{2 +} ]_j 30 sec later (average ±_ SD, 20 cells each of 5 cell clones). Of thirty G418-resistant cell lines obtained from transfecting Ltk- cells with M5 receptor plus the six antisense trp cDNAs, 9 responded to carbachol. All cells expressing the M5 receptor, identified by their response to carbachol (CCh), were assumed to express also the co-transfected cDNA (Hftp3 or Hftpl ) or antisense cDNA fragments. Measurement of Changes in Intracelluiar Ca²⁺ ([Ca²⁺],) Intracelluiar Ca^{2 +} transients were measured in individual cells by fluorescence videomicroscopy using the Attof luor Digital Imaging and Photometry attachment of a Carl Zeiss Axiovert inverted microscope. Cells (COS-M6 or L) were grown on circular coverslips, rinsed and incubated with 5 pM Fura2/AM (Molecular Probes) in Hepes buffered saline (HPSS: 120 mM NaCI, 5.3 mM KCl, 0.8 mM MgS0₄, 1.8 mM CaCI₂, 1 1 .1 mM glucose, 20 mM Hepes-Na, pH 7.4) at

37 °C for 30 min and then washed with HPSS twice at room temperature. The coverslips with the cells were then clamped into a circular open-bottom chamber and mounted onto the stage of the microscope. [Ca^{2 +} ]_j in individual cells was monitored at room temperature exciting Fura2 alternatingly at 334 and 380 nm and recording emitted fluorescence at 520 nm. All reagents were diluted to their final concentrations in HPSS and applied to the cells by surface perfusion. The duration of exposure to each reagent mixture is indicated by the horizontal lines above the graphs depicting the changes in [Ca^{2 +}]_j as a function of time. The system allows data acquisition from up to 99 user-defined variably-sized regions of interest per field of view. Data from 15 to 30 individual cells were thus collected per experiment and experiments were repeated until data from sufficient cells were collected to generate an ensemble average that was calculated after transfer into Microsoft Excel 5.0. Data acquisition was typically at 1.2 to 1.5 sec intervals and lasted for 500-800 seconds. For assessment of the rate at which [Ca^{2 +} ]_j falls after an initial stimulation with agonist, t = 0 is the time of agonist addition; for assessment of rate of influx of Ca^{2 +} into cells in which Ca^{2 +} stores had been depleted by agonist, t = 0 is the time of Ca^{2 +} readdition. t_{1 /2}/ values were obtained by fitting the function A = A°exp(-t«ln2/t_{1 /2}) + B to the data points shown. Membrane potential measurement

The resting membrane potential of transfected murine L cells was measured using the patch clamp technique. On-cell patches were obtained in the - -

voltage clamp configuration. Before going to the whole cell configuration, the amplifier was switched to current clamp mode so that the resting membrane potential could be measured at the moment access was gained to the cell interior. The pipette solution was composed of the following (in mM): potassium gluconate 140, KCl 5, CaCI₂ 0.5, MgCI₂, EGTA 5, Hβpβs 5, ATP 5, pH 7.1. The bath solution was the same as that used for [Ca^{2 +} ]_j measurements by digital videomicroscopy. Functional Expression of Hftpl and Hftp3

The demonstration that ftp proteins are components of CCE requires that their activity be determined in intact cells and recognized in a background of existing agonist-stimulated Ca^{2 +} influx. Two complementing approaches were used. The first was to express full length trp cDNAs in a mammalian cell and test whether they would increase CCE. The second was to expand our knowledge on the molecular complexity of the mammalian ftp gene family and test whether expression of partial cDNAs of several members of this family in antisense direction would interfere with CCE. We reasoned that if both conditions could be met, we would be justified in concluding that the trp having this activity is a component CCE, i.e., the capacitative Ca ⁺ entry pathway.

The Hftp3 cDNA was transfected into C0S-M6 cells together with a marker gene that would identify cells that had taken up DNA from non-transfected cells. The marker gene used was the G_q-coupled M5 muscarinic receptor (M5R) (31 ). This receptor stimulates phospholipase C (PLC) (31 ,32) and served as a trigger to activate CCE. Our initial experiments characterized Ca^{2 +} transients in C0S-M6 cells transfected only with the M5 receptor. Stimulation of the PLC/IP3 pathway through the M5 receptor by addition of carbachol (CCh) caused an immediate fast rise in cytosolic Ca ⁺ ([Ca ⁺ ]_j) to a peak level that fell with an approximate t₁ of 30 sec to a plateau that was above the starting resting level. Maintenance of this plateau was dependent on both continuous Ca^{2 +} entry from the extracellular medium and on the continuous stimulation of the M5 receptor/G protein/PLC/IP3 pathway by the receptor agonist, as it was blocked upon addition of the receptor antagonist atropine. Although this was not assessed specifically in COS cells, we believe that the initial fast rise in [Ca^{2 +} ]_j is due to IP3-stimulated release of Ca^{2 +} from intracelluiar stores (33). In agreement with this interpretation, the fast rise in [Ca^{2 +} ]_j in response to CCh occurred also in the absence of extracellular Ca^{2 +} (Ca ⁺-free medium plus 0.5 mM EGTA), but rather than falling to an above-basal plateau, fell to levels very close to basal. Addition of Ca^{2 +} to cells that had undergone the initial agonist-induced [Ca^{2 +} ]j increase in the absence of Ca^{2 +} , then resulted in a rise in [Ca^{2 +}]_j. This entry of Ca^{2 +} is a measure of agonist-activated CCE. Under these conditions, Ca^{2 +} influx was dependent on expression of the M5 receptor. Addition of Ca^{2 +} to cells kept for up to 10 minutes in Ca ⁺ -free medium in the absence of CCh also failed to show Ca ⁺ influx. These features of agonist activated Ca ⁺ transients have been shown previously for the M5 receptor expressed in stable form in murine L cells (32).

We next tested whether Hftp3 would affect M5 receptor induced capacitative Ca^{2 +} transients. We expected the putative ftp-mediated Ca^{2 +} entry to reduce the rate at which [Ca^{2 +} 1; falls after the initial effect of IP3, and possibly to increase the steady state (plateau) level of [Ca^{2 +} ]j. We expected also that cells stimulated in the absence of extracellular Ca^{2 +} would show, upon Ca ⁺ re- addition, a faster Ca^{2 +} influx leading to a higher [Ca^{2 +}]j.

Cells that had been transfected with expression vectors carrying the M5 receptor and, as appropriate, either the newly cloned Hftp3 cDNA or the previously cloned Hftpl cDNA (29), were grown on coverslips, loaded with the florescent Ca^{2 +} indicator dye Fura2 and tested for a response to CCh 40-48 hours after transfection. For purpose of analysis the cells that responded to carbachol were assumed to be expressing not only the receptor but also the co- transfected trp cDNA. Changes in [Ca^{2 +}]_j as a function of time were recorded from individual cells, averaged and fitted by a first order decay function plus an offset.

The decay of the carbachol/IP3-induced peak [Ca ⁺ ]_j in the presence of extracellular Ca^{2 +} was well fit by the first order decay function, and the rate of return was slower in cells transfected with Hftp3 than in cells transfected with the M5R only: t_{1 /2} = 27 ±_ 3 sec for cells with M5R only (mean _±. SEM; number of individual M5R positive cells analyzed (n) = 81 ) vs. 37 ±_ 4 sec for cells transfected with M5R plus Hftp3 (n = 81 ; p<0.01 ). In contrast, the decay in cells transfected with M5R plus Hftpl (t_{1 2} = 24 ±_ 3 sec, n = 104) was not significantly different from that seen in cells transfected with M5R alone. Furthermore, the fit required an offset or plateau of [Ca^{2 +} ]_j that was 2.2 to 2.5 times that of the [Ca^{2 +}]_j at the time of CCh addition. This plateau showed a small, but significant difference between control and Hftp3 transfected cells (88 nM (95% confidence limits: 77-101 nM) vs. 1 17 nM (95% confidence limits: 105-130 nM). The plateau derived from the fit for Hftpl -transfected cells did not differ significantly from that of either control or Hftp3-transfected cells. The effect of readdition of Ca^{2 +} to cells that had been stimulated with

CCh in the absence of Ca^{2 +} showed that Ca^{2 +} influx into cells transfected with Hftp3 was faster and lasted longer than in control cells causing lCa^{2 +} l; to increase to levels that were 200% to 230% above those seen in cells transfected without Hftp3. It is noteworthy that while co-expression of Hftpl had no measurable effect on the rate of decay of the IP3-induced peak [Ca^{2 "1"}],, it did cause a significant increase in Ca^{2 +} influx when measured by the Ca ⁺ readdition protocol. The magnitude of the effect of Hftpl , a maximum of 75% over control, was smaller than that of Hftp3. Thus, the Ca ⁺ readdition protocol is a more sensitive way of measuring changes in Ca^{2 +} influx than assessing changes in the kinetics of the IP3-induced [Ca^{2 +} ], transient or changes in plateau [Ca^{2 +} 1; as seen in the continuous presence of extracellular Ca^{2 +} .

