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Paxilline Inhibits BK Channels by an Almost Exclusively Closed-Channel Block Mechanism Yu Zhou Washington University School of Medicine in St

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Paxilline Inhibits BK Channels by an Almost Exclusively Closed-Channel Block Mechanism Yu Zhou Washington University School of Medicine in St Washington University School of Medicine Digital Commons@Becker Open Access Publications 2014 Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism Yu Zhou Washington University School of Medicine in St. Louis Christopher J. Lingle Washington University School of Medicine in St. Louis Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Recommended Citation Zhou, Yu and Lingle, Christopher J., ,"Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism." Journal of General Physiology.144,5. 415-440. (2014). https://digitalcommons.wustl.edu/open_access_pubs/3548 This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. Published October 27, 2014 Research Article Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism Yu Zhou and Christopher J. Lingle Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110 Paxilline, a tremorogenic fungal alkaloid, potently inhibits large conductance Ca2+- 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 inhibi- tion 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 inhibi- Downloaded from tion 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 conforma- tion. The rate of inhibition of closed channels was linear up through 2 µM paxilline, with a slope of 2 × 106 M1s1. 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. jgp.rupress.org INTRODUCTION Since the early days of investigation of ion channel 2005; Uysal et al., 2009). First, in closed K+ channels, function to the present day in which functional studies the so-called crossing of the S6 helices provides a physi- on December 18, 2014 of ion channels are also guided by structural informa- cal barrier to access of small molecules to an inner aque- tion, molecules that inhibit ion channels continue to be ous cavity preceding the selectivity filter. Second, in open essential tools for probing state-dependent conforma- K+ channels, separation of the S6 helices creates an tional changes in binding-site accessibility. For potas- aperture (Perozo et al., 1999) that allows access not sium channels (K+ channels), some of the earliest work only of permeant ions but also of quaternary blockers with channel blockers insightfully established that state- (Lenaeus et al., 2005) and peptides (Zhou et al., 2001) The Journal of General Physiology dependent conformational changes on the cytosolic to positions within the inner cavity. Together, these side of voltage-dependent K+ channels are required to types of functional and structural tests have provided a allow charged quaternary blockers to reach their block- compelling picture of a category of channel inhibition ing position within the pore (Armstrong, 1969; Armstrong that exclusively involves open-channel block; that is, and Hille, 1972; Choi et al., 1991). Furthermore, for binding sites for particular blockers only become avail- many Kv channels, channel closure can only occur after able when the channel is open. However, ion channel exit of the blocker from its blocking position (Armstrong inhibition can also occur by a variety of other mecha- and Hille, 1972; Choi et al., 1993). This idea that there nisms, which can also be informative about channel is gated access to the permeation pathway from the cy- function and its structural components. One particularly tosolic side of K+ channels was subsequently born out in rich source of interesting channel blockers are naturally structures of K+ channels (Doyle et al., 1998; Long et al., occurring toxins. Evolutionary pressures for species to optimize their survival have resulted in a vast, useful, and only partially Correspondence to Christopher J. Lingle: c l i n g l e @ m o r p h e u s . w u s t l . e d u exploited array of naturally occurring compounds that Abbreviations used in this paper: bbTBA, N-(4-[benzoyl]benzyl)-N,N,N- tributylammonium; BK, large conductance Ca2+- and voltage-activated K+; ChTX, charybdotoxin; C-O, closed–open; MTSEA, 2-aminoethyl MTS; © 2014 Zhou and Lingle This article is distributed under the terms of an Attribution– MTSES, sodium (2-sulfanoethyl) MTS; MTSET, [2-(trimethylammonium) Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publi- cation date (see http://www.rupress.org/terms). After six months it is available under a ethyl] MTS bromide; NPo, open probability times the number of chan- Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, nels; Pc, closed probability; Po, open probability. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Gen. Physiol. Vol. 144 No. 5 415–440 www.jgp.org/cgi/doi/10.1085/jgp.201411259 415 Published October 27, 2014 may kill or incapacitate prey or limit predation (Bush 2009) that are potent nonpeptidergic blockers of BK et al., 1997; Han et al., 2008; Liang, 2008). Such com- channels, generally acting at low nanomolar concentra- pounds often target ion channels, and the high selectiv- tions (Knaus et al., 1994; Imlach et al., 2009). Because ity and strong affinity for particular ion channels have some BK channels containing particular auxiliary sub- proven of enormous value for the identification of par- units exhibit resistance to block by the BK-blocking ticular subtypes of ion channels and their physiological scorpion toxins, iberiotoxin and ChTX (Xia et al., 1999; roles. Many such toxins are thought to act simply by Meera et al., 2000; Xia et al., 2000), paxilline, as a more binding to the extracellular face of an ion channel, per- general BK inhibitor, is often used in tests of physiologi- haps in a largely state-independent fashion, thereby cal roles of BK channels (Shao et al., 1999; Raffaelli occluding ion permeation or preventing channel open- et al., 2004; Tammaro et al., 2004; Essin et al., 2009). ings. This would include toxins such as tetrodotoxin Although inhibition by paxilline has been termed allo- acting on Na+ channels (Narahashi et al., 1964, 1967), steric and shows relief by elevations of Ca2+ (Sanchez charybdotoxin (ChTX) acting on large conductance and McManus, 1996), the mechanism and site of action Ca2+- and voltage-activated K+ (BK)-type K+ channels of the lipophilic paxilline are unknown. Recently, we (Smith et al., 1986; Anderson et al., 1988; MacKinnon observed that mutation of the so-called hinge glycine and Miller, 1988), and agitoxin acting on voltage- position (G311) in the S6 helix thought to line the BK dependent K+ channels (Gross and MacKinnon, 1996). channel inner cavity totally abolishes paxilline inhibi- Recent structural work has now specifically defined the tion (Zhou et al., 2010). This suggests that paxilline Downloaded from complex of ChTX in association with the Kv2.1 paddle- block is associated in some way with the BK pore-gate Kv1.2 chimeric channel (Banerjee et al., 2013). Over domain, perhaps dependent in some way on the confor- the past several years, another category of naturally oc- mational status of the inner pore. Here, we establish curring toxin has been identified that selectively targets that paxilline inhibition can best be viewed as an alloste- voltage-sensor domains of K+ channels, inhibiting chan- ric effect on the BK channel closed–open (C-O) equi- nels by stabilizing voltage-sensor domains in resting librium, stabilizing channels in closed conformations, jgp.rupress.org conformations (Swartz and MacKinnon, 1997a,b; Lee while having no discernible direct blocking effect on and MacKinnon, 2004; Milescu et al., 2009; Wang et al., open BK channels and no effect on voltage sensors. Oc- 2011). The ability of toxins to target not only specific cupancy of the paxilline-binding site
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