A propofol binding site in the voltage sensor domain mediates inhibition of HCN1 channel activity | Science Advances

Hyperpolarization-activated and cyclic nucleotide–gated (HCN) ion channels are members of the cyclic nucleotide–binding family and are crucial for regulating cellular automaticity in many excitable cells. HCN channel activation contributes to pain perception, and propofol, a widely used anesthetic, acts as an analgesic by inhibiting the voltage-dependent activity of HCN channels. However, the molecular determinants of propofol action on HCN channels remain unknown. Here, we use a propofol-analog photoaffinity labeling reagent to identify propofol binding sites in the human HCN1 isoform. Mass spectrometry analyses combined with molecular dynamics simulations show that a binding pocket is formed by extracellularly facing residues in the S3 and S4 transmembrane segments in the resting voltage-sensor conformation. Mutations of residues within the putative binding pocket mitigate or eliminate voltage-dependent modulation of HCN1 currents by propofol. Together, these findings reveal a conformation-specific propofol binding site that underlies voltage-dependent inhibition of HCN currents and provides a framework for identifying highly specific modulators of HCN channel gating.