Understanding the mechanism by which ion channel modulators act is critical for interpretation of their physiological effects and can provide insight into mechanisms of ion channel gating. The small molecule RY785 is a potent and selective inhibitor of Kv2 voltage-gated K+ channels that has a use-dependent onset of inhibition. Here, we investigate the mechanism of RY785 inhibition of rat Kv2.1 (Kcnb1) channels heterologously expressed in CHO-K1 cells. We find that 1 µM RY785 block eliminates Kv2.1 current at all physiologically relevant voltages, inhibiting ≥98% of the Kv2.1 conductance. Both onset of and recovery from RY785 inhibition require voltage sensor activation. Intracellular tetraethylammonium, a classic open-channel blocker, competes with RY785 inhibition. However, channel opening itself does not appear to alter RY785 access. Gating current measurements reveal that RY785 inhibits a component of voltage sensor activation and accelerates voltage sensor deactivation. We propose that voltage sensor activation opens a path into the central cavity of Kv2.1 where RY785 binds and promotes voltage sensor deactivation, trapping itself inside. This gated-access mechanism in conjunction with slow kinetics of unblock supports simple interpretation of RY785 effects: channel activation is required for block by RY785 to equilibrate, after which trapped RY785 will simply decrease the Kv2 conductance density.

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