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 SOCE);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 three 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 plasma membrane 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 of 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 previous two years we discovered that Orai is able to confer the so far missing store depletion responsiveness to store-insensitive TRPCs stably expressed in HEK cells and, in collaboration with Drs. David Armstrong and Christian Erxleben from the Laboratory of Neurobiology, that co-expression of Orai and TRPC leads to reconstitution of TRPC-dependent Icrac. We are also performing a general screening 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 go on to map the interaction surface between Orai and TRPC. Finally, and importantly, we found that two Orai mutants (Orai1-R91W, and Orai1-G98A) have dominant negative properties and interfere not only with store operated Ca entry and receptor operated Ca entry, but also with diacylglycerol-activated Ca entry which is mediated by TRPC3 and C6, and does not involve either store depletion or PLC activation. This last finding strengthened our belief that TRPCs and Orai form functional units. As a second approach to answer the question whether TRPC channels are essential in SOCE we have begun a program in which by breeding generate compound knockout mice, i.e. mice deficient in ever more TRPC channels. In previous years we had generated mice deficient in TRPC1, TRPC3, TRPC5, TRPC6 and TRPC7 which have been used to study physiological roles of TRPC channels. Further, we now obtained from other investigators TRPC2 and TRPC4 and are creating more complex compound KO mice. As of this writing we have live TRPC1,C2,C3,C5,and C6 Quintuple KO breeding pairs. Also TRPC1,C3,C6 and C7 quadruple KO breeding pairs. In this year we hope to be able to generate if not a mouse, at least acell line that lacks all TRPCs. This cell line will empower us to determine precisely which functions TRPCs have in cells and the minimum molecular makeup of the SOCE channel. 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, vascular tension, intestinal motility and in cardiac hypertrophy. Thus, our KO mice will serve to further investigate the physiological roles of the TRPC family of cation 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 with glutamic acid.

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Liao, Yanhong; Abramowitz, Joel; Birnbaumer, Lutz (2014) The TRPC family of TRP channels: roles inferred (mostly) from knockout mice and relationship to ORAI proteins. Handb Exp Pharmacol 223:1055-75
Seo, Kinya; Rainer, Peter P; Shalkey Hahn, Virginia et al. (2014) Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy. Proc Natl Acad Sci U S A 111:1551-6
Tano, Jean-Yves; Solanki, Sumeet; Lee, Robert H et al. (2014) Bone marrow deficiency of TRPC3 channel reduces early lesion burden and necrotic core of advanced plaques in a mouse model of atherosclerosis. Cardiovasc Res 101:138-44
Kochukov, Mikhail Y; Balasubramanian, Adithya; Abramowitz, Joel et al. (2014) Activation of endothelial transient receptor potential C3 channel is required for small conductance calcium-activated potassium channel activation and sustained endothelial hyperpolarization and vasodilation of cerebral artery. J Am Heart Assoc 3:
Ilatovskaya, Daria V; Palygin, Oleg; Chubinskiy-Nadezhdin, Vladislav et al. (2014) Angiotensin II has acute effects on TRPC6 channels in podocytes of freshly isolated glomeruli. Kidney Int 86:506-14
Xia, Yang; Yang, Xiao-Ru; Fu, Zhenzhen et al. (2014) Classical transient receptor potential 1 and 6 contribute to hypoxic pulmonary hypertension through differential regulation of pulmonary vascular functions. Hypertension 63:173-80
Solanki, Sumeet; Dube, Prabhatchandra R; Tano, Jean-Yves et al. (2014) Reduced endoplasmic reticulum stress-induced apoptosis and impaired unfolded protein response in TRPC3-deficient M1 macrophages. Am J Physiol Cell Physiol 307:C521-31
Seo, Kinya; Rainer, Peter P; Lee, Dong-Ik et al. (2014) Hyperactive adverse mechanical stress responses in dystrophic heart are coupled to transient receptor potential canonical 6 and blocked by cGMP-protein kinase G modulation. Circ Res 114:823-32
Du, Juan; Ma, Xin; Shen, Bing et al. (2014) TRPV4, TRPC1, and TRPP2 assemble to form a flow-sensitive heteromeric channel. FASEB J 28:4677-85
Phelan, Kevin D; Shwe, U Thaung; Abramowitz, Joel et al. (2014) Critical role of canonical transient receptor potential channel 7 in initiation of seizures. Proc Natl Acad Sci U S A 111:11533-8

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