(-)-Englerin A binds a conserved lipid site of TRPC5 and exposes a Met-aromatic motif in channel activation

TRPC4/5 cation channels are polymodal cellular sensors that play key roles in signal transduction/integration and have been implicated in various human pathologies, including anxiety, pain and cardiometabolic disease1-3. The plant natural product (-)-englerin A (EA)4 is a potent, selective TRPC4/5 agonist5,6 that has transformed fundamental and translational research on TRPC4/5 channels. However, the structural basis of interactions between EA and TRPC4/5 proteins has remained elusive, limiting our ability to fully understand and exploit mechanisms of TRPC4/5 channel activation by this intriguing natural product. Here, we present nine high-resolution cryo-EM structures (2.4-3.2 [A]) of human TRPC5 - representing different states and ligand occupancies - which show that EA binds to a conserved lipid binding site between transmembrane domains of adjacent TRPC5 subunits. Our structural models are consistent with the effects of mutagenesis of nearby residues on EAs potency, efficacy and activation kinetics, and allow us to rationalise competitive inhibition by other TRPC4/5 modulators as well as EAs selectivity profile within the TRPC family. Comparison of structures containing various TRPC5:EA stoichiometries revealed key structural and molecular determinants of EA-mediated TRPC5 activation - most notably the aromatic interaction network around Phe520 - underscoring the critical function of Met-aromatic motifs in ion channel structure and function. Binding of EA causes conformational changes of nearby amino acid residues, resulting in rearrangement of the pore helices into a pre-open state. Collectively, we provide structural insight into the mode-of-action of the most widely used TRPC4/5 agonist, which will underpin fundamental TRPC4/5 channel research as well as ongoing drug discovery programmes.