A new photochromic sterol controls TRPC3 activity by actuating a hub of regulatory lipid coordination

Transient receptor potential canonical 3 (TRPC3) is a paradigm of primary lipid-gated ion channels. TRPC3 activity is controlled via regulatory lipid-channel interactions involving multiple lipid coordination sites. Sterols are coordinated as non-annular lipids within TRPC3 channels, while their functional impact is still elusive. Here we utilized a new photoswitchable cholesterol derivative (OptoChol-1) and computational analysis to explore the regulatory impact of sterols on TRPC3. Molecular modeling of OptoChol-1 binding to the L1 lipid coordination site identified a cysteine (C350) as pivotal for sterol coordination and a methionine (M586) as critical for control of TRPC3 activity by photoisomerization of OptoChol-1. The predicted roles of these residues were confirmed by site-directed mutagenesis and electrophysiology. Importantly, our experiments demonstrate that OptoChol-1-induced modulation of channel function is dependent on the structure of activating diacylglycerols (DAGs) bound to the L2 regulatory site. Bond-to-bond propensity analysis supported the concept of allosteric communication between the sterol-occupied L1 site and the DAG-sensing L2 domain. In aggregate, we introduce a photochromic sterol (OptoChol-1) as a valuable molecular probe for ion channel-sterol interactions. We suggest OptoChol-1 governs TRPC3 function by operating within a critical hub of multimodal lipid sensing.