Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel

Visualizing conformational changes during ion channel gating is crucial to understand their regulation. Here, Gao et al. capture multiple gating intermediates in a CNG channel as well as subtle, lateral voltage sensor movements necessary for channel opening. Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the intracellular gate, pointing to an inhibitory VSD at rest. We propose that in wild-type SthK, depolarization leads to such VSD displacements resulting in release of inhibition. In summary, we report conformational transitions along the activation pathway that reveal allosteric couplings between key sites integrating to open the intracellular gate.

Automated summary

This study reveals the importance of visualizing conformational changes and voltage sensor movements during ion channel gating. Multiple gating intermediates and subtle voltage sensor movements were captured in a CNG channel, providing insights into the regulation of ion channels. The ligand-gating trajectory of SthK was defined, showing allosteric couplings between voltage sensor, cyclic nucleotide-binding, and pore domains. The findings shed light on the physiological roles of ion channels and their potential as therapeutic targets in pathophysiological conditions.