Impact of C-Terminal PKC Phosphorylation on TRPC6 Current Kinetics.

Transient receptor potential canonical 6 (TRPC6) channels are promising drug targets for kidney, lung, and neurological diseases, making a detailed understanding of their regulation crucial to developing novel channel modulators with more precise modes of action. TRPC6 channels are commonly accepted as calcium-permeable, receptor-operated cation channels activated by diacylglycerol (DAG) downstream of phospholipase C (PLC) signaling. DAG, the endogenous activator of TRPC channels, also activates protein kinase C (PKC), which can phosphorylate TRPC6 and potentially modify its function. This study examined whether five putative PKC phosphorylation sites located in the C-terminus of TRPC6 affect channel gating. Using whole-cell patch-clamp recordings and utilizing photopharmacology with photoswitchable TRPC6 activators (OptoBI-1 and OptoDArG), we analyzed the activation, inactivation, and deactivation kinetics. Pharmacological modulation of PKC activity and strategic mutation of the phosphorylation sites-either to prevent or mimic phosphorylation-altered the current kinetics as well as the normalized slope conductances that were used to quantify differences in the curve progression of current-voltage relations, even when maximally induced current density amplitudes were unchanged. Our findings reveal activator-specific differences in TRPC6 current kinetics associated with C-terminal amino acid exchanges and PKC-dependent signaling, suggesting that phosphorylation-related mechanisms may fine-tune channel activity.