TRPC2) Channel: Alternative Splicing and Calmodulin Binding to Its N Terminus

The mouse C-type transient receptor potential 2 (TRPC2) channel: Alternative splicing and calmodulin binding to its N terminus

Eda Yildirim, Alexander Dietrich*, and Lutz Birnbaumer†

Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, 111 T. W. Alexander Drive, Research Triangle Park, NC 27709

Contributed by Lutz Birnbaumer, December 31, 2002 Channels of the C-type transient receptor potential (TRPC) are in- although previous studies on the binding of Ca2ϩ͞calmodulin volved in agonist-stimulated and capacitative calcium entry. There are (CaM) concentrated only on presence of such sites in the C termini, seven TRPCs, all of which have a Ca2؉-dependent calmodulin (CaM)- we now extended these studies to the N termini. TRPC2a and binding domain in their C termini. We now tested binding of CaM to TRPC2b, but none of the other TRPCs, contain Ca2ϩ-dependent TRPC N termini and show that only that of TRPC2 binds CaM in a CaM-binding sites at their N terminus. Thus, TRPC2a and TRPC2b ؉ Ca2 -dependent manner. Four TRPC2 cDNAs have been reported: a join TRPC4␣ (11, 12) in having more than one CaM-binding site. (also clone 14), b (also clone 17), ␣, and ␤. Sequences responsible for CaM binding in TRPC2 a and b are absent from the ␣ and ␤ isoforms. Materials and Methods The ␣ and ␤ cDNAs of TRPC2 were reported as alternative forms, Protein–Protein Interaction Tests. Standard procedures reported when recloning of TRPC2 a and b proved impossible. Here we previously were used to construct plasmids encoding CaM and analyzed total RNA samples from brain and testis for presence of N termini as GST fusion proteins for expression in Escherichia TRPC2 a and b and describe the splicing patterns responsible for their coli or under the control of the T7 promoter for expression and formation, as well as those leading to the ␣ and ␤ forms of TRPC2. We labeling with [35S]Met and [35S]Cys in reticulocyte lysates by re-assert existence of RNA encoding the TRPC2 a and b, encoded in 21 using Promega’s coupled transcription and translation reagents exons with an initiator ATG in exon 2 for TRPC2a and in exon 4 for (TNT) (11, 13, 14). Protein–protein interactions were then ␣ ␤ TRCP2b. The analysis of and TRPC2 cDNAs indicates that although performed as described in these same reports. the TRPC2␤ mRNA may exist, the TRPC2␣ cDNA is derived from an incompletely processed TRPC2a mRNA: It includes in its presumed RT-PCR. Total RNA from either mouse brain or mouse testis were ؅ 5 -untranslated sequence, 713 nt of TRPC2a cDNA fused to 291 nt of isolated by using reagents and protocols of the RNeasy RNA an incompletely excised intron. While encoding an active channel in isolation kit (Qiagen). Reverse transcripts were prepared by a the mouse, the human TRPC2 appears to be a pseudogene. We two-step procedure. In the first step, 0.2 ␮g of total RNA was searched for the human gene in the data bank and located approx- incubated with 50 ng of random hexamers in a final volume of 12 imately one-half of it in a chromosomal region syntenic to that of the ␮l at 65°C for 5 min, followed by a chill on ice. For the second step, mouse, with similar intron–exon structure. We conclude that the the product of the first step was incubated at 25°C for 10 min in a human TRPC2 gene may never have been an active gene because