Rubidium Efflux as a Tool for the Pharmacological Characterisation of Compounds with BK Channel Opening Properties

Large-conductance Ca2+- and voltage-activated K+ channel (BK) open probability is enhanced by depolarization, increasing Ca2+ concentration, or both. These stimuli activate modular voltage and Ca2+ sensors that are allosterically coupled to channel gating. Here, we report a point mutation of a phenylalanine (F380A) in the S6 transmembrane helix that, in the absence of internal Ca2+, profoundly hinders channel opening while showing only minor effects on the voltage sensor active–resting equilibrium. Interpretation of these results using an allosteric model suggests that the F380A mutation greatly increases the free energy difference between open and closed states and uncouples Ca2+ binding from voltage sensor activation and voltage sensor activation from channel opening. However, the presence of a bulky and more hydrophobic amino acid in the F380 position (F380W) increases the intrinsic open–closed equilibrium, weakening the coupling between both sensors with the pore domain. Based on these functional experiments and molecular dynamics simulations, we propose that F380 interacts with another S6 hydrophobic residue (L377) in contiguous subunits. This pair forms a hydrophobic ring important in determining the open–closed equilibrium and, like an integration node, participates in the communication between sensors and between the sensors and pore. Moreover, because of its effects on open probabilities, the F380A mutant can be used for detailed voltage sensor experiments in the presence of permeant cations.

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Heme Regulates Allosteric Activation of the Slo1 BK Channel

The Journal of General Physiology ◽

10.1085/jgp.200509262 ◽

2005 ◽

Vol 126 (1) ◽

pp. 7-21 ◽

Cited By ~ 66

Author(s):

Frank T. Horrigan ◽

Stefan H. Heinemann ◽

Toshinori Hoshi

Keyword(s):

Divalent Cations ◽

Ionic Currents ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

Gating Currents ◽

Calcium Dependent ◽

Channel Opening ◽

Activation Gate ◽

Voltage Dependent

Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531–535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G–V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G–V simulated by weakening the coupling of both Ca2+ binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca2+- and voltage-dependent gating as well as intrinsic stability of the open state.

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Broken symmetry in the human BK channel

10.1101/494385 ◽

2018 ◽

Cited By ~ 4

Author(s):

Lige Tonggu ◽

Liguo Wang

Keyword(s):

Ion Channels ◽

Binding Sites ◽

Bk Channel ◽

Excitable Cells ◽

Local Conformation ◽

Channel Opening ◽

Significant Movement ◽

Em Method ◽

The Common ◽

Functional States

ABSTRACTVoltage-gated and ligand-modulated ion channels play critical roles in excitable cells. To understand the interplay among voltage-sensing, ligand-binding and channel opening, the structures of ion channels in various functional states need to be determined. Here, the “random spherically constrained” (RSC) single-particle cryo-EM method was employed to study the human large conductance voltage- and calcium-activated potassium (hBK or hSlo1) channels reconstituted into liposomes. The hBK structure has been determined at 3.5 Å resolution in the absence of Ca2+. Instead of the common four-fold symmetry observed in ligand-modulated ion channels, a two-fold symmetry was observed in hBK. Two opposing subunits in the Ca2+ sensing gating ring rotate around the center of each subunit, which results in the movement of the assembly and flexible interfaces and Ca2+ binding sites. Despite the significant movement, the local conformation of the assembly interfaces and Ca2+ binding sites remains the same among the four subunits.

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Novel oxime and oxime ether derivatives of 12,14-dichlorodehydroabietic acid: Design, synthesis, and BK channel-opening activity

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2008.10.078 ◽

2008 ◽

Vol 18 (24) ◽

pp. 6386-6389 ◽

Cited By ~ 13

Author(s):

Yong-Mei Cui ◽

Eriko Yasutomi ◽

Yuko Otani ◽

Takashi Yoshinaga ◽

Katsutoshi Ido ◽

...