Various aspects of the Hftp3-induced Ca^{2 +} influx are set forth below. The first was to determine that increased Ca^{2 +} influx was not merely a non-specific leak that developed in response to protein overexpression. This was addressed by testing whether Ca^{2 +} influx in the presence of Hftp3 could be inhibited by lanthanum and nickel, which both inhibit capacitative Ca^{2 +} influx (34,35). For lanthanum, the Hftp3-stimulatβd Ca^{2 +} influx is fully inhibited by 1 mM La^{3 +} , as is the CCE endogenous to COS cells. Hftp3-mediated Ca^{2 +} influx differed from agonist-stimulated COS cell CCE in that it was significantly less sensitive to low concentrations of La^{3 +} . At 250 pM, endogenous Ca^{2 +} influx was 80-90% blocked while the difference due to Hftp3 influx was blocked only 30-40%. In another set of examples we found that endogenous CCh-stimulated CCE was blocked > 90% by 2 mM Ni^{2 +} , while CCh-stimulated influx due to Hftp3 was inhibited by only 20%; 10 mM Ni^{2 +} blocked Ca^{2 +} influx in Hftp3 cells 85%. Although it still needs to be determined whether part of the endogenous COS cell CCE is Hfrp3-like, the above results demonstrate that Ca^{2 +} entry stimulated by expression of Hftp3 is not due to appearance of a non-specific leak.

We also tested whether Ca^{2 +} influx in Hftp3 transfected cells allowed passage of Mn^{2 +} . Some forms of CCE channels allow passage of Mn^{2 +} while others do not (36,37). We thus depleted internal stores in Ca^{2 +} free medium by addition of CCh, allowed [Ca^{2 +} ]_j to return to baseline levels (range: 40 and 60 nM) and then added 25 pM MnCI₂ so as to monitor Mn^{2 +} entry by its effect to quench the fluorescence signal of Fura2 excited at 380 nm. In Hftp3-transfected cells the Fura2 signal was quenched at a rate of 0.14%/sec, which was 3-times faster than quenching observed in control cells (0.05%/sec, data not shown). These finding indicated that in control cells as well as in Hftp3-transfected cells, Ca^{2 +} enters through channels that allow passage of Ca^{2 +} and Mn^{2 +} .

We tested whether the Hftp3-induced influx is regulated by store depletion in the absence of agonist. Cells were placed into Ca^{2 +} free medium plus 500 nM TG to inhibit internal Ca pumps and thus promote agonist-independent store depletion. Ca ⁺ (1.8 mM) was then added to measure Ca^{2 +} influx. The store depletion-activated increase in [Ca^{2 +} l_j was larger in Hftp3-transfectθd cells than in control cells indicating that Hftp3 dependent Ca^{2 +} influx can be activated by store depletion independent of prior activation of the G-protein/PLC/IP3 pathway. As in control experiments with agonist-stimulated Ca^{2 +} entry, TG-stimulated Ca^{2 +} entry was also blocked > 80% by 250 pM La^{3 +} while Ca ⁺ entry into Hftp3 transfected cells showed a significant residual Ca^{2 +} entry confirming stimulation of a distinct type of Ca^{2 +} entry pathway. We noted that the increase in TG-stimulated Ca^{2 +} influx due to expression of Hftp3 is of a more transient nature than the endogenous TG-stimulated Ca^{2 +} influx. The above tests demonstrate that Hftp3- and Hft l -mediated CCE is subject to regulation by store depletion and does not require simultaneous stimulation by an agonist, and also, that there are differences with respect to the endogenous COS cell CCE. It appears also that Hftp3-mediated Ca^{2 +} influx may be more sensitive to agonist- promoted store depletion than thapsigargin-mediated store depletion.

The above description shows that mammalian homologues of insect channels that were expressed in mammalian cells could permeate Ca^{2 +} in response to a manipulation that activates endogenous CCE. These results did not rule out the possibility that while expression of these homologues mimicked CCE, they were not the type of molecules that naturally fulfilled this function in mammalian cells. We thus investigated the molecular diversity of mammalian trp genes, cloned partial cDNA fragments and expressed these in the antisense direction in a mammalian cell line (murine L cells) to determine whether they would interfere with natural CCE. Molecular Diversity of the trp Family.

We found by Northern analysis that Hft l is expressed human tissues with higher amounts in ovary, testis, heart and brain. Hftpl is not expressed in liver. Since agonist-stimulated calcium influx is readily demonstrable in liver (38,39), this suggested strongly that if ftp-related proteins participated in or were to be responsible for this type of Ca^{2 +} influx, the mRNA encoding the particular trp carrying out this function in liver should be represented in liver RNA. Using mouse liver polyA ⁺ RNA as template and degenerate sets of primers based on the amino acids known to be conserved in Drosophila trp (Dtrp), Drosophila ftp-like (Dftpl ),

Caenorhabditis elegans trp (Ceftp) and Hftpl , we amplified and cloned a PCR fragment of 405 bp that had a continuous open reading frame of 135 codons encoding an amino acid sequence very similar to that encoded in the human pseudogene-derived EST T67673 (ΨHftp2), with two exceptions: 1 . that alignment of the murine sequence with other trp sequences did not require introduction of a 31 amino acid gap and 2. that where EST T67673 has a Stop codon we found the CGA codon for Arg.

Using a second set of sense and antisense primers, we amplified and cloned another PCR fragment which, except for beginning 93 nt downstream from the first, had the same nucleotide sequence as the first and hence encoded the same murine ftp-homologue, Mftp2. Using mouse brain polyA ⁺ RNA as template and other mixtures of degenerate oligonucleotides we identified cDNA fragments that potentially encoded five additional murine ftp-related proteins. Published data (40, 41) and a query of dbEST had predicted that including the human pseudogene we should have found only three additional murine ftp-related gene products. A comparison of the predicted amino acid sequences of the cDNA fragments obtained by RT-PCR to known ftp-related sequences showed that we had obtained in addition to Mfrpi , Mftp2 and Mftp3, the murine equivalents of their human counterparts, Mftp4, a murine sequence described by (40), and two new sequences, Mftp5 and Mtrpβ. Compared to Mftp5, Mfrpi , -2, -3, -4 and -6 differ at the nucleotide level by 53, 46, 40, 22 and 39 percent, respectively. Ignoring gaps, the same comparison at the amino acid level shows Mtrpλ , -2, -3, -

4 and -6 to differ from Mftp5 in this region of the proteins by 57, 49, 45, 7, and 56% percent, respectively.

Murine genomic DNA was tested for the presence of six distinct trp genes using a PCR approach. All the trp cDNA sequences reported here lie immediately upstream of a highly conserved EWKFAR motif. Using as 3' PCR primers, antisense oligonucleotides based on this motif, and as 5' PCR primers, exact sense oligonucleotides specific for each of the six trp transcripts, it was possible to amplify genomic fragments from four of the six murine trp genes. The length of these fragments exceeded by 600 bp to 2.8 kb that of the 180 bp product predicted if there would have been no iritron between the primers, indicating that the primers spanned introns that varied in length in the separate genes. The PCR fragments were cloned and their identity was confirmed by sequencing the intron- exon boundaries. One explanation for our failure to amplify a fragment of the Mtrpλ and Mftp5 genes is that in these genes the introns are tόδlarge to amplify under the conditions used. Another explanation could be that for these genes the

EWKFAR motif on which the 3' primers were based is not absolutely conserved in these genes — in the C. elegans trp it is EKWFHR — which could make our primers ineffective in the PCR reaction. Absence of an intron between the primers would have yielded a 180 bp fragment, which was not obtained. The identification of distinct genomic fragments for four of the trp sequences found by RT-PCR provides independent confirmation for the existence of four of the six trp genes inferred from by analyzing the RT-PCR products. The fact that these genes have conserved intron/exon boundaries is further proof of the evolutionary relatedness of the sequences identified by RT-PCR. Inhibition of Endogenous CCE by trp Antisense Sequences.