of final volume of 20 ␮l by addition of 4 ␮lof5ϫ cDNA synthesis incomplete ancestral duplication or, if it was complete at one point, buffer (250 mM Tris-acetate, pH 8.4͞375 mM K acetate͞40 mM that it became inactive upon loss of chromosomal sequences. Mg acetate), 1 ␮l 100 mM DDT, 1 ␮l of RNaseOUT (40 units), 2 ␮l 10 mM dNTPs, 1 ␮l of RNase HϪ reverse transcriptase cation channel ͉ store-operated channel ͉ gene structure ͉ splice (ThermoScript, Invitrogen, 15 units), and 1 ␮l of diethyl pyrocar- variants ͉ pseudogene bonate-treated H20 followed by 50 min at 55°C, 5 min at 85°C, cooling to room temperature, and a final incubation with 1 ␮lof hannels of the C-type transient receptor potential (TRPC) are RNase H (2 units) at 37°C for 20 min. Reverse transcripts were Cthe founding subfamily of the superfamily of TRP channels. amplified by PCR by adding 2 ␮l of the previous reaction products Other subfamilies include the TRPV and TRPM channels (1). TRPs to 48 ␮l of PCR reaction mixture containing 5 ␮lof10ϫ PCR buffer encode cation channels with widely varying cation selectivity and (100 Tris⅐HCl,pH8.3͞500 mM KCl͞0.01% gelatin), 1.5 ␮lof50 ͞͞ widely differing mechanisms of activation (ref. 2 and http: skte. mM MgCl ,1␮lof10mMdNTPs,1␮lof10␮M sense and 10 ␮M ͞ ͞ ͞ ͞ ͞ ͞ 2 sciencemag.org cgi content full OC_sigtrans;2001 90 re1). antisense primers, 0.4 ␮lofTaq polymerase (5 units͞␮l), and diethyl Some of the TRPC channels, e.g., TRPC1 (3, 4), TRPC2 (5), and pyrocarbonate-treated water. The cycling protocol used was 60 sec TRPC4 (6, 7), but not all, e.g., TRPC3 (8), are involved in mediating at 94°C, then 60 sec at 94°C, 60 sec at 52°C, and 120 sec at 72°C for capacitative entry triggered by depletion of endogenous calcium 34 cycles, and finally 5 min at 72°C and a minimum of 10 min at stores without concomitant activation of a phospholipase C, be it 15°C. Reaction products were separated by standard agarose gel through activation ofaGproteinorsecondary to activation of a electrophoresis (15) and cloned into pCR-2.1 TOPO (Invitrogen) tyrosine kinase. Studies on the mechanism of TRPC activation are by using a protocol supplied by the vendor. The identity of the complex, not only because of the nature of the putative regulatory inserts was then confirmed by dideoxy sequencing (16). mechanism under investigation, but also because of existence of splice variants. Moreover, in some cases the reported splice variants PCR Primer Composition. The TRPC2 primers used to test for (9) appear to be difficult to be verified by other laboratories, one of presence of specific transcripts in total brain and testis RNA which reported instead the identification by molecular cloning of two novel alternate variants that appear to be selectively retained in the endoplasmic reticulum (10). In the present report, we Abbreviations: CaM, calmodulin; TRP, transient receptor potential; S, short; M, medium; L, concentrated on defining the existence and mode of origination of long; TRPC, C-type TRP. the clone 14 (1,172 aa) and clone 17 (1,072 aa) N-terminal variants *Present address: Institute of Pharmacology and Toxicology, Philips University of Marburg, of TRPC2, cloned by Vannier et al. (9) and the origins of the ␣ and 35043 Marburg, Germany. ␤ TRPC2 transcripts cloned by Hofmann et al. (10). Furthermore, †To whom correspondence should be addressed. E-mail: [email protected].

