scholarly journals Alcohol modulation of BK channel gating depends on β subunit composition

2016 ◽  
Vol 148 (5) ◽  
pp. 419-440 ◽  
Author(s):  
Guruprasad Kuntamallappanavar ◽  
Alex M. Dopico
Keyword(s):  
Single Channel ◽  
Ionic Current ◽  
Limited Range ◽  
Bk Channels ◽  
Bk Channel ◽  
Experimental Conditions ◽  
Allosteric Coupling ◽  
Channel Opening ◽  
Mammalian Tissues ◽  
Voltage Dependent

In most mammalian tissues, Ca2+i/voltage-gated, large conductance K+ (BK) channels consist of channel-forming slo1 and auxiliary (β1–β4) subunits. When Ca2+i (3–20 µM) reaches the vicinity of BK channels and increases their activity at physiological voltages, β1- and β4-containing BK channels are, respectively, inhibited and potentiated by intoxicating levels of ethanol (50 mM). Previous studies using different slo1s, lipid environments, and Ca2+i concentrations—all determinants of the BK response to ethanol—made it impossible to determine the specific contribution of β subunits to ethanol action on BK activity. Furthermore, these studies measured ethanol action on ionic current under a limited range of stimuli, rendering no information on the gating processes targeted by alcohol and their regulation by βs. Here, we used identical experimental conditions to obtain single-channel and macroscopic currents of the same slo1 channel (“cbv1” from rat cerebral artery myocytes) in the presence and absence of 50 mM ethanol. First, we assessed the role five different β subunits (1,2,2-IR, 3-variant d, and 4) in ethanol action on channel function. Thus, two phenotypes were identified: (1) ethanol potentiated cbv1-, cbv1+β3-, and cbv1+β4-mediated currents at low Ca2+i while inhibiting current at high Ca2+i, the potentiation–inhibition crossover occurring at 20 µM Ca2+i; (2) for cbv1+β1, cbv1+wt β2, and cbv1+β2-IR, this crossover was shifted to ∼3 µM Ca2+i. Second, applying Horrigan–Aldrich gating analysis on both phenotypes, we show that ethanol fails to modify intrinsic gating and the voltage-dependent parameters under examination. For cbv1, however, ethanol (a) drastically increases the channel’s apparent Ca2+ affinity (nine-times decrease in Kd) and (b) very mildly decreases allosteric coupling between Ca2+ binding and channel opening (C). The decreased Kd leads to increased channel activity. For cbv1+β1, ethanol (a) also decreases Kd, yet this decrease (two times) is much smaller than that of cbv1; (b) reduces C; and (c) decreases coupling between Ca2+ binding and voltage sensing (parameter E). Decreased allosteric coupling leads to diminished BK activity. Thus, we have identified critical gating modifications that lead to the differential actions of ethanol on slo1 with and without different β subunits.

scholarly journals Heme Regulates Allosteric Activation of the Slo1 BK Channel

2005 ◽  
Vol 126 (1) ◽  
pp. 7-21 ◽  
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.

scholarly journals Mechanism of β4 Subunit Modulation of BK Channels

2006 ◽  
Vol 127 (4) ◽  
pp. 449-465 ◽  
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.

Peroxynitrite reversibly inhibits Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

2000 ◽  
Vol 278 (6) ◽  
pp. H1883-H1890 ◽  
Author(s):  
Anna K. Brzezinska ◽  
Debebe Gebremedhin ◽  
William M. Chilian ◽  
Balaraman Kalyanaraman ◽  
Stephen J. Elliott
Keyword(s):  
Single Channel ◽  
Cerebral Arteries ◽  
Ionic Current ◽  
Inhibitory Effect ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Cell Current