The results presented in the preceding paragraphs increased the number of possible f p-related proteins that could be involved in agonist- and store- operated CCE to six. The murine f p-related sequences were cloned in their antisense direction downstream of the CMV promoter of the eukaryotic expression vector pGWI H and transfected together with the M5 receptor (in pcDNA3) into murine L cells. Cells transformed by pcDNA3 DNA were isolated by growing in G418-containing medium. pcDNA3, but not pGWI H, carries the neomycin resistance gene. Transfection of L cells with human genomic DNA has shown that these cells are able to incorporate in stable form as much as 1 .5 million base pairs (42). On the basis of this we assumed that cells selected for transformation by the pcDNA3 vector were likely to have incorporated also the pGWI H vectors with the six antisense trp sequences and hence to be co- expressing the M5 receptor and the anti-ftp sequences. Cells from the isolated cell clones that were positive for M5 receptor expression as seen by their ability to respond to CCh with an IP3-induced rise and fall in [Ca²*];, were then tested for their ability to mount a capacitative Ca^{2 +} influx response. In six of the nine M5 receptor positive cell lines that been transfected with both the M5 receptor and antisense cDNA fragments, the expression of antisense sequences fully prevented activation of CCE. As determined for cells from two cell lines transfected with antisense cDNAs and showing no agonist-stimulated CCE, the loss of CCE was not due to a collapse their resting membrane potentials. Thus, the resting membrane potentials (mean ±_ SEM) of cells from clones a6.19 and a6.5, which had their CCE responses suppressed, were -30_£4 mV (n = 8) and -35 t.4 mV (n = 8), respectively; and those of cells from clones c.1 and c.4, which expressed the M5 receptor alone and showed agonist-activated CCE, were -11 ± 7. mV (n = 8) and -34.±4 mV (n = 8), respectively. None of these membrane potentials differed significantly from the other (p>0.01 ). This indicated that loss of CCE was not a non-specific effect of the antisense sequences causing a collapse of the membrane potential. These examples further demonstrate that one or more of the mammalian trp homologues Hftpl and Hftp3 are components of the CCE pathway, and vice versa that CCE is totally dependent on one or more ftp-related gene products.

Primary structure, tissue expression and model of topology of Hftp3. Northern analysis detected an Hftp3 mRNA of ca. 4 Kb predominantly in brain, and at much lower levels also in ovary, colon, small intestine, lung, prostate, placenta and testis. A larger size mRNA present at a lower level in brain, could be composed of incompletely processed mRNA or alternatively spliced products. A Kyte-Doolittle analysis revealed a core of eight hydrophobic regions of which six could encode transmembrane segments based on degree of hydrophobicity and length ( ≥ 16 amino acids). This core is 320 amino acids long and is delimited, in analogy to other ion channels, by putative cytosolic N- and C- termini that are 350 and 200 amino acids long, respectively. The above results show that Hftp3 is a protein that enhances CCE in COS cells and that Hftpl show a similar activity. The activity of these gene products was best observed when CCE was measured following agonist-stimulated depletion of intracelluiar stores in Ca^{2 +}-free medium. This protocol is similar to that used by Peterson et al. (40) showing that expression of Drosophila frp in a vertebrate cell, the Xenopus oocyte, causes an increase in capacitative Ca^{2 +} influx of 66% in excess of the oocyte 's endogenous CCE. The activities of Hftpl and Ηfrp3, increasing Ca^{2 +} entry into COS cells by 75% and 230%, respectively, compare favorably to that of the insect channel.

In accordance with the present invention, the Ca^{2 +} influx due to Hfrp3 was less sensitive to inhibition by La^{3 +} and Ni^{2 +} than Ca^{2 +} entry through the endogenous COS cell CCE channel(s). The CCE channel formed in Hfrp3- expressing cells was found to permeate Ca^{2 +}and Mn^{2 +} . Several reports during the last years have emphasized that hormones, growth factors and other cellular activators stimulate more than one Ca ⁺ influx pathway (44,38,44a), and expression of the Drosophila frp and frp-like in Sf9 cells showed formation of two different type of channels. One is highly selective for Ca^{2 +} (trp) and activated upon TG-induced store depletion. The other, frp-like, shows no-selectivity for Ca^{2 +} , is insensitive to store depletion, permeates mono-and divalent cations alike, is activated by IP3 and has a tendency for spontaneous agonist-independent activation (45,46,47,48). It is not known whether CCE channels with properties of insect frp and frp-like exist in vertebrate cells. The existence of a family of mammalian trp proteins described here, of which two members (Hftpl and Hftp3) have the ability to increase Ca^{2 +} influx, and the effect of anti-ftp sequences suppressing CCE in a fibroblast cell line, provide a formal link between the activity of Hfrp3/Hftp1 and CCE.

As is apparent from the preceding description, mammalian ftp proteins are a required component of capacitative calcium ion entry into mammalian cells.

Accordingly, control of the amount of active frp protein in a cell provides a way to control the calcium ion level of the cell. Methods for controlling the amount of active protein expressed by a cell are well-known. For example the cells can be treated with nucleotides which are anti-sense to the gene which expresses the protein. This type of treatment prevents expression of the trp protein. Anti-sense treatment protocols are used when it is desired to reduce frp protein present in the cell and thereby reduce calcium ion entry. The nucleotide sequences may also be introduced into the cell in order to increase the expression of trp proteins and thereby increase calcium ion entry. These two procedures allow one to control calcium ion levels in the cell by either increasing or decreasing the level of trp protein expressed by the cell.

In addition to controlling frp protein expression, calcium ion entry can be contfoUed-by treating the cell with an inhibitory agent which binds to or otherwise denatures the trp protein. Suitable types of inhibitory agents include imidazole derivatives such as SKF 96365, econazole, micozolβ, clotrimazole, and calmidazolium [Merrit et al. (52); Daly et al. (53)] plant alkaloids such as tetrandine and hernandezine (Low et al., 1996). The activity of trp may also be regulated by cellular substances known to affect CCE. Such substances include an unidentified diffusible messenger (CIF), inositol phosphates (IP3 and IP4), cyclic GMP, or by covalent modification by enzymes such as protein kinases, protein phosphatases, small GTPases and cytochrome P450. It has been suggested that maitotoxin may stimulate CCE channels [Worley et al. (54)]. Monoclonal antibodies may also be used as inhibitory agents. Suitable monoclonal and polyclonal antibodies could be obtained by standard techniques using purified GST-fusion proteins as antigens, which are also made by standard procedures and where the fusion aspect of the complex is a portion of the ectodomain of the trp protein. For Hfrp3 this could be any stretch between amino acid 350 and 650. It is anticipated that such antibodies could modulate the CCE and be of therapeutic use.

Treatment of the mammalian cells with sense and anti-sense trp nucleotides and/or ftp inhibitory agents can be accomplished in accordance with any of the known procedures for treating cells to control the production of a selected protein. The various dosages and amounts of selected agents which are required to achieve desired levels of calcium ion entry can be established by routine experimentation.

Examples of treatment protocols in accordance with the present invention involving the use of anti-sense nucleotides to reduce calcium ion levels are as follows:

Cellular Trp levels in cells can be regulated by introduction of antisense sequences by inserting partial or complete trp cDNAs in the antisense direction into viral expression vectors based on retroviruses or adenoviruses using protocols that are being applied for purposes of gene therapy as summarized in Chapter 5:

Gene Based Therapy of Goodman and Gilman's Ninth Edition of The Pharmacological Basis of Therapeutics McGraw-Hill, pp. 77-101 (1996). Alternatively, oligonucleotides complementary to the coding region of trp molecules can be administered in to humans in pharmaceutical formulations such as aerosols or creams, if epithelia of the airways or cells in the dermis and epidermis are to be targeted. The same technique can be used to suppress trp expression in cultured cells in vitro.

Examples of treatment protocols in accordance with the present invention involving the use of frp control agents to control calcium ion levels are as follows: - inhibition of airway smooth muscle CCE to treat asthma inhibition of vascular endothelial CCE to treat hypertension stimulation of pancreatic CCE to stimulate insulin secretion in type II (non-insulin-de pendent) diabetes inhibition of osteociast CCE to prevent osteoporosis stimulation of osteoblast CCE to promote bone formation - inhibition of platelet CCE as an antithrombotic therapy gene therapy of primary immunodeficiencies if they are due to mutations in trp genes (see references 55 and 56).

The dosage levels and treatment regimens for all of the above-mentioned uses for the present invention can be established using routine experimentation.

The discovery of the importance of Hfrp protein in the control of calcium ion entry into the cell also provides a basis in accordance with the present invention to screen a large number of compounds to determine if they may be useful in controlling cellular calcium ion levels. In its simplest form, the screening method involves exposing the cell to a potential drug or other compound and determining if the level of trp protein is reduced. If the compound is effective in reducing trp protein levels, then it is considered a good candidate for use in reducing calcium ion entry into the cell.

The type of compounds which can be screened according to this aspect of the present invention are unlimited. The screening procedures which may be used to test compounds for their ability to inhibit trp protein are well-known to those skilled in the art. The same screening procedures which have been used to screen compounds for inhibitory properties with respect to other proteins and enzymes expressed by celts may be used. An exemplary screening protocol is set forth as follows.