Keyword(s):

Bk Channel ◽

Oxime Ether ◽

Design Synthesis ◽

Channel Opening ◽

Derivatives Of

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Mechanism of β4 Subunit Modulation of BK Channels

The Journal of General Physiology ◽

10.1085/jgp.200509436 ◽

2006 ◽

Vol 127 (4) ◽

pp. 449-465 ◽

Cited By ~ 80

Author(s):

Bin Wang ◽

Brad S. Rothberg ◽

Robert Brenner

Keyword(s):

Calcium Binding ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

Type Ii ◽

Allosteric Coupling ◽

Channel Opening ◽

Sensor Activation ◽

Compensatory Effect ◽

Allosteric Model

Large-conductance (BK-type) Ca2+-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca2+. BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (β1–β4). Biophysical characterization has shown that the β4 subunit confers properties of the so-called “type II” BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the β4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca2+ sensitivity. Specifically, channel activity at low Ca2+ is inhibited, while at high Ca2+, activity is enhanced. The goal of this study is to understand the mechanism underlying β4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that β4's most profound effect is a decrease in Po (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, β4 promotes channel opening by increasing voltage dependence of Po-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of β4 on BK channels. β4 reduces channel opening by decreasing the intrinsic gating equilibrium (L0), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, β4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vho) to more negative membrane potentials. The consequence is that β4 causes a net positive shift of the G-V relationship (relative to α subunit alone) at low calcium. At higher calcium, the contribution by Vho and an increase in allosteric coupling to Ca2+ binding (C) promotes a negative G-V shift of α+β4 channels as compared to α subunits alone. This manner of modulation predicts that type II BK channels are downregulated by β4 at resting voltages through effects on L0. However, β4 confers a compensatory effect on voltage sensor activation that increases channel opening during depolarization.

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Closed state-coupled C-type inactivation in BK channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1607584113 ◽

2016 ◽

Vol 113 (25) ◽

pp. 6991-6996 ◽

Cited By ~ 2

Author(s):

Jiusheng Yan ◽

Qin Li ◽

Richard W. Aldrich

Keyword(s):

Conformational Changes ◽

Bk Channels ◽

Bk Channel ◽

Membrane Potentials ◽

Selectivity Filter ◽

Open Probability ◽

Closed State ◽

State Dependent ◽

Channel Opening ◽

Activation Gate

Ion channels regulate ion flow by opening and closing their pore gates. K+ channels commonly possess two pore gates, one at the intracellular end for fast channel activation/deactivation and the other at the selectivity filter for slow C-type inactivation/recovery. The large-conductance calcium-activated potassium (BK) channel lacks a classic intracellular bundle-crossing activation gate and normally show no C-type inactivation. We hypothesized that the BK channel’s activation gate may spatially overlap or coexist with the C-type inactivation gate at or near the selectivity filter. We induced C-type inactivation in BK channels and studied the relationship between activation/deactivation and C-type inactivation/recovery. We observed prominent slow C-type inactivation/recovery in BK channels by an extreme low concentration of extracellular K+ together with a Y294E/K/Q/S or Y279F mutation whose equivalent in Shaker channels (T449E/K/D/Q/S or W434F) caused a greatly accelerated rate of C-type inactivation or constitutive C-inactivation. C-type inactivation in most K+ channels occurs upon sustained membrane depolarization or channel opening and then recovers during hyperpolarized membrane potentials or channel closure. However, we found that the BK channel C-type inactivation occurred during hyperpolarized membrane potentials or with decreased intracellular calcium ([Ca2+]i) and recovered with depolarized membrane potentials or elevated [Ca2+]i. Constitutively open mutation prevented BK channels from C-type inactivation. We concluded that BK channel C-type inactivation is closed state-dependent and that its extents and rates inversely correlate with channel-open probability. Because C-type inactivation can involve multiple conformational changes at the selectivity filter, we propose that the BK channel’s normal closing may represent an early conformational stage of C-type inactivation.

