BPS2026 - Conserved cation-π interactions in voltage-sensing domains prevent pathogenic gating-pore currents across ion channel families

Pathological gating-pore currents through voltage-sensing domains (VSDs) underlie numerous channelopathies. While our previous work demonstrated that gating-charge mutations promote aberrant gating-pore opening in cardiac and brain sodium channels, the complementary protective mechanisms within the hydrophobic constriction site (HCS) remained unclear. In this computational study, we identified evolutionarily conserved cation-π interactions between HCS aromatic residues (Phe/Tyr) and voltage-sensing gating charges as a universal “gatekeeper” mechanism preventing pathological gating-pore formation in voltage-gated ion channels (VGICs). Our multiple sequence alignments across Na v , Ca v , K v , HCN, and CNG families revealed > 95% conservation of the HCS aromatic residue. Then our comprehensive structural survey of 390 ion channel structures in the PDB database identified cation-π interactions in 96% of these channels, spanning diverse channel subtypes and conformational states. Such a high conservation underscores the fundamental importance of this cation-π interaction. Our microsecond-scale molecular dynamics (MD) simulations further showed that weakening cation-π interactions destabilizes the VSD seal and promotes water/ion permeation. In the K v channel Shaker , substitution of Phe290 with pentafluorophenylalanine reduced cation-π occupancy ∼1.5-fold and increased gating-pore leakage ∼25-fold due to reduced π-electron density from fluorine substituents. Likewise, aromatic-to-leucine substitutions at HCS in Na v 1.5 enhanced gating-pore currents, revealing the molecular mechanism of pathological phenotypes in previous cardiac and brain sodium channels. This work provides the first systematic computational characterization of a putative cation-π interaction at HCS as an evolutionarily conserved safeguard against pathological gating pore currents across VGIC families.