Trp proteins can be expressed in cells by standard recombinant means such as described in Innamoratti et al. (57), Gudermann et al. (58), Zhu et al. (59) and Ca^{2 +} influx monitored in single cells as described in Zhu et al. (30) or in a population of cells as described in Liao et al. (32). By doing this in the absence and presence of test compounds of which the effect on frp-mediated CCE can then be determined. An example is shown below (FIGS. 1 and 2) where human embryonic kidney cells (HEK-293 cells) expressing Hfrp3 in stable form (HEKt3-9) are stimulated with carbachol and CCE is measured upon readdition of Ca^{2 +} to the extracellular medium. In the example, 25 pM SKF 96365 blocks selectively CCE due to Hftp3. It should be noted that CCE endogenous to the HEK 293 cell (control), presumably mediated by frp's other than Hfrp3 is much less sensitive to this concentration of SKF 96365. Not only agents that block calcium entry due to trp expression but also agents that stimulate calcium entry due to trp can be monitored in this way. The second example, FIG. 3, below shows maitotoxin- stimulated Ca^{2 +} influx into HEK 293 ceils that is several fold larger in cells expressing Hftp3 than in control cells. In the above examples, cell were suspended in extracellular solution at a concentration of 20 x 10^β cells/ml, loaded with Fura2AM (5pM, 30 min), washed with solution nominally free of calcium, twice, and suspended at 2 x 10^β cells/ml. Intracelluiar Ca^{2 +} concentrations were then monitored as described in Liao et al (32). Times and concentrations of additions are depicted by the bars in the Figures. Control cells were HEK 293 cells expressing an unrelated protein. For further details see references 32 and 30. Note in FIG. 1 that expression of Hfrp3 in HEKt3-9 cells potentiates carbachol (CCh)-stimulated CCE and that the "extra- CCE" due to Hfrp3 expression is blocked by 25 pM SKF 96365 in FIG. 2.

Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the disclosures herein are exemplary only and that various other alternations, adaptations and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Birnbaumer, Lutz Zhu, Xi

(ii) TITLE OF INVENTION: Method And Compounds For Controlling

Capacitative Calcium Ion Entry Into Mammalian Cells Essential for Agonist-Activated Capacitative Ca2+ Entry

(iii) NUMBER OF SEQUENCES: 32

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Poms, Smith, Lande & Rose

(B) STREET: 2029 Century Park East, Suite 3800

(C) CITY: Los Angeles

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 90067

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: WordPerfect 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/025,111

(B) FILING DATE: August 29, 1996

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Oldenkamp, David J.

(B) REGISTRATION NUMBER: 29,421

(C) REFERENCE/DOCKET NUMBER: 120186

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (310) 788-5000

(B) TELEFAX: (310) 277-1297

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2922 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ACCAGATTGC AACTTTGCGG AGATGATGAT GGACTGACAT GGCCTGAAGC -50

ATG GCT CAG TTC TAT TAC AAA AGA AAT GTC AAC GCC CCC TAC AGA GAC 48 Met Ala Gin Phe Tyr Tyr Lye Arg Asn Val Asn Ala Pro Tyr Arg Asp 1 5 10 15

CGC ATC CCA CTG AGG ATT GTC AGA GCA GAA TCT GAG CTC TCA CCA TCA 96 Arg lie Pro Leu Arg lie Val Arg Ala Glu Ser Glu Leu Ser Pro Ser 20 25 30

GAG AAA GCC TAC TTG AAT GCT GTG GAG AAG GGG GAC TAT GCA AGC GTC 144 Glu Lye Ala Tyr Leu Asn Ala Val Glu Lys Gly Asp Tyr Ala Ser Val 35 40 45

AAG AAG TCT CTG GAG GAA GCT GAG ATT TAT TTT AAA ATC AAC ATT AAC 192 Lys Lys Ser Leu Glu Glu Ala Glu lie Tyr Phe Lys lie Asn lie Asn 50 55 60

TGC ATC GAC CCC CTG GGA AGG ACC GCC CTC CTC ATT GCC ATT GAA AAT 240 Cys lie Asp Pro Leu Gly Arg Thr Ala Leu Leu lie Ala lie Glu Asn 65 70 75 80

GAG AAT CTG GAG CTT ATT GAA CTA TTG TTG AGT TTC AAT GTC TAT GTA 288 Glu Asn Leu Glu Leu lie Glu Leu Leu Leu Ser Phe Asn Val Tyr Val 85 90 95

GGC GAT GCG CTG CTT CAC GCC ATC AGA AAA GAG GTG GTT GGA GCC GTG 336 Gly Asp Ala Leu Leu His Ala lie Arg Lys Glu Val Val Gly Ala Val 100 105 110

GAG CTA CTG CTG AAC CAC AAA AAG CCA AGT GGA GAG AAG CAG GTG CCT 384 Glu Leu Leu Leu Asn His Lys Lys Pro Ser Gly Glu Lys Gin Val Pro 115 120 125

CCC ATT CTC CTT GAT AAA CAG TTC TCT GAA TTC ACT CCG GAC ATC ACA 432 Pro lie Leu Leu Asp Lys Gin Phe Ser Glu Phe Thr Pro Asp lie Thr 130 135 140

CCC ATC ATC TTG GCT GCA CAT ACA AAT AAT TAC GAG ATA ATC AAA CTT 480 Pro lie lie Leu Ala Ala His Thr Asn Asn Tyr Glu lie lie Lys Leu 145 150 155 160

TTG GTT CAG AAA GGT GTC TCA GTG CCC AGA CCC CAC GAG GTC CGC TGT 528 Leu Val Gin Lys Gly Val Ser Val Pro Arg Pro His Glu Val Arg Cys 165 170 175

AAC TGT GTT GAG TGT GTC TCC AGC TCG GAT GTG GAC AGC CTC AGG CAT 576 Asn Cys Val Glu Cys Val Ser Ser Ser Asp Val Asp Ser Leu Arg His 180 185 190

TCA CGG TCC AGG CTC AAC ATC TAC AAG GCC TTG GCC AGC CCC TCG CTC 624 Ser Arg Ser Arg Leu Asn lie Tyr Lys Ala Leu Ala Ser Pro Ser Leu 195 200 205 ATT GCC CTG TCA AGC GAA GAC CCT TTC CTT ACT GCC TTT CAG TTA AGT 672 lie Ala Leu Ser Ser Glu Asp Pro Phe Leu Thr Ala Phe Gin Leu Ser 210 215 220

TGG GAG CTG CAA GAA CTC AGC AAG GTG GAG AAC GAA TTC AAG TCG GAG 720 Trp Glu Leu Gin Glu Leu Ser Lys Val Glu Asn Glu Phe Lys Ser Glu 225 230 235 240

TAT GAG GAG CTG TCT AGA CAG TGC AAA CAA TTT GCC AAG GAC CTC CTA 768 Tyr Glu Glu Leu Ser Arg Gin Cys Lys Gin Phe Ala Lys Asp Leu Leu 245 250 255

GAT CAG ACA CGG AGT TCC AGA GAG CTG GAA ATC ATT CTT AAT TAC CGT 816 Asp Gin Thr Arg Ser Ser Arg Glu Leu Glu lie lie Leu Asn Tyr Arg 260 265 270

GAT GAC AaT AGT CTG ATC GAA GAA CAG AGT GGA AAT GAT CTT GCA AGG 864 Asp Asp Asn Ser Leu lie Glu Glu Gin Ser Gly Asn Asp Leu Ala Arg 275 280 285

CTA AAA TTA GCC ATT AAG TAC CGT CAA AAA GAG TTT GTT GCT CAG CCC 912 Leu Lys Leu Ala lie Lys Tyr Arg Gin Lys Glu Phe Val Ala Gin Pro 290 295 300

AAC TGC CAG CAG CTG CTC GCT TCC CGC TGG TAC GAT GAG TTC CCA GGC 960 Asn Cyβ Gin Gin Leu Leu Ala Ser Arg Trp Tyr Asp Glu Phe Pro Gly 305 310 315 320

TGG AGG AGA AGA CAC TGG GCG GTG AAG ATG GTG ACG TGT TTC ATA ATA 1008 Trp Arg Arg Arg His Trp Ala Val Lys Met Val Thr Cys Phe lie lie 325 330 335

GGA CTA CTC TTC CCC GTC TTC TCC GTG TGC TAC CTG ATA GCT CCC AAA 1056 Gly Leu Leu Phe Pro Val Phe Ser Val Cyβ Tyr Leu lie Ala Pro Lye 340 345 350

AGC CCA CTT GGA CTG TTC ATC AGa AAG CCA TTT ATC AAG TTT ATC TGC 1104 Ser Pro Leu Gly Leu Phe lie Arg Lys Pro Phe lie Lys Phe lie Cyβ 355 360 365

CAC ACA GCC TCC TAT CTG ACC TTT TTG TTT CTG CTG CTG CTA GCC TCT 1152 His Thr Ala Ser Tyr Leu Thr Phe Leu Phe Leu Leu Leu Leu Ala Ser 370 375 380