2220–2225 ͉ PNAS ͉ March 4, 2003 ͉ vol. 100 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0438036100 Downloaded by guest on September 29, 2021 Fig. 1. Kyte and Doolittle plots and predicted transmembrane topology of TRPC2a and TRPC3. The ﬁgure highlights eight hydrophobic regions of which the regions 2–5 and 8 form the transmembrane segments. The seventh encodes the pore region and the ﬁrst is wholly intracellular. ᮀ, Location of EWKFAR motif. The hydrophobic domain clusters extend from amino acids 626 to 921 in TRPC2a and from amino acids 350 to 671 in TRPC3.

(vide infra) were designed with the help of PRIMER3 software TRPC2␣, and TRPC2␤ (10). The a and b forms correspond to (www-genome.wi.mit.edu͞cgi-bin͞primer͞primer3.cgi). The the cDNAs originally reported as clones 14 and 17, respectively. Ј composition of PCR primers used was: primer A, 5 -TGGAG- They code for proteins of 1,172 and 1,072 aa, respectively, BIOCHEMISTRY TTTCAGCTGGAGAGG; primer B, 5Ј-GCTAATGTCCCG- differing in their N termini. The shorter, TRPC2b (clone 17), CACTGACT; primer C, 5Ј-GTTCTCCGTGGCTGTTGTTT; differs from TRPC2a in its first 11 aa, with amino acids 12 –1,072 primer D, 5Ј-ACGTCATCAGTCCTTTGGCCT; and primer E, being identical to amino acids 112–1,172 of TRPC2a. Thus, both 5Ј-GGTTCTCCGTGGCTGTTGTTT. TRPC2a and TRPC2b have the N-terminal CaM-binding site. The WISCONSIN PACKAGE (Version 10.3, Accelrys, San Diego) There are examples in the literature of proteins with differing was used for standard nucleotide and amino acid sequence N termini, each regulated by a separate promoter, i.e., each exon manipulations and analysis. 1 being preceded by an independent promoter. This allows for different tissue specific or developmentally conditioned expres- Results sion of a single gene product. To determine whether this might A sequence similarity analysis of mammalian TRPC channels yields be the case for the a and b forms of TRPC2, we mapped the a phylogenetic tree that groups these channels into four subgroups respective mRNAs onto the genomic sequence to delineate their (9): TRPC1 and TRPC2 belong to subgroups I and II, TRPC3, intron–exon organization. Genome analysis software accessible TRPC6, and TRPC7 belong to subgroup III, and TRPC4 and from the National Center for Biotechnology Information web TRPC5 belong to subgroup IV. Fig. 1 shows typical Kyte and site predicts not only the exons as they are defined by the cDNA Doolittle plots of TRPC2 and TRPC3, highlighting eight hydro- sequence, but its MODEL MAKER subroutine predicts one addi- phobic domains. Of these, the first was shown to be intracellular by tional exon (complement to the region 19,937,673–19,937,815 of glycosylation scanning mutagenesis and epitope insertion mapping the supercontig NW_000328), where splice donor site is 2,570 nt (17). One of the remaining seven comprises a region that forms the 5Ј to the exon encoding Met-1 of TRPC2a. We, thus tested for selectivity pore of the channel. The remaining six, five prior and one the presence of sequences in reverse transcripts of brain and after the pore region, form the transmembrane domain of these testis total RNA that would start in either putative exon 1 or in channels. Previous studies had shown that TRPC1–7 have a con- exon 2 by PCR analysis where partnering reverse primer is served Ca2ϩ-dependent CaM-binding site in their C termini (11– designed to anchor in exon 5, the first exon common to TRPC2a 13), and that TRPC4 also has three CaM-binding sites in its C and TRPC2b (Fig. 4A). terminus. Among these three CaM-binding sites, two are encoded Sequencing of the A-C and B-C PCR products (Fig. 4B Left) in the facultative exon 12 (252 nt) of TRPC4␣ variant. Binding of revealed that putative exon 1 indeed existed (A-C products were CaM to the N termini of TRPCs on the other hand has not been extensions of B-C products) and, furthermore, that each set systematically evaluated. Fig. 2 shows, that among TRPC1–6, contained three distinct products that differ in the length of exon TRPC2 is unique in that its N terminus strongly binds CaM and 4. Direct PCR amplification of exon 4 with primers D and E doessoinaCa2ϩ-dependent manner. Subdividing the TRPC2 N anchored in exons 3 and 5, respectively, yielded three distinct terminus, showed that its CaM-binding activity is located in a region bands (Fig. 4B Right). These bands coincided with their length formed by amino acids 104–195, possibly just 109–161 (Fig. 3, as predicted from A-C and B-C PCR products and defined exons TRPC2a numbering), a little more than 400 aa from the first 4S, 4M, and 4L, for short, medium, and long, all arising from the hydrophobic region. We failed to find amino acid sequences similar use of a common splice acceptor site at the beginning of exon 4, to those comprising the TRPC2 N-terminal CaM-binding domain and either 2 different splice donor sites in exon 4, or, in the case in the N (or C) termini of other TRPC channels, indicating that they of exon 4L, from a failure to excise the intron between exon 4 and are unique to TRPC2. 5. As shown in Fig. 5AI, the ORF initiated by the first Met of Four variants, apparently having their origin in alternative exon 2 (MLM motif) is preserved to give that of clone 14 in the splicing of the primary transcripts have been reported for transcripts with exon 4S, but terminate either 73 codons into TRPC2. They have been named TRPC2a, TRPC2b (9), exon 4L or 32 codons into exon 5 in transcripts made with exon

Yildirim et al. PNAS ͉ March 4, 2003 ͉ vol. 100 ͉ no. 5 ͉ 2221 Downloaded by guest on September 29, 2021 Fig. 3. The CaM-binding activity of the TRPC2 N terminus resides in a fragment encoded in exon 5. (A) Diagram of exons encoding the N terminus of TRPC2. (B) Binding of CaM synthesized in a reticulocyte lysate in the presence of 35S-labeled cysteine and methionine to fragments of the N terminus of TRPC2. Bound CaM was separated from free by washing and SDS͞PAGE. When present Ca2ϩ was 1 mM; 1 ␮g of fragment B fused to GST retained Ϸ5% of the [35S]CaM.