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State-independent Block of BK Channels by an Intracellular Quaternary Ammonium

The Journal of General Physiology ◽

10.1085/jgp.200609579 ◽

2006 ◽

Vol 128 (3) ◽

pp. 347-364 ◽

Cited By ~ 71

Author(s):

Christina M. Wilkens ◽

Richard W. Aldrich

Keyword(s):

Steady State ◽

Quaternary Ammonium ◽

Bk Channels ◽

Bk Channel ◽

Fold Change ◽

Time Dependent ◽

Kinetic Measurements ◽

State Dependent ◽

Channel Opening ◽

High Concentrations

Intracellular blockade by quaternary ammonium (QA) molecules of many potassium channels is state dependent, where the requirement for channel opening is evidenced by a time-dependent component of block in the macroscopic record. Whether this is the case for Ca2+- and voltage-activated potassium (BK) channels, however, remains unclear. Previous work (Li, W., and R.W. Aldrich. 2004. J. Gen. Physiol. 124:43–57) tentatively proposed a state-dependent, trapping model, but left open the possibility of state-independent block. Here, we found BK channel blockade by a novel QA derivative, bbTBA, was time dependent, raising the possibility of state-dependent, open channel block. Alternatively, the observed voltage dependence of block could be sufficient to explain time-dependent block. We have used steady-state and kinetic measurements of bbTBA blockade in order to discriminate between these two possibilities. bbTBA did not significantly slow deactivation kinetics at potentials between −200 and −100 mV, suggesting that channels can close unhindered by bound bbTBA. We further find no evidence that bbTBA is trapped inside BK channels after closing. Measurements of steady state fractional block at +40 mV revealed a 1.3-fold change in apparent affinity for a 33-fold change in Po, in striking contrast to the 31-fold change predicted by state-dependent block. Finally, the appearance of a third kinetic component of bbTBA blockade at high concentrations is incompatible with state-dependent block. Our results suggest that access of intracellular bbTBA to the BK channel cavity is not strictly gated by channel opening and closing, and imply that the permeation gate for BK channels may not be intracellular.

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Characteristics of single large-conductance Ca2+-activated K+ channels and their regulation of action potentials and excitability in parasympathetic cardiac motoneurons in the nucleus ambiguus

AJP Cell Physiology ◽

10.1152/ajpcell.00423.2012 ◽

2014 ◽

Vol 306 (2) ◽

pp. C152-C166 ◽

Cited By ~ 7

Author(s):

Min Lin ◽

Jeff T. Hatcher ◽

Robert D. Wurster ◽

Qin-Hui Chen ◽

Zixi (Jack) Cheng

Keyword(s):

Single Channel ◽

Cell Voltage ◽

Bk Channels ◽

Bk Channel ◽

Spike Frequency ◽

Half Width ◽

Nucleus Ambiguus ◽

Whole Cell ◽

Channel Opening ◽

Channel Open Time

Large-conductance Ca2+-activated K+ channels (BK) regulate action potential (AP) properties and excitability in many central neurons. However, the properties and functional roles of BK channels in parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus (NA) have not yet been well characterized. In this study, the tracer X-rhodamine-5 (and 6)-isothiocyanate (XRITC) was injected into the pericardial sac to retrogradely label PCMNs in FVB mice at postnatal 7–9 days. Two days later, XRITC-labeled PCMNs in brain stem slices were identified. Using excised patch single-channel recordings, we identified voltage-gated and Ca2+-dependent BK channels in PCMNs. The majority of BK channels exhibited persistent channel opening during voltage holding. These BK channels had a conductance of 237 pS and a 50% opening probability at +27.9 mV, the channel open time constant was 3.37 ms at +20 mV, and dwell time increased exponentially as the membrane potential depolarized. At the +20-mV holding potential, the [Ca2+]50 was 15.2 μM with a P0.5 of 0.4. Occasionally, some BK channels showed a transient channel opening and fast inactivation. Using whole cell voltage clamp, we found that BK channel mediated outward currents and afterhyperpolarization currents ( IAHP). Using whole cell current clamp, we found that application of BK channel blocker iberiotoxin (IBTX) increased spike half-width and suppressed fast afterhyperpolarization (fAHP) amplitude following single APs. In addition, IBTX application increased spike half-width and reduced the spike frequency-dependent AP broadening in trains and spike frequency adaption (SFA). Furthermore, BK channel blockade decreased spike frequency. Collectively, these results demonstrate that PCMNs have BK channels that significantly regulate AP repolarization, fAHP, SFA, and spike frequency. We conclude that activation of BK channels underlies one of the mechanisms for facilitation of PCMN excitability.

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