CAG CAC ATC GAC AGG TCA GAC TTG AAC AGG CAA GGT CCA CCA CCA ACC 1200 Gin His lie Asp Arg Ser Asp Leu Asn Arg Gin Gly Pro Pro Pro Thr 385 390 395 400

ATC GTG GAG TGG ATG ATA TTA CCG TGG GTC CTG GGT TTT ATA TGG GGA 1248 lie Val Glu Trp Met lie Leu Pro Trp Val Leu Gly Phe lie Trp Gly 405 410 415

GAG ATT AAA CAG ATG TGG GAT GGC GGA CTC CAG GAT TAC ATC CAT GAC 1296 Glu lie Lys Gin Met Trp Asp Gly Gly Leu Gin Asp Tyr lie His Asp 420 425 430

TGG TGG AAT CTA ATG GAC TTT GTG ATG AAC TCC TTG TAT CTG GCA ACA 1344 Trp Trp Asn Leu Met Asp Phe Val Met Asn Ser Leu Tyr Leu Ala Thr 435 440 445

ATC TCC TTG AAG ATT GTC GCG TTT GTA AAG TAC AGT GCT CTG AAC CCA 1392 lie Ser Leu Lye lie Val Ala Phe Val Lye Tyr Ser Ala Leu Asn Pro 450 455 460 CGG GAA TCA TGG GAC ATG TGG CAC CCC ACC CTG GTG GCA GAG GCA TTA 1440 Arg Glu Ser Trp Asp Met Trp His Pro Thr Leu Val Ala Glu Ala Leu 465 470 475 480

TTT GCT ATT GCA AAC ATC TTC AGT TCC CTC CGC CTG ATC TCT CTG TTC 1488 Phe Ala lie Ala Asn lie Phe Ser Ser Leu Arg Leu lie Ser Leu Phe 485 490 495

ACT GCC AAT TCT CAC CTG GGG CCT CTG CAG ATA TCT CTG GGA AGG ATG 1536 Thr Ala Asn Ser His Leu Gly Pro Leu Gin lie Ser Leu Gly Arg Met 500 505 510

CTT CTG GAC ATC CTG AAG TTC TTG TTC ATC TAC TGC CTC GTG CTG CTA 1584 Leu Leu Asp lie Leu Lys Phe Leu Phe lie Tyr Cys Leu Val Leu Leu 515 520 525

GCT TTT GCA AAT GGC CTA AAT CAG CTG TAC TTT TAC TAT GAA GAA ACA 1632 Ala Phe Ala Asn Gly Leu Asn Gin Leu Tyr Phe Tyr Tyr Glu Glu Thr 530 535 540

AAG GGG CTA AGC TGC AAA GGC ATC CGG TGC GAG AAA CAG AAC AAC GCG 1680 Lys Gly Leu Ser Cys Lys Gly lie Arg Cys Glu Lys Gin Asn Asn Ala 545 550 555 560

TTT TCC ACG TTA TTC GAG ACA CTA CAG TCC CTG TTT TGG TCA ATA TTT 1728 Phe Ser Thr Leu Phe Glu Thr Leu Gin Ser Leu Phe Trp Ser lie Phe 565 570 575

GGA CTC ATC AAT CTC TAT GTT ACC AAT GTC AAG GCC CAG CAC GAG TTC 1776 Gly Leu lie Asn Leu Tyr Val Thr Asn Val Lys Ala Gin His Glu Phe 580 585 590

ACT GAG TTT GTT GGG GCC ACC ATG TTT GGC ACA TAT AAT GTC ATC TCT 1824 Thr Glu Phe Val Gly Ala Thr Met Phe Gly Thr Tyr Asn Val He Ser 595 600 605

CTG GTT GTC CTG CTG AAC ATG TTA ATT GCT ATG ATG AAT AAT TCT TAC 1872 Leu Val Val Leu Leu Asn Met Leu He Ala Met Met Asn Asn Ser Tyr 610 615 620

CAA CTA ATT GCC GAC CAT GCA GAT ATA GAA TGG AAA TTT GCT CGA ACA 1920 Gin Leu He Ala Asp His Ala Asp He Glu Trp Lys Phe Ala Arg Thr 625 630 635 640

AAG CTT TGG ATG AGC TAC TTT GAA GAA GGA GGT ACC CTG CCT ACA CCT 1968 Lys Leu Trp Met Ser Tyr Phe Glu Glu Gly Gly Thr Leu Pro Thr Pro 645 650 655

TTC AAT GTC ATC CCA AGC CCC AAG TCC CTG TGG TAC CTG GTC AAG TGG 2016 Phe Asn Val He Pro Ser Pro Lys Ser Leu Trp Tyr Leu Val Lys Trp 660 665 670

ATA TGG ACA CAC TTA TGT AAG AAA AAA ATG AGA AGG AAG CCA GAA AGC 2064 He Trp Thr His Leu Cyβ Lys Lys Lys Met Arg Arg Lys Pro Glu Ser 675 680 685

TTC GGG ACA ATT GGG CGG CTT GCT GCT GAT AAC TTG AGA AGA CAT CAC 2112 Phe Gly Thr He Gly Arg Leu Ala Ala Asp Asn Leu Arg Arg His His 690 695 700

CAA TAC CAA GAG GTG ATG AGG AAC CTG GTG AAG CGG TAC GTG GCT GCC 2160 Gin Tyr Gin Glu Val Met Arg Asn Leu Val Lys Arg Tyr Val Ala Ala 705 710 715 720 ATG ATC AGA GAG GCA AAA ACC GAA GAA GGC TTG ACG GAG GAG AAT GTT 2208 Met He Arg Glu Ala Lys Thr Glu Glu Gly Leu Thr Glu Glu Asn Val 725 730 735

AAG GAA CTA AAG CAA GAC ATT TCT AGC TTC CGC TTC GAA GTT CTG GGA 2256 Lys Glu Leu Lye Gin Asp He Ser Ser Phe Arg Phe Glu Val Leu Gly 740 745 750

TTG CTC AGA GGA AGC AAG CTC TCT ACA ATA CAG TCA GCC AAC GCG GCG 2304 Leu Leu Arg Gly Ser Lys Leu Ser Thr He Gin Ser Ala Asn Ala Ala 755 760 765

AGT TCA GCG GAC TCC GAC GAG AAG AGC CAG AGC GAA GGT AAT GGC AAG 2352 Ser Ser Ala Asp Ser Asp Glu Lys Ser Gin Ser Glu Gly Asn Gly Lys 770 775 780

GAC AAG AGA AAG AAT CTC AGC CTC TTT GAT TTA ACC ACT CTG ATC TAC 2400 Asp Lys Arg Lys Asn Leu Ser Leu Phe Asp Leu Thr Thr Leu He Tyr 785 790 795 800

CCG CGG TCG GCA GCC ATT GCC TCC GAG AGA CAT AAC CTA AGC AAT GGT 2448 Pro Arg Ser Ala Ala He Ala Ser Glu Arg Hie Asn Leu Ser Asn Gly 805 810 815

TCC GCC CTG GTG GTG CAG GAG CCG CCC AGG GAG AAG CAG AGG AAA GTG 2496 Ser Ala Leu Val Val Gin Glu Pro Pro Arg Glu Lye Gin Arg Lys Val 820 825 830

AAT TTT GTG GCT GAT ATC AAA AAC TTC GGG TTA TTT CAT AGA CGG TCA 2544 Asn Phe Val Ala Asp He Lye Asn Phe Gly Leu Phe His Arg Arg Ser 835 840 845

AAA CAA AAT GCT GCT GAG CAA AAC GCA AAC CAA ATC TTC TCT GTT TCA 2592 Lys Gin Asn Ala Ala Glu Gin Asn Ala Aβn Gin He Phe Ser Val Ser 850 855 860

GAA GAA ATT ACT CGT CAA CAG GCG GCA GGA GCA CTT GAG CGA AAT ATC 2640 Glu Glu He Thr Arg Gin Gin Ala Ala Gly Ala Leu Glu Arg Asn He 865 870 875 880

GAA CTG GAA TCC AAA GGA TTA GCT TCA CTG GGT GAC CGC AGC ATT CCT 2688 Glu Leu Glu Ser Lys Gly Leu Ala Ser Leu Gly Asp Arg Ser He Pro 885 890 895

GGT CTC AAT GAA CAG TGT GTG CTA GTA GAC CAT AGA GAA AGG AAT ACG 2736 Gly Leu Aβn Glu Gin Cys Val Leu Val Asp His Arg Glu Arg Asn Thr 900 905 910

GAC ACT TTG GGT TTA CAG GTA GGC AAG AGA GTG TGC TCC ACC TTC AAG 2784 Asp Thr Leu Gly Leu Gin Val Gly Lys Arg Val Cys Ser Thr Phe Lys 915 920 925

