We are focusing our studies on proving or disproving the hypothesis that TRPC type cation channels are the mediators of store depletion activated calcium entry. In earlier studies we had discovered six of the seven TRPC channels, cloned full length cDNAs of four (TRPC1, TRPC2, TRPC3 and TRPC6) and shown them to be activated by maneuvers that stimulate the Gq-PLCb-IP3 mediated depletion of calcium stores, and showed that peptides of the IP3 receptor that interact in vitro with TRPC3 segments (GST pull-down) affect store depletion activated calcium entry. But direct activation of TRPC3 upon thapsigargin stimulated store depletion independent of G protein-PLC-beta activation failed to show the classical store operated calcium entry response. Moreover, electrophysiological measurements only revealed the appearance of non-selective cation channels when TRPCs were expressed in model cells, which, while permeant to Ca, lacked the required Ca selectivity exhibited if they were the sole responsible molecules forming the Icrac channels. ? ? During this year, we focused on the role of the newly disovered Orai molecules in SOCE. Others showed that co-expression of Orai plus an also newly discovered membrane protein, STIM, which spans the membranes of internal stores and communicates with the SOCE channels of the plasma membrane, ? results in giantSOCE (also monsterSOCE). These other studies also showed that mutations in Orai changed the permeation properties of SOCE and Icrac leading to the new proposal that Orai is the SOCE channel proper activated by the Ca sensing STIM molecule. Yet previous data connecting TRPCs to SOCE, while not conclusive, had clearly indicated that TRPCs are at least in part involved in SOCE. We now found that Orai is able to confer the so far missing store depletion responsiveness to STORE-insensitive TRPCs stably expressed in HEK cells. We therefore are pursuing the hypothesis that TRPCs are pore-forming subunits of alpha-beta channels, where beta is a regulatory subunit embodied in Orai. ? ? In support of our hypothesis, we found, in collaboration with Dr. David Armstrong, Head of the Membrane Signaling Group of the Laboratory of Neurobiology, and Dr. Christian Erxleben, member of Dr. Armstrongs group, that Orai not only confers store depletion sensitivity to stably expressed TRPCs, but also reconstitutes Icrac in a TRPC dependent manner. As mentioned, Icrac is highly Ca selective, while expressed TRPCs for the most part form non-selective cation channels instead of the expeted Ca selective channels one would expect if TRPCs alone were the SOCE channels. Future experiments will concentrate in characterizing by mass-spectrometry the components of a TRPC based channel immunoprecipitated from extracts of normal non-transfected cells. We hope for co-immunopecipitation of TRPC and Orai. Maybe also STIM, and perhaps one or more unexpected components of this important calcium influx pathway. ? ? WE are continiun with a breeding program by which we are obtaining singely and combined TRPC1, TRPC3, TRPC5, TRPC6 and TRPC7 KO mice. Of these, TRPC3, TRPC5 and TRPC7 are of the conditional type. The mice will serve to further investigate the physiological roles of the TRPC family of TRP channels.? ? In collaboration with Dr. Indu Ambudkar from NIDCR, we discovered using TRPC1 deficient mice, that TRPC1 is responsible for 80% of SOCE and Icrac in acinar salivary glands, and also for secretion of saliva in response to cholinergic stimulants such as pilocarpine. The drop in SOCE and Icrac in TRPC1 deficient cells is quite compelling for our hypothesis that TRPCs are part of SOCE channels and that Orai alone cannot be the STIM-responsive SOCE channels.? ? Other collaborations, based on the availability of TRPC deficient mice, are exploring roles of TRPCs in hearing, in circadian rhythms, in slow postsynaptic currents of glutamate synapses involved in learning and memory, and in hypertension.
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