onwards (stop codon in exon 21). Transcripts made with exon 4M and 4L encode in addition two shortened forms of the TRPC2a, peptides M (mRNA with exon 4M) and L (mRNA with exon 4L; Fig. 5AI). Peptides M and L are predicted to have 169 and 155 aa, respectively (Fig. 5B). Amino acids that form the N-terminal CaM-binding domain of Fig. 3 are encoded in exon 5 and are thus present in TRPC2a and TRPC2b. Amino acids encoding the C-terminal CaM- binding site of TRPC2 are encoded in exon 20. The ␣ and ␤ forms of TRPC2, reported by Hofmann et al. (10), toward the end of 2000, are identical to amino acids 287–1,172 of the TRPC2a reported by us at the beginning of 1999. Codon 283 codes for the first P of the PQP motif located at the 5Ј-end of exon ␣ ␤ Fig. 2. The N terminus of TRPC2 associates with CaM in a Ca2ϩ-dependent 10. Thus, the , , and a forms share sequences encoded in exons manner. (A) N termini of TRPC1–6 were translated in vitro in a reticulocyte 10–21, differing upstream of exon 10. Amino acids upstream lysate in the presence of 35S-labeled methionine and cysteine. Aliquots were of PQP in TRPC2␣ and ␤ are Met- and Met-Asp-Pro-Leu-Ser subjected to SDS͞PAGE and the gels were autoradiographed. (B)Ca2ϩ- (MDPLS), respectively. The TRPC2␣ cDNA was reported with dependent binding of the TRPC2 N terminus, but not that of other TRPCs, to 1,004 nt of 5Ј-untranslated sequence. Sequence alignment of the 2ϩ CaM fused to GST. When present, Ca was 1 mM. TRPC2a and TRPC2␣ cDNAs, revealed that the first 713 nt of the 5Ј-untranslated sequence of TRPC2␣ are identical to those sections of the TRPC2a cDNA that are derived from exons 3–8, the 4M. Transcripts with exon 4S therefore encode TRPC2a. Exon remaining 291 nt just before the initiator ATG of TRPC2␣ are Ј 4M, on the other hand, has in its 3 extension (with respect to 4S) identical to the 5Ј-end of the intron H between exons 8 and 9 (Fig. an ORF with a Met-encoding ATG codon (MGT motif), which 5AII). We conclude that the TRPC2␣ cDNA, rather than repre- upon joining exon 5 uses as its extension the same ORF used by senting an independent splice variant of the murine TRPC2 gene, the MLM of exon 2 in transcripts made with exon 4M. Thus, the resulted from the molecular cloning of a misprocessed TRPC2a ORF initiated in 4M continues throughout exons 5–21 to encode mRNA, due to use of the ATGgt sequence in intron H as a splice TRPC2b. TRPC2a and TRPC2b are thus identical from exon 5 donor site. An in-frame TAA stop codon in the intronic sequence

2222 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0438036100 Yildirim et al. Downloaded by guest on September 29, 2021 Fig. 4. RT-PCR of brain and testis RNA confirms existence of predicted exon 1 upstream of the exon harboring Met-1 of TRPC2 and reveals existence of three forms of exon 4. (A Upper) Scheme of TRPC2a mRNA with location of exon boundaries. Open boxes, exons; filled boxes, untranslated sequences. The numbers above the cDNA are exons as predicted by National Center for Biotechnology Information’s MAP VIEWER software by using the complement to BIOCHEMISTRY region 19,929,800–19,960,915 of supercontig NW_000328. Diagonally hatched areas denote transmembrane segments 1–6 and pore region (P). (Lower) Design of RT-PCR analysis for identification of putative exon 1 (pink box) and presumed composition of exon 4 variants between exons 3 and 5 (graded blue boxes) (B) Agarose gel electrophoresis of RT-PCR products. (Left) Products obtained with primer pairs AC and BC. (Right) Agarose gel electrophoresis of products obtained with primer pairs D and E. Note that instead of the two bands with exon 4 predicted by clones 17 and 14, there exist three distinct forms of exon 4: 4S, 4M, and 4L.