TCG GAG AAG GTG GTG GTG GAA GAC ACC GTC CCT ATT ATA CCA AAG GAG 2832 Ser Glu Lye Val Val Val Glu Asp Thr Val Pro He He Pro Lys Glu 930 935 940

AAA CAC GCC CAT GAG GAG GAC TCG AGC ATA GAC TAT GAC TTA AGC CCC 2880 Lys Hie Ala His Glu Glu Asp Ser Ser He Asp Tyr Asp Leu Ser Pro 945 950 955 960

ACG GAC ACA GCT GCC CAT GAA GAT TAT GTG ACC ACA AGA TTG 2922

Thr Asp Thr Ala Ala His Glu Asp Tyr Val Thr Thr Arg Leu 965 970 974 TGACCCTTGG AGGAGTGTTT ACCATACCTA TACATATTTT CCATAGTGCT CTGAGCAGGC -60 AAAATGTTTG AAATCCCATT ATCAAATGCT AATTTCCACT TTCTAATGTT TATCTGTTGT -120 GGCATATTAA CCTGTAATAT GTTTGAACAA AGCAGAGGTA ATATGAACCC TTCTCTTTTG -180 TAGCCTGCTT TTGCTTTCAC CGTTTATTTT ACAAGTGTTT CTGTTAAATA AACGCACCTT -240 TTCTCCTTGT ACTGTTACAA TAACCCACAG AAAACTTTTA GCTATCTTTT TTCAATTAAA -300 ACCAATGCAA TTGTTTTC -318

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2786 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

CGCCTGTGC CCTCTGCCTG GGAGCCTGGG GCCGCCTGTC TGCGCGGTCC GGATGCGCTC -60

AGGTCAAGG TTCCTTTCGC GGCTGTCTCC CAAGCCCCTA ACTAGTGACT TCCACTGTGG -120

CGGGCAGGG AAGCCATTGG CAGAACCTAG CCAGTCAGGA ATCTGCATCT CTTCCCTCAT -180

TATCCTCTC CCTGGCATTG CTTTGCTCGG GTCCAGCTCA GTTGGTGACG CGGCCCCTTC -240

TCCCCAGGT TCCCATCCAC GGAAGCAGGG GTGCAGGCCG GCCAGGCACT GTGCC -295 AGC CCG AGG TTC GTG ACC CGG AGG GGC GGC TCT CTA AAG 48 Met Ser Gin Ser Pro Arg Phe Val Thr Arg Arg Gly Gly Ser Leu Lys 5 10 —-15

GCT GCC CCT GGA GCC GGC ACC CGG CGC AAC GAG AGC CAG GAC TAT TTG 96 Ala Ala Pro Gly Ala Gly Thr Arg Arg Aβn Glu Ser Gin Asp Tyr Leu 20 25 30

CTG ATG GAC GAG CTG GGA GAC GAC GGC TAC CCG CAG CTC CCG CTG CCA 144 Leu Met Asp Glu Leu Gly Aβp Asp Gly Tyr Pro Gin Leu Pro Leu Pro 35 40 45

CCG TAT GGC TAC TAC CCC AGC TTC CGG GGT AAT GAA AAC AGA CTG ACT 192 Pro Tyr Gly Tyr Tyr Pro Ser Phe Arg Gly Asn Glu Asn Arg Leu Thr 50 55 60

CAC CGG CGG CAG ACG ATT CTT CGT GAG AAG GGA AGA AGG TTA GCT AAT 240 His Arg Arg Gin Thr He Leu Arg Glu Lye Gly Arg Arg Leu Ala Aβn 65 70 75 80 CGA GGA CCA GCA TAC ATG TTT AAT GAT CAT TCA ACA AGC CTG TCT ATT 288 Arg Gly Pro Ala Tyr Met Phe Aβn Aβp Hie Ser Thr Ser Leu Ser He 85 90 95

GAG GAA GAA CGC TTT CTA GAT GCA GCT GAA TAT GGC AAC ATC CCA GTG 336 Glu Glu Glu Arg Phe Leu Asp Ala Ala Glu Tyr Gly Aβn He Pro Val 100 105 110

GTG CGG AAG ATG CTA GAA GAG TGT CAT TCC CTC AAT GTT AAC TGT GTG 384 Val Arg Lys Met Leu Glu Glu Cyβ His Ser Leu Asn Val Asn Cyβ Val 115 120 125

GAT TAC ATG GGC CAG AAT GCC CTA CAG CTG GCT GTG GCC AAT GAG CAC 432 Asp Tyr Met Gly Gin Asn Ala Leu Gin Leu Ala Val Ala Aβn Glu His 130 135 140

TTG GAA ATC ACA GAG CTG CTA CTC AAG AAG GAA AAC TTG TCT CGA GTT 480 Leu Glu He Thr Glu Leu Leu Leu Lys Lys Glu Asn Leu Ser Arg Val 145 150 155 160

GGG GAT GCT TTA CTT TTA GCC ATT AGT AAA GGT TAT GTA CGG ATT GTG 528 Gly Asp Ala Leu Leu Leu Ala He Ser Lys Gly Tyr Val Arg He Val 165 170 175

GAG GCA ATC CTC AAC CAT CCA GCT TTT GCT GAA GGC AAA AGG TTA GCG 576 Glu Ala He Leu Asn His Pro Ala Phe Ala Glu Gly Lys Arg Leu Ala 180 185 190

ACA AGC CCC AGC CAG TCT GAA CTT CAG CAA GAT GAC TTT TAT GCC TAT 624 Thr Ser Pro Ser Gin Ser Glu Leu Gin Gin Asp Asp Phe Tyr Ala Tyr 195 200 205

GAT GAA GAT GGG ACG CGG TTC TCC CAT GAT GTG ACC CCA ATC ATT CTC 672 Asp Glu Aβp Gly Thr Arg Phe Ser Hie Aβp Val Thr Pro He He Leu 210 215 220

GCT GCA CAT TGC CAG GAA TAT GAA ATT GTG CAT ACC CTC CTG AGA AAG 720 Ala Ala Hie Cyβ Gin Glu Tyr Glu He Val Hie Thr Leu Leu Arg Lys 225 230 235 240

GGT GCC CGG ATT GAG CGG CCT CAT GAT TAC TTC TGC AAG TGT ACA GAA 768 Gly Ala Arg He Glu Arg Pro His Aβp Tyr Phe Cys Lys Cyβ Thr Glu 245 250 255

TGC AGC CAG AAG CAG AAG CAT GAT TCC TTC AGC CAC TCT AGA TCC AGG 816 Cys Ser Gin Lys Gin Lys His Aβp Ser Phe Ser His Ser Arg Ser Arg 260 265 270

ATC AAT GCA TAC AAA GGT CTG GCA AGT CCA GCA TAC CTG TCA TTG TCC 864 He Asn Ala Tyr Lys Gly Leu Ala Ser Pro Ala Tyr Leu Ser Leu Ser 275 280 285

AGT GAA GAT CCA GTC ATG ACT GCT TTA GAA CTT AGC AAT GAG CTG GCA 912 Ser Glu Asp Pro Val Met Thr Ala Leu Glu Leu Ser Asn Glu Leu Ala 290 295 300

GTG CTT GCC AAC ATT GAG AAA GAG TTC AAG AAT GAC TAC AGG AAG CTG 960 Val Leu Ala Aβn He Glu Lye Glu Phe Lys Asn Aβp Tyr Arg Lye Leu 305 310 315 320

TCT ATG CAG TGC AAG GAT TTC GTT GTT GGT CTC TTG GAC CTC TGC AGA 1008 Ser Met Gin Cyβ Lye Aβp Phe Val Val Gly Leu Leu Asp Leu Cys Arg 325 330 335 AAC ACA GAG GAA GTG GAG GCC ATC CTG AAT GGG GAT GCA GAG ACT CGC 1056 Aβn Thr Glu Glu Val Glu Ala He Leu Aβn Gly Aβp Ala Glu Thr Arg 340 345 350

CAG CCC GGG GAC TTC GGC CGT CCA AAT CTC AGC CGT TTA AAA CTT GCT 1104 Gin Pro Gly Asp Phe Gly Arg Pro Asn Leu Ser Arg Leu Lys Leu Ala 355 360 365

ATT AAG TAT GAA GTA AAA AAA TTT GTG GCT CAT CCA AAC TGT CAG CAA 1152 He Lys Tyr Glu Val Lys Lys Phe Val Ala His Pro Asn Cyβ Gin Gin 370 375 380

CAG CTC CTG TCC ATA TGG TAT GAG AAC CTC TCT GGT TTA CGG CAG CAG 1200 Gin Leu Leu Ser He Trp Tyr Glu Asn Leu Ser Gly Leu Arg Gin Gin 385 390 395 400

