Critical contributions of pre-S1 shoulder and distal TRP box in DAG-activated TRPC6 channel by PIP₂ regulation

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂ or PIP₂) regulates the activities of numerous membrane proteins, including diacylglycerol(DAG)-activated TRPC3/6/7 channels. Although PIP₂ binding is known to support DAG-activated TRP channel activity, its binding site remains unknown. We screened for PIP₂ binding sites within TRPC6 channels through extensive mutagenesis. Using voltage-sensitive phosphatase (DrVSP), we found that Arg437 and Lys442, located in the channel's pre-S1 domain/shoulder, are crucial for interaction with PIP₂. To gain structural insights, we conducted computer protein-ligand docking simulations with the pre-S1 domain/shoulder of TRPC6 channels. Further, the functional significance of PIP₂ binding to the pre-S1 shoulder was assessed for receptor-operated channel functions, cross-reactivity to DAG activation, and the kinetic model simulation. These results revealed that basic residues in the pre-S1 domain/shoulder play a central role in the regulation of PIP₂-dependent gating. In addition, neutralizing mutation of K771 in the distal TRP box reversed the effect of PIP₂ depletion from inhibiting to potentiating channel activity. A similar effect was seen in TRPV1 channels, which suggests that TRPC6 possesses a common but robust polarity switch mediating the PIP₂-dependent effect. Overall, these mutagenesis studies reveal functional and structural insights for how basic residues and channel segments in TRP channels are controlled through phosphoinositides recognition.