Closed State Coupled C-Type Inactivation in BK Channels

Abstract

C-type inactivation of voltage-gated ion channels commonly occurs from the open state (open-sate inactivation, OSI) at strongly positive membrane potentials or from pre-open closed states (closed-state inactivation, CSI) at modestly depolarized membrane potentials. Slow recovery from inactivation requires hyperpolarized membrane potentials. C-type inactivation is known to result from structural rearrangement or collapse at selectivity filter. BK channels are known to be different from most voltage-gated K+ channels due to the absence of a classic intracellular cross-bundle activation gate, prompting suggestions of an opening mechanism due to rearangements of the selectivity filter. We studied C-inactivation in BK channels to explore the hypothesis that BK channel's activation and C-inactivation may have closely related gating mechanisms. The BK channel doesn't have C-inactivation under physiological conditions because of its high affinity to external K+. We observed prominent slow C-inactivation and recovery upon providing two stimulating factors of C-inactivation: (1) mutation Y294E/K/Q/S or Y279F, whose equivalent in Shaker channels(T449E/K/D/Q/S or W434F) caused greatly increased rates of C-inactivation or constitutive inactivation; (2) a very low concentration of extracellular K+ by replacement of K+ with NMDG+. However, BK channels showed a distinct state-dependency of C-inactivation which occurs at negative membrane potentials or decreased [Ca2+]i that cause channels to close, whereas recovery from inactivation requires positive membrane potentials or elevated [Ca2+]i that promote channel opening. Constitutively open mutatations prevented BK channel from C-inactivation, suggesting the absence of an open- inactivated state. We conclud that channel closing is a prerequisite although not an equivalent to the C-inactivation in the BK channels, indicating a strong coupling between these two processes. Because C-inactivation can involve multiple conformational changes at selectivity filter, the BK channel closing which can recover (open) relatively quickly might represent an early conformational stage of C-inactivation.