Identification Of An Essential Structural Element Of Lipid Gating Mechanism In The Transient Receptor Potential Canonical Channel Type 3 (Trpc3)

Transient receptor potential canonical 3 (TRPC3), is highly expressed in neuronal and cardiac tissues, and its importance in development and (patho)physiology of these tissues is well established. So far, our understanding of structure-function relations, specifically in terms of the lipid sensing machinery in this channel protein is incomplete.Using a homology model of TRPC3, based on the recently available structural information on TRPV1, we performed structure-guided mutagenesis and identified a single residue within the transmembrane domain 6 (G652) as pivotal for lipid-mediated gating of TRPC3. Increasing the residue size in position 652 eliminated lipid sensitivity. TRPC3G652A expressed in HEK293 cells was found resistant to activation via the phospholipase C pathway or to direct administration of diacylglycerols. On the contrary, a synthetic agonist of TRPC3/6/7 channels (GSK1702934A) activated both wild-type and TRPC3G652A channels, generating even larger membrane conductances in the lipid-insensitive mutant.To gain further insight into the mechanisms of TRPC3 activation by GSK1702934A and diacylglycerols, we synthetized and characterized a series of structurally related analogs of GSK1702934A. Our results identify two groups of TRPC3 activators, one of which includes the putative physiological activator diacylglycerol and is largely inactive in TRPC3G652A channels.We suggest that lipid gating of TRPC3 strictly requires a hinge-point or a certain level of flexibility within transmembrane segment S6 provided by G652. Lipids and synthetic activators of TRPC3 may be capable of initiating divergent gating movements in the channel.