Peroxynitrite (ONOO−) is a contractile agonist of rat middle cerebral arteries. To determine the mechanism responsible for this component of ONOO−bioactivity, the present study examined the effect of ONOO− on ionic current and channel activity in rat cerebral arteries. Whole cell recordings of voltage-clamped cells were made under conditions designed to optimize K+ current. The effects of iberiotoxin, a selective inhibitor of large-conductance Ca2+-activated K+ (BK) channels, and ONOO− (10–100 μM) were determined. At a pipette potential of +50 mV, ONOO− inhibited 39% of iberiotoxin-sensitive current. ONOO− was selective for iberiotoxin-sensitive current, whereas decomposed ONOO− had no effect. In excised, inside-out membrane patches, channel activity was recorded using symmetrical K+solutions. Unitary currents were sensitive to increases in internal Ca2+ concentration, consistent with activity due to BK channels. Internal ONOO− dose dependently inhibited channel activity by decreasing open probability and mean open times. The inhibitory effect of ONOO− could be overcome by reduced glutathione. Glutathione, added after ONOO−, restored whole cell current amplitude to control levels and reverted single-channel gating to control behavior. The inhibitory effect of ONOO− on membrane K+ current is consistent with its contractile effects in isolated cerebral arteries and single myocytes. Taken together, our data suggest that ONOO− has the potential to alter cerebral vascular tone by inhibiting BK channel activity.

scholarly journals 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

2014 ◽  
Vol 306 (2) ◽  
pp. C152-C166 ◽  
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.

scholarly journals Intracellular Mg2+ Enhances the Function of Bk-Type Ca2+-Activated K+ Channels

2001 ◽  
Vol 118 (5) ◽  
pp. 589-606 ◽  
Author(s):  
Jingyi Shi ◽  
Jianmin Cui
Keyword(s):  
Binding Sites ◽  
Neurotransmitter Release ◽  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Dependent Manner ◽  
Physiological Conditions ◽  
Channel Function ◽  
Open Conformation ◽  
Voltage Dependent

BK channels modulate neurotransmitter release due to their activation by voltage and Ca2+. Intracellular Mg2+ also modulates BK channels in multiple ways with opposite effects on channel function. Previous single-channel studies have shown that Mg2+ blocks the pore of BK channels in a voltage-dependent manner. We have confirmed this result by studying macroscopic currents of the mslo1 channel. We find that Mg2+ activates mslo1 BK channels independently of Ca2+ and voltage by preferentially binding to their open conformation. The mslo3 channel, which lacks Ca2+ binding sites in the tail, is not activated by Mg2+. However, coexpression of the mslo1 core and mslo3 tail produces channels with Mg2+ sensitivity similar to mslo1 channels, indicating that Mg2+ sites differ from Ca2+ sites. We discovered that Mg2+ also binds to Ca2+ sites and competitively inhibits Ca2+-dependent activation. Quantitative computation of these effects reveals that the overall effect of Mg2+ under physiological conditions is to enhance BK channel function.

scholarly journals Coupling and cooperativity in voltage activation of a limited-state BK channel gating in saturating Ca2+

2010 ◽  
Vol 135 (5) ◽  
pp. 461-480 ◽  
Author(s):  
Christopher Shelley ◽  
Xiaowei Niu ◽  
Yanyan Geng ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Coupling Factor ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Channel Kinetics ◽  
Voltage Sensors ◽  
Gating Mechanisms ◽  
Voltage Dependent ◽  
Single Channel Currents

Voltage-dependent gating mechanisms of large conductance Ca2+ and voltage-activated (BK) channels were investigated using two-dimensional maximum likelihood analysis of single-channel open and closed intervals. To obtain sufficient data at negative as well as positive voltages, single-channel currents were recorded at saturating Ca2+ from BK channels mutated to remove the RCK1 Ca2+ and Mg2+ sensors. The saturating Ca2+ acting on the Ca2+ bowl sensors of the resulting BKB channels increased channel activity while driving the gating into a reduced number of states, simplifying the model. Five highly constrained idealized gating mechanisms based on extensions of the Monod-Wyman-Changeux model for allosteric proteins were examined. A 10-state model without coupling between the voltage sensors and the opening/closing transitions partially described the voltage dependence of Po but not the single-channel kinetics. With allowed coupling, the model gave improved descriptions of Po and approximated the single-channel kinetics; each activated voltage sensor increased the opening rate approximately an additional 23-fold while having little effect on the closing rate. Allowing cooperativity among voltage sensors further improved the description of the data: each activated voltage sensor increased the activation rate of the remaining voltage sensors approximately fourfold, with little effect on the deactivation rate. The coupling factor was decreased in models with cooperativity from ∼23 to ∼18. Whether the apparent cooperativity among voltage sensors arises from imposing highly idealized models or from actual cooperativity will require additional studies to resolve. For both cooperative and noncooperative models, allowing transitions to five additional brief (flicker) closed states further improved the description of