ACC ATG GCA GTG AAG TTC CTC GTG GTC CTT GCT GTT GCC ATT GGA TTG 1248 Thr Met Ala Val Lys Phe Leu Val Val Leu Ala Val Ala He Gly Leu 405 410 415

CCC TTC CTG GCT CTC ATA TAC TGG TGT GCT CCT TGC AGC AAG ATG GGG 1296 Pro Phe Leu Ala Leu He Tyr Trp Cyβ Ala Pro Cys Ser Lys Met Gly 420 425 430

AAG ATA TTG CGA GGA CCG TTC ATG AAG TTT GTA GCA CAC GCA GCC TCC 1344 Lys He Leu Arg Gly Pro Phe Met Lys Phe Val Ala His Ala Ala Ser 435 440 445

TTC ACC ATT TTC CTG GGG CTG CTC GTC ATG AAT GCA GCT GAC AGA TTT 1392 Phe Thr He Phe Leu Gly Leu Leu Val Met Aβn Ala Ala Aβp Arg Phe 450 455 460

GAA GGC ACC AAG CTC CTC CCT AAT GAA ACC AGC ACA GAT AAT GCA AGG 1440 Glu Gly Thr Lys Leu Leu Pro Asn Glu Thr Ser Thr Aβp Aβn Ala Arg 465 470 475 480

CAG CTG TTC AGG ATG AAA ACA TCC TGT TTC TCA TGG ATG GAG ATG CTC 1488 Gin Leu Phe Arg Met Lys Thr Ser Cyβ Phe Ser Trp Met Glu Met Leu 485 490 495

ATT ATA TCC TGG GTA ATA GGC ATG ATA TGG GCT GAA TGT AAA GAA ATC 1536 He He Ser Trp Val He Gly Met He Trp Ala Glu Cyβ Lye Glu He 500 505 510

TGG ACT CAA GGC CCC AAA GAA TAC TTA TTT GAG TTG TGG AAT ATG CTT 1584 Trp Thr Gin Gly Pro Lye Glu Tyr Leu Phe Glu Leu Trp Aβn Met Leu 515 520 525

GAC TTT GGA ATG CTG GCA ATC TTT GCA GCA TCA TTC ATT GCA AGA TTT 1632 Aβp Phe Gly Met Leu Ala He Phe Ala Ala Ser Phe He Ala Arg Phe 530 535 540

ATG GCG TTC TGG CAT GCA TCC AAA GCT CAG AGC ATC ATT GAT GCA AAT 1680 Met Ala Phe Trp Hie Ala Ser Lys Ala Gin Ser He He Asp Ala Aβn 545 550 555 560

GAT ACT TTA AAG GAT TTG ACA AAA GTC ACA CTG GGG GAC AAC GTT AAA 1728 Aβp Thr Leu Lys Asp Leu Thr Lys Val Thr Leu Gly Aβp Asn Val Lys 565 570 575

TAC TAC AAT CTG GCC AGG ATA AAG TGG GAC CCT ACT GAT CCT CAG ATC 1776 Tyr Tyr Asn Leu Ala Arg He Lye Trp Asp Pro Thr Aβp Pro Gin He 580 585 590 ATC TCT GAA GGT CTT TAT GCA ATC GCT GTG GTT TTA AGT TTC TCC AGA 1824 He Ser Glu Gly Leu Tyr Ala He Ala Val Val Leu Ser Phe Ser Arg 595 600 605

ATA GCT TAC ATT TTA CCA GCA AAT GAA AGC TTT GGA CCT CTG CAG ATT 1872 He Ala Tyr He Leu Pro Ala Asn Glu Ser Phe Gly Pro Leu Gin He 610 615 620

TCA CTT GGA AGA ACA GTG AAA GAT ATC TTC AAA TTC ATG GTC ATA TTC 1920 Ser Leu Gly Arg Thr Val Lye Aβp He Phe Lys Phe Met Val He Phe 625 630 635 640

ATC ATG GTG TTT GTA GCC TTT ATG ATT GGA ATG TTC AAC CTT TAC TCC 1968 He Met Val Phe Val Ala Phe Met He Gly Met Phe Asn Leu Tyr Ser 645 650 655

TAC TAC ATT GGC GCA AAA CAG AAT GAA GCA TTC ACA ACA GTT GAG GAA 2016 Tyr Tyr He Gly Ala Lys Gin Aβn Glu Ala Phe Thr Thr Val Glu Glu 660 665 670

AGT TTT AAG ACA CTG TTC TGG GCT ATC TTT GGT CTT TCT GAA GTG AAG 2064 Ser Phe Lys Thr Leu Phe Trp Ala He Phe Gly Leu Ser Glu Val Lys 675 680 685

TCA GTG GTC ATT AAC TAC AAT CAC AAG TTC ATT GAA AAC ATC GGC TAC 2112 Ser Val Val He Asn Tyr Asn His Lys Phe He Glu Aβn He Gly Tyr 690 695 700

GTT CTG TAT GGT GTC TAT AAT GTC ACA ATG GTC ATT GTT TTG CTA AAT 2160 Val Leu Tyr Gly Val Tyr Asn Val Thr Met Val He Val Leu Leu Asn 705 710 715 720

ATG TTA ATT GCG ATG ATC AAT AGT TCA TTC CAG GAA ATT GAG GAT GAT 2208 Met Leu He Ala Met He Asn Ser Ser Phe Gin Glu He Glu Asp Aβp 725 730 735

GCG GAC GTG GAG TGG AAG TTT GCA AGG GCC AAA TTG TGG TTT TCC TAC 2256 Ala λβp Val Glu Trp Lye Phe Ala Arg Ala Lys Leu Trp Phe Ser Tyr 740 745 750

TTT GAG GAG GGG AGA ACA CTT CCT GTC CCC TTC AλT CTT GTλ CCA AGT 2304 Phe Glu Glu Gly Arg Thr Leu Pro Val Pro Phe Asn Leu Val Pro Ser 755 760 765

CCA AAA TCC TTG CTT TAT CTC CTA TTG AAA TTT AλG λAA TGG ATG TGT 2352 Pro Lye Ser Leu Leu Tyr Leu Leu Leu Lye Phe Lye Lye →rp-Met Cyβ 770 775 780

GAG CTC ATC CAG GGT CAA AAG CAA GGC TTC CAA GAA GAT GCA GAG ATG 2400 Glu Leu He Gin Gly Gin Lye Gin Gly Phe Gin Glu λβp Ala Glu Met 785 790 795 800

AAC AAG AGA AAT GAA GAA AAG AAA TTT GGA ATT TCA GGA AGT CAC GAA 2448 Aβn Lye Arg Aen Glu Glu Lye Lye Phe Gly He Ser Gly Ser Hie Glu 805 810 815

GAC CTT TCA AAA TTT TCA CTT GAC AAA AAT CAG TTG GCA CAC AAC AAA 2496 Aβp Leu Ser Lye Phe Ser Leu λβp Lye Aβn Gin Leu Ala His λβn Lys 820 825 830

CAA TCA AGT ACA AGG AGC TCA GAA GAT TAT CAT TTA AλT λGT TTC λGT 2544 Gin Ser Ser Thr Arg Ser Ser Glu Aβp Tyr Hie Leu Aβn Ser Phe Ser 835 840 845 λλC CCT CCλ λGλ CλA TAT CAG AAA ATC ATG λλG λGλ CTC λTT AλA λGA 2592 Aβn Pro Pro Arg Gin Tyr Gin Lys He Met Lys λrg Leu He Lye Arg 850 855 860

TAT GTλ TTG CλG GCC CλG λTT GλT λλG GλG λGC GλT GλG GTG λAT GAA 2640 Tyr Val Leu Gin Ala Gin He Aβp Lye Glu Ser λβp Glu Val Aβn Glu 865 870 875 880

GGG GAA TTG AλG Gλλ λTT λλG CAA GAC ATC TCA AGT CTC CGT TAT GAA 2688 Gly Glu Leu Lye Glu He Lye Gin Aβp He Ser Ser Leu Arg Tyr Glu 885 890 895

CTC CTT GAA GAG AAA TCA CAG AAC TCλ GAA GAC CTA GCA GAG CTC ATT 2736 Leu Leu Glu Glu Lys Ser Gin Asn Ser Glu Asp Leu Ala Glu Leu He 900 905 910

AGA AAA CTC GGG GAG AGλ CTG TCG TTA GAG CCλ λλG CTG GλG Gλλ λGC 2784 Arg Lye Leu Gly Glu Arg Leu Ser Leu Glu Pro Lye Leu Glu Glu Ser 915 920 925

