TRPC5 channel has recently emerged as a novel potential target for the treatment of peripheral and visceral pain, and its abnormal functioning has been linked to pathophysiologically increased levels of lysophosphatidylcholine (LPC) [1, 2]. Therefore, a deep understanding of the underlying molecular mechanism of TRPC5 regulation by LPC has become urgent for the design of new effective analgesics. Like several other members of the large family of Transient Receptor Potential (TRP) channels, TRPC5 can be also tightly regulated by membrane voltage that dampens its activity at negative potentials and enhances it at positive potentials. The structural bases for these modes of regulation are unclear. We measured whole-cell membrane currents from recombinant human TRPC5 channels expressed in HEK293T cells in response to LPC 18:1 at various membrane potentials ranging from -100 to +200 mV. Using molecular docking and molecular dynamics analysis, we identified a pocket within the outer pore region (dubbed lipid/xanthine binding domain) as a potential site from which the activity of the channel can be effectively up-regulated by LPC 18:1 and through which strongly depolarizing voltage may act to promote the transition of the pore loop toward the open channel conformation. Replacing the highly conserved tryptophan residue 577 from the pore helix by alanine (W577A) rendered TRPC5 completely insensitive to depolarizing voltage. Surprisingly, LPC activated this mutant to a similar extent as the wild-type channel but only at highly depolarizing potentials. Substitution of conserved glycine 606, located in the sixth transmembrane segment directly opposite W577, with tryptophan (G606W) enhanced activation by voltage and LPC but abolished activation by xanthine. The W577A/G606W double mutation partially rescued the voltage-dependent activation of the channel, which was further enhanced by LPC. These results may help identify sites of effective pharmacological targeting of TRPC5 and, given the considerable degree of homology between TRP proteins and the similar role of lipids in their regulation, may provide insights in the search for a general mechanism of TRP voltage and lipid activation.
This work was supported by the Czech Science Foundation Grant (22-13750S) and the Grant Agency of the Charles University (297921).