Fig. 5. (A) Diagrams of ORF in mature TRPC2 transcripts based on RT-PCR at position Ϫ108 from this ATG gave the impression of having results of Fig. 4 and on ␣ and ␤ cDNAs reported by Hofmann et al. (10). (B) isolated a complete cDNA. In contrast to TRPC2␣, there is no Alignment of deduced amino acid sequences of ORFs encoded in TRPC2 evidence in favor of or against TRPC2␤ being derived from a transcripts with exons 4S, 4M, and 4L. Top line of alignment is master se- correctly spliced mRNA. The 5Ј-end of its ORF codes for MDPLS quence; -, amino acids identical to TRPC2a in TRPC2b, peptide L, and peptide M; ⅐, gap; , stop; ͉, exon boundaries. proceeded by 57 nt of presumptive 5Ј-untranslated sequence. This * sequence is wholly represented within the intron between exons 9 and 10, the latter beginning with codons coding for the PQP motif larly, it will be of interest to determine whether, as suggested by the ␤ that marks the beginning of TRPC2 ’s identity to the TRPC2a (or data of Liman et al. (18), this form is expressed exclusively in the ␤ b) sequence. Moreover, the mouse TRPC2 is nearly identical to auxiliary olfactory bulb where loss of the channel it encodes causes the rat TRPC2 cDNA, which was reported to be expressed in the loss of sexual discrimination and promotes aggressive behavior in vomeronasal auxiliary olfactory bulb (18). The ORF of the rat male mice (19). TRPC2␤ cDNA codes for a protein that is highly homologous to TRPC2a from amino acid 289–1,172, beginning with MDPLSP, Discussion where the P corresponds to the second P of the PQP motif of mouse In this study, we have concentrated on the intron–exon organi- exon 10. As is the case for mouse TRPC2␤, the upstream nucleo- zation of murine TRPC2 and the available evidence for the tides reported for rat TRPC2 are wholly represented within the expression of splice variants as deduced from RT-PCR analysis genomic sequence upstream of exon 10, giving no further infor- of mRNA extracted from two tissues, brain and testis. At the mation as to whether the cDNA is derived from a bonafide mRNA amino acid level, a comparison of TRPC2 to the other six or not. Further tests will be required to settle this issue. It is members of the TRPC subfamily of TRP channels shows TRPC2 noteworthy to comment here that, neither the murine TRPC2␣ nor to be unique in that it has an extension, N-terminal to the the murine TRPC2␤ cDNAs encodes a protein capable of forming location of the three ankyrin repeats found in all TRPCs. This active channels. Instead, when observed by confocal microscopy, extension shows no evolutionary relation to sequences elsewhere TRPC2␣ and TRPC2␤ failed to reach the plasma membrane and in the other TRPCs. Sequence similarities that define TRPCs as to form active influx channels (10). Finally, analysis of the same such, stretch from the beginning of the ankryin repeats through batch of reverse transcripts that allowed for PCR amplification of the region of the six transmembrane segments and an immedi- the fragments shown in Fig. 4, failed to yield amplified fragments ately following group of six amino acids, EWKFAR. This group with primers based on the nucleotide sequence preceding that of amino acids is referred to as the TRP motif, and is present not encoded in exon 10 (data not shown). Further studies are therefore only in the C-type but, in a slightly degenerated form, also in the required to determine the molecular origin of TRPC2␤. Particu- V- and M-type TRPs. Although being clear structural land-

