BPS2026 – Tracking the hydrophobic gate in BK channels using ANAP

Large-conductance, Ca 2+ - and voltage-activated K + (BK) channels are essential regulators of cellular excitability in neuronal and muscle tissues. Unlike other K + channels, BK channels do not rely on pore constriction for closure, raising the possibility that their gating mechanism depends on hydration changes rather than steric occlusion. Structural evidence and molecular dynamics simulations have proposed a hydrophobic gating process, where the pore dewets in the closed state and rehydrates upon opening. However, direct experimental demonstrations are lacking. To test this hypothesis, we employed the fluorescent unnatural amino acid ANAP. ANAP’s emission spectrum is significantly influenced by the hydrophobicity of its surrounding environment, making it a reliable indicator of the pore’s hydration state. We incorporated ANAP into specific residues along the pore region of the S6 helix, a structural segment implicated in hydrophobic gating, and we recorded fluorescence and ionic currents using cut-open voltage clamp fluorimetry. We focused on two positions suggested by structural and computational studies: A313 and F315. Signals at A313 and F315 indicate that the inner vestibule is the relevant hydrophobic-gating region, where wetting-dewetting transitions occur as predicted by molecular simulations. We recorded robust fluorescence signals at both positions. In particular, at position 313, depolarization induces a decrease in fluorescence signal, indicating a transition from a hydrophobic to a hydrophilic environment, as predicted by the hydrophobic gating hypothesis. Together, our direct experimental evidence suggests that the BK channel pore becomes more hydrophilic as it opens, consistent with the hydrophobic gating hypothesis. Beyond BK, this methodology establishes a generalizable framework to investigate hydration-dependent gating in other ion channels, offering a minimally invasive optical approach to resolve the role of wetting and dewetting in permeation. Supported by NIH R01-GM030376, NSF QuBBE QLCI (NSF OMA-2121044), and FONDECYT Regular #1230265.