Molecular cloning of TRPC3a, an N-terminally extended, store-operated variant of the human C3 transient receptor potential channel

Abstract

AK032317 is the GenBank accession no. of a full-length RIKEN mouse cDNA. It encodes a putative variant of the C3-type TRPC (transient receptor potential channel) that differs from the previously cloned murine TRPC3 cDNA in that it has a 5 extension stemming from inclusion of an additional exon (exon 0). The extended cDNA adds 62 aa to the sequence of the murine TRPC3. Here, we report the cloning of a cDNA encoding the human homologue of this extended TRPC3 having a highly homologous 73-aa N-terminal extension, referred to as hTRPC3a. A query of the GenBank genomic database predicts the existence of a similar gene product also in rats. Transient expression of the longer TRPC3a in human embryonic kidney (HEK) cells showed that it mediates Ca 2؉ entry in response to stimulation of the Gq-phospholipase C ␤ pathway, which is similar to that mediated by the shorter hTRPC3. However, after isolation of HEK cells expressing hTRPC3 in stable form, TRPC3a gave rise to Ca 2؉ -entry channels that are not only activated by the Gq-phospholipase C ␤ pathway (receptor-activated Ca entry) but also by thapsigargin triggered store depletion. In conjunction with findings from our and other laboratories that TRPC1, TRPC2, TRPC4, TRPC5, and TRPC7, can each mediate storedepletion-activated Ca 2؉ entry in mammalian cells, our findings with hTRC3a support our previous proposal that TRPCs form capacitative Ca-entry channels.capacitative Ca entry ͉ cation channel ͉ store-operated Ca entry

Orai proteins interact with TRPC channels and confer responsiveness to store depletion

“…Thus, when TRPC3 channels are expressed in excess, they respond differently to receptor activation of phospholipase C and to store depletion. These results have also been interpreted as evidence for the idea that homomeric (over expressed) TRPC3 channels are insensitive to store depletion (18,19,23,43). Alternatively, we hypothesized that the regulatory sites with which diacylglycerol and protein kinase C need to interact to affect Ca 2ϩ entry are not accessible under physiologic conditions because binding of Orai occludes them.…”

“…COS7 cells were a gift from Savio Woo (Baylor College of Medicine, Houston, TX). T3-9, T3-65, and T3a-10 are HEK-293 cell-derived cell clones that stably overexpress TRPC3 or TRPC3a tagged at their N termini with the HA epitope (MYPYDVPDYA) (12,19). T6-11 is a HEK-293 cell-derived clone that overexpresses the murine HA-tagged (N terminus) TRPC6 in stable form (56).…”

“…The protocols to measure changes in intracellular concentrations of cytosolic Ca 2ϩ in single cells by videomicroscopy using the fluorescent indicator-dye Fura-2 have been described (12,19). The concentrations shown are the averages of the indicated number of cells on two to three coverslips that resulted from single transfections.…”

“…When expressed in model cells they form calcium-permeable, largely nonselective cation channels (12,13). Most of the TRPCs have been shown either to be activated by store depletion or to be essential components of SOCE, including TRPC1 (14,15), TRPC2 (16,17), TRPC3 (18,19), TRPC4 (20), TRPC5 (21), and TRPC7 (22,23). Gene inactivation (24) and RNAi (25) studies also support a role for TRPCs in SOCE.…”

“…However, the dependence of TRPCs on store depletion requires special conditions. For TRPCs of the 3-6-7 subfamily, the overriding condition appears to be that they be expressed at low densities (18,19,26,27). The reason for this has not been clarified.…”