Yildirim et al. PNAS ͉ March 4, 2003 ͉ vol. 100 ͉ no. 5 ͉ 2223 Downloaded by guest on September 29, 2021 Fig. 6. Comparison of exon boundary locations in TRPC cDNAs highlighting in addition transmembrane and pore regions, the TRP motifs and exons coding for ankyrin repeats and CaM-binding domains. All cDNAs are human except TRPC2, which is murine. The spliced (␤) form of TRPC1 differs from the Fig. 7. Comparison of mouse chromosome 7 region, harboring the TRPC2 unspliced (␣) form in that it lacks exon 3 (turquoise) and its attendant middle gene, to the syntenic region of human chromosome 11, harboring the TRPC2 ankyrin repeat. The ␤ form of TRPC4, able to form active channels, is shown; pseudogene. (A) Intron–exon distribution along the chromosome is shown the ␣ form, which does not form active channels, incorporates the 252-nt exon above the diagram of the cDNA with its exon boundaries. (B) Intron–exon 12 (21). This exon harbors two additional CaM-binding domains (11, 12). distribution along the human chromosome is shown above the diagram of the human cDNA. Note that we leave as undecided (?) the existence of an additional exon with a separate promoter located 5Ј to exon ␤ (between exons marks, neither the role of the ankyrin repeats nor that of the TRP 9 and 10). The human TRPC2 sequences can be found in the complements to motif have been elucidated. Ankyrin repeats are presumed to regions 63,880–74,537 of genomic contig NT_035090 and 2,272,990– play a role in protein–protein interactions, the TRP motif may 2,336,104 of contig NT_033927. play no practical role. The highest sequence similarity among the TRP-C, -V, and -M subfamilies lie within their transmembrane full-length ␣ form is inactive and plays a dominant negative role regions, especially sequences associated with S3–S6 regions. (11, 12, 19). These, more than the TRP motif, contribute to the structural The TRPC2 channel is not only important in male sexual and definition of TRP channels. Indeed, the TRPP subfamily, that social behavior (20, 21), but also in the process of fertilization, includes polycystic kidney disease 2, lacks the TRP motif, but in which it is an intermediary step of the signaling pathway shows sequence similarity at the level of the transmembrane triggered by the oocyte’s zona pellucida to elicit the sperm’s segments that classifies it and its congeners as TRP channels. C-terminal to the TRP motif, TRPC channels share very limited acrosome reaction (4). Despite these central roles in the mouse, sequence identity, except for the presence of a CaM and inositol the sole human cDNA resembling TRPC2 (GenBank accession trisphosphate receptor-binding domain, that has been implied in no. X89067) only codes for a protein that begins in the pre- both feedback inhibition of channel activity by calcium, and a sumptive third transmembrane segment, lacks the fifth and half mechanisms for activation associated with store depletion. A of the pore region, resuming thereafter to end with a C terminus comparison of the intron–exon organization of the seven TRPC highly similar to that of the mouse TRPC2. In addition, the ORF channels and relative location of exons coding for ankyrin of this TRPC2-like sequence is interrupted by three stop codons. repeats, the TRP motif and the various CaM-binding domains is Thus, the human TRPC2-like cDNA appears to be derived from shown in Fig. 6. They are aligned along the EWKFAR TRP motif a pseudogene. We asked whether the TRPC2-like gene was found after transmembrane segment 6. Exons encoding the unique or whether there might exist also a bonafide TRPC2 gene common C-terminal CaM-binding domains are in cyan, and that had so far eluded detection through the molecular cDNA exons containing the three ankyrin motifs are in green. A careful cloning approach. If there were such a true orthologous human inspection of the boundaries (not shown) reveals no overriding TRPC2 gene, one would expect to find it in a chromosomal conservation of boundaries associated with certain transmem- location that is syntenic to that in which the mouse gene is brane segments, the pore, or the EWKFAR motif. For example located. The mouse TRPC2 gene is on chromosome 7 in a region although the end of the S6 segment and the EWKFAR are in the that is syntenic to human chromosome 11p15.3–15.4. Rather same exon in TRPC1, the TRP motif is found in a dedicated exon than finding a full TRPC2 gene in this location of the human in all other TRPCs; although the three ankyrin repeats are in a chromosome 11, we found the genomic region encoding the single exon in TRPC2–7, in TRPC1 they are in three exons, and human TRPC2-like transcript. Mapping the human cDNA to the one of the TRPC1 splice variants, TRPC1␤, lacks the middle of human chromosome 11 sequence revealed an intron–exon struc- these exons. The transmembrane segments on the other hand are ture similar to that of the mouse gene spanning from exons encoded in four exons in all TRPCs except in TRPC2 and 14–21, with the exception that the sequence corresponding to TRPC1, in which they are encoded in five and six exons, exon 16, coding for the fifth transmembrane segment and half of respectively. The most intriguing C-terminal splice variants is the pore region, is absent (Fig. 7). A search for TRPC2 sequences that of TRPC4. The ␣ form results from failure to excise the elsewhere in the human genome was negative, except for locating intron between exons 11 and 12 so that it’s last exon spans from sequences similar to that of corresponding to a fusion of mouse the beginning of exon 11 to the end of the transcript. The ␤ form exons 2 and 3 Ϸ70 Mbp upstream of the sequences coding for is made up of 12 exons as shown in Fig. 6. The spliced out the TRPC2-like transcript (Fig. 7). This reduces the number of segment codes for 84 aa with two CaM-binding sites. RT-PCR functional human TRPC genes to 6. Further studies should analysis showed that both splice forms are coexpressed; the clarify the involvement of TRPC2 channel(s) in mouse.

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