The functionally relevant site for paxilline inhibition of BK channels

Abstract

Significance Paxilline (PAX) is a specific BK channel inhibitor that has been widely used in various biophysical and physiological studies involving BK channels. It is also one of a few nonpeptidergic toxins with binding affinity comparable to that of peptidergic toxins. The current study identifies a binding pose of PAX in the closed BK channel that fits all known functional characteristics of PAX inhibition and is confirmed by additional functional tests. This result provides critical information to thoroughly understand the mechanism underlying BK channel inhibition by PAX, which will provide not only important insight into BK channel function, but also valuable guidance in the development of novel ion channel modulators. The tremorgenic fungal alkaloid paxilline (PAX) is a commonly used specific inhibitor of the large-conductance, voltage- and Ca2+-dependent BK-type K+ channel. PAX inhibits BK channels by selective interaction with closed states. BK inhibition by PAX is best characterized by the idea that PAX gains access to the channel through the central cavity of the BK channel, and that only a single PAX molecule can interact with the BK channel at a time. The notion that PAX reaches its binding site via the central cavity and involves only a single PAX molecule would be consistent with binding on the axis of the permeation pathway, similar to classical open channel block and inconsistent with the observation that PAX selectively inhibits closed channels. To explore the potential sites of interaction of PAX with the BK channel, we undertook a computational analysis of the interaction of PAX with the BK channel pore gate domain guided by recently available liganded (open) and metal-free (closed) Aplysia BK channel structures. The analysis unambiguously identified a preferred position of PAX occupancy that accounts for all previously described features of PAX inhibition, including state dependence, G311 sensitivity, stoichiometry, and central cavity accessibility. This PAX-binding pose in closed BK channels is supported by additional functional results.