.We are focusing our studies 1. on proving or disproving the hypothesis that TRPC type cation channels are, together with Orai molecules components of the channels that mediate of store depletion activated calcium entry; and 2. on determining their roles in health and disease. ? ? 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, also store operated calcium entry or SOCE. 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 the last two years, we focused on the role of the newly discovered Orai molecules in SOCE. Others showed that co-expression of Orai plus an also newly discovered membrane protein, STIM, which is the calcium sensor and responsible for activation of plasmam mebrane SOCE channel complex, 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, 2006-2007 proposal that Orai is the SOCE channel proper without involvement og TRPC channels. However, previous data connecting TRPCs to SOCE, while not conclusive, had clearly indicated that TRPCs are at least in part involved in SOCE. During the previou year (2007) we discovered that Orai is able to confer the so far missing store depletion responsiveness to STORE-insensitive TRPCs stably expressed in HEK cells. These experiments were expanded to include, in collaboration with Drs. David Armstrong and Christian Erxleben from the Laboratory of neurobiology, the reconstitution of Icrac in a TRPC dependent manner by co-expression of Orai. We are also performing a general screen to determine which TRPCs can be shown to interact functionally with Orai. So far we have found that TRPC1, C3, C6, and C7 become sensitive to store depletion when provided with Orai. New stable cell lines have been generated that express TRPC3 and Orai. With these cells we hope to be able, by crosslinking, to characterize the interaction of TRPCs with Orai in intact cells. If this were successful, we would goon to map the interaction surface between Orai and TRPC.? ? At the physiologic level, we found in collaboration with Drs. Arthur Konnert (Technical University of Munich,Germany) and Veit Flockerzi (University of the Saarland, Germany) that TRPC3 is required for the development of slow postsynaptic excitatory currents that are generated in cerebellar Purkinje cells when stimulated by climbing fibers - indicating an essential role for TRPCs in synaptic transmission. ? ? As we did with G protein alpha subunits, we have created a panel of mouse lines in which TRPC genes have been disrupted. For three (C3, C5, and C7) we have generated conditional alleles that will aid in exploring the roles of TRPCs. The panel includes, in addition, the disruption of TRPC1 and TRPC6. TRPC2 is of very restricted expression i mice and is in humans a pseudogene and will not be studied. TRPC4 was disrupted by Veit Flockerz in 2001. We therefore have a complete panel of TRPC KO mice available for studies in our laboratory or in collaboration with others. ? ? 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. Thus, our KO mice will serve to further investigate the physiological roles of the TRPC family of TRP channels.? ? One focus of interest is the role of TRPCs in excitotoxicity. Preliminary data from others have shown that TRPC KO mice are more resistant to the deleterious effects of electroshock, or massive activation of excitatory receptors wit glutamic acid.
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