CGC AGλ 2790

Arg Arg 930

AGCλGλGCCC CTCλGAAGTG CATλTTTλTT TCTCCλCTTG λλGCCλTλTT λTTTTCTGAC -60

TTATTTTTTT AAGTGTCAAT GATλλAAλGT ATGTTAλCTG λTλλCTTGGλ TCλTTTλGλG -120

TCCTAATλTC λλGCTTTTTG GGλGATTAλA TTGCATTGCT GAGGGCTλλC λλTTGCTG -178

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(C) INDIVIDUλL ISOLλTE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TTGAλCATAλ ATTGCGTλGA TGTGCTTGGG AGλλATGCTG TTACC 45

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

TCGCACGCCA GCAAGAAAAG 20

(2) INFORMλTION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

CGATGλGCAG CTAλλλTGλC 20

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

TGTCAGTCCA ATTGTGAAAG A 21

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

TGACTTCCGT TGTGCTCλλλ TATGATCACA AATTCλTλG 39

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (λ) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

ATGGAATATA CAATGTAACT ATGGTGGTCG 30

(2) INFORMATION FOR SEQ ID NO: :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUλL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

GGACTλGGλA CTAGλCTGλλ AGGTGGAGGT AλTGTTTTTC CλTCλTCλ 48 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERIST CS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINλL SOURCE: (λ) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CGAGCAλλCT TCCλTTCTλC λTCACTGTC 29

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GCNGARGGNC TCTTNGC 17

(2) INFORMλTION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS: (λ) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNλ

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUλL ISOLλTE: Mtrp4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CGNGCRλλYT GCARRT 16

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHλRACTERISTICS: (λ) LENGTH: 16 base paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) λNTI-SENSE: NO

(vi) ORIGINλL SOURCE: (λ) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

TGGGNCCNYT GCARRT 16

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) λNTI-SENSE: NO

(vi) ORIGINλL SOURCE: (λ) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

CGNGCRλλYT TCCλYTC 17

(2) INFORMλTION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO

(iv) λNTI-SENSE: NO

(vi) ORIGINλL SOURCE: (λ) ORGANISM: (C) INDIVIDUλL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

ACCTCTCAGG CCTAλGGGλG 20

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(C) INDIVIDUλL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CCTTCTGAλG TCTTCTCCTT CTGC 24

(2) INFORMλTION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS: (λ) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(Vi) ORIGINAL SOURCE: (λ) ORGANISM: (C) INDIVIDUλL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

TCTGCλGATλ TCTCTGGGλA GGATGC 26

(2) INFORMλTION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(C) INDIVIDUλL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: AAGCTTTGTT CGAGCAAATT TCCATTC 27

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINλL SOURCE: (λ) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

AMRCCNTTYλ TGλλRTT 17

(2) INFORMλTION FOR SEQ ID NO: 20:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 22 base paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

CCACTCCACG TCCGCATCλT CC 22 (2) INFORMλTION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) λNTI-SENSE: NO

(vi) ORIGINλL SOURCE: (λ) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

GGTTTAGCTλ TGGGGλλGλG C 21

(2) INFORMλTION FOR SEQ ID NO: 22:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICλL: NO

(iv) λNTI-SENSE: NO

(vi) ORIGINλL SOURCE: (λ) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

TTTCCANTCT TTATCCTCλT G 21

(2) INFORMλTION FOR SEQ ID NO: 23:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 21 base paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: TGGACλTGCC TCλGTTCCTG G 21

(2) INFORMλTION FOR SEQ ID NO: 24:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 21 base paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) λNTI-SENSE: NO

(vi) ORIGINλL SOURCE: (A) ORGANISM: (C) INDIVIDUAL ISOLλTE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

TTTCCλNTCC ACATCλGCλT C 21

(2) INFORMλTION FOR SEQ ID NO:25:

(i) SEQUENCE CHλRACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNλ (iii) HYPOTHETICAL: NO (iv) λNTI-SENSE: NO

(vi) ORIGINλL SOURCE:

(A) ORGANISM:

(C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: GGCTλTGTTC TTTλTGGGλT λT 22

(2) INFORMλTION FOR SEQ ID NO: 26:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICλL: NO

(iv) λNTI-SENSE: NO

(vi) ORIGINλL SOURCE: (λ) ORGANISM: (C) INDIVIDUλL ISOLATE: Mtrp4

( i) SEQUENCE DESCRIPTION: SEQ ID NO:26:

CCATCATCλλ AGTAGGAGAG CC 22

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(C) INDIVIDUAL ISOLATE: Mtrp4

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: ATGTCλλλGC CCAGCλCGλG T 21

(2) INFORMλTION FOR SEQ ID NO: 28:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICλL: NO

(iv) λNTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

AAGCTTTGTT CGλGCAAATT TCCATTC 27

(2) INFORMλTION FOR SEQ ID NO: 29:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 21 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) λNTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

ATGTGλλGGC CCGACATGAG T 21

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) λNTI-SENSE: NO

(vi) ORIGINλL SOURCE: (λ) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

TTTCCATTCλ λTλTCλGCλT G 21

(2) INFORMλTION FOR SEQ ID NO: 31:

(i) SEQUENCE CHλRACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (λ) ORGANISM: (C) INDIVIDUAL ISOLλTE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: λTCGGCTλCG TTCTGTλTGG TGTC 24 (2) INFORMλTION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: (C) INDIVIDUAL ISOLATE: Mtrp4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

GGAAAACCAC λATTTGGCCC TTGC 24

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Claims

CLAIMSWe Claim:

1. A method for controlling capacitative calcium ion entry into a mammalian cell, wherein said cell expresses a transient receptor potential (trp) protein which is necessary for capacitative calcium ion entry into said mammalian cell, said ceil expressing said trp protein to provide a naturally occurring level of biologically active trp protein associated with said cell, said method comprising the step of treating said cell with a sufficient amount of ftp-control agent to either raise or lower the amount of biologically active trp protein associated with said cell to thereby control capacitative calcium ion entry into said cell.

2. A method for increasing the capacitative calcium ion entry into a mammalian cell according to claim 1 wherein said cell is treated with a frp-control agent which increases the expression of trp protein by said cell.

3. A method for decreasing the capacitative calcium ion entry into a mammalian cell according to claim 1 wherein said cell is treated with a frp-control agent which decreases the expression of trp protein by said cell.

4. A method for decreasing the capacitative calcium ion entry into a mammalian cell according to claim 1 wherein said cell is treated with a frp-control agent which binds to and biologically inactivates trp protein expressed by said cell.

5. A method for controlling capacitative calcium ion entry into a mammalian cell according to claim 1 wherein said trp protein is trpλ or Htrp3.

6. A method for controlling capacitative calcium ion entry into a mammalian cell according to claim 2 wherein said frp-control agent comprises a nucleotide sequence which codes for the expression of trp protein when said nucleotide sequence is introduced into said cell.

7. A method for controlling capacitative calcium ion entry into a mammalian cell according to claim 3 wherein said frp-control agent comprises an anti-sense nucleotide sequence which is anti-sense to a nucleotide sequence which codes for the expression of trp protein.

8. A method for controlling capacitative calcium ion entry into a mammalian cell according to claim 4 wherein said frp-control agent comprises a trp inhibitor which binds to and biologically inactivates said trp protein.

9. A transient receptor potential (trp) protein which has the amino acid sequence set forth in SEQ. ID. NO: 1 .

10. A transient receptor potential (trp) protein which has the amino acid sequence set forth in SEQ. ID. NO: 2.

1 1. An oligonucleotide sequence which codes for a transient receptor potential (trp) protein, said oligonucleotide having the nucleotide sequence set forth in SEQ. ID. NO: 1.

12. An oligonucleotide sequence which codes for a transient receptor potential (trp) protein, said oligonucleotide having the nucleotide sequence set forth in SEQ. ID. NO: 2.

13. A method for screening a compound to determine the compounds potential for use in controlling capacitative calcium ion entry into mammalian cells, said method comprising the steps of: providing a cell culture which expresses a transient receptor potential (trp) protein which is necessary for capacitative calcium ion entry into said mammalian cell, said cell expressing said trp protein to provide a naturally occurring level of biologically active trp protein associated with said cell; exposing said ceil culture to said compound; and determining if the exposure of said cell culture to said compound increases or decreases the expression of said trp protein to thereby provide an indication of the compounds potential use in controlling capacitative calcium ion entry into mammalian cells.

14. A method for screening a compound to determine the compounds potential for use in controlling capacitative calcium ion entry into mammalian cells according to claim 13 wherein the trp protein is selected from the group consisting of Hfrpl and Hfrp3.

15. A method for screening a compound to determine the compounds potential for use in controlling capacitative calcium ion entry into mammalian cells according to claim 13 wherein said compound is a nucleotide sequence.

16. A method for screening a compound to determine the compounds potential for use in controlling capacitative calcium ion entry into mammalian cells according to claim 13 wherein said compound is an inhibitor.