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The functionally relevant site for paxilline inhibition of BK channels.

ABSTRACT: 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.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC6969516 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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The functionally relevant site for paxilline inhibition of BK channels.

Zhou Yu Y Xia Xiao-Ming XM Lingle Christopher J CJ

Proceedings of the National Academy of Sciences of the United States of America 20191226 2

The tremorgenic fungal alkaloid paxilline (PAX) is a commonly used specific inhibitor of the large-conductance, voltage- and Ca<sup>2+</sup>-dependent BK-type K<sup>+</sup> 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 it ...[more]

PMID: 31879339

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Project description:Paxilline, a tremorogenic fungal alkaloid, potently inhibits large conductance Ca(2+)- and voltage-activated K(+) (BK)-type channels, but little is known about the mechanism underlying this inhibition. Here we show that inhibition is inversely dependent on BK channel open probability (Po), and is fully relieved by conditions that increase Po, even in the constant presence of paxilline. Manipulations that shift BK gating to more negative potentials reduce inhibition by paxilline in accordance with the increase in channel Po. Measurements of Po times the number of channels at negative potentials support the idea that paxilline increases occupancy of closed states, effectively reducing the closed-open equilibrium constant, L(0). Gating current measurements exclude an effect of paxilline on voltage sensors. Steady-state inhibition by multiple paxilline concentrations was determined for four distinct equilibration conditions, each with a distinct Po. The IC50 for paxilline shifted from around 10 nM when channels were largely closed to near 10 µM as maximal Po was approached. Model-dependent analysis suggests a mechanism of inhibition in which binding of a single paxilline molecule allosterically alters the intrinsic L(0) favoring occupancy of closed states, with affinity for the closed conformation being >500-fold greater than affinity for the open conformation. The rate of inhibition of closed channels was linear up through 2 µM paxilline, with a slope of 2 × 10(6) M(-1)s(-1). Paxilline inhibition was hindered by either the bulky cytosolic blocker, bbTBA, or by concentrations of cytosolic sucrose that hinder ion permeation. However, paxilline does not hinder MTSET modification of the inner cavity residue, A313C. We conclude that paxilline binds more tightly to the closed conformation, favoring occupancy of closed-channel conformations, and propose that it binds to a superficial position near the entrance to the central cavity, but does not hinder access of smaller molecules to this cavity.

Project description:BackgroundOuter hair cells are the specialized sensory cells that empower the mammalian hearing organ, the cochlea, with its remarkable sensitivity and frequency selectivity. Sound-evoked receptor potentials in outer hair cells are shaped by both voltage-gated K(+) channels that control the membrane potential and also ligand-gated K(+) channels involved in the cholinergic efferent modulation of the membrane potential. The objectives of this study were to investigate the tonotopic contribution of BK channels to voltage- and ligand-gated currents in mature outer hair cells from the rat cochlea.Methodology/principalFindings In this work we used patch clamp electrophysiology and immunofluorescence in tonotopically defined segments of the rat cochlea to determine the contribution of BK channels to voltage- and ligand-gated currents in outer hair cells. Although voltage and ligand-gated currents have been investigated previously in hair cells from the rat cochlea, little is known about their tonotopic distribution or potential contribution to efferent inhibition. We found that apical (low frequency) outer hair cells had no BK channel immunoreactivity and little or no BK current. In marked contrast, basal (high frequency) outer hair cells had abundant BK channel immunoreactivity and BK currents contributed significantly to both voltage-gated and ACh-evoked K(+) currents.Conclusions/significanceOur findings suggest that basal (high frequency) outer hair cells may employ an alternative mechanism of efferent inhibition mediated by BK channels instead of SK2 channels. Thus, efferent synapses may use different mechanisms of action both developmentally and tonotopically to support high frequency audition. High frequency audition has required various functional specializations of the mammalian cochlea, and as shown in our work, may include the utilization of BK channels at efferent synapses. This mechanism of efferent inhibition may be related to the unique acetylcholine receptors that have evolved in mammalian hair cells compared to those of other vertebrates.

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The functionally relevant site for paxilline inhibition of BK channels.

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The functionally relevant site for paxilline inhibition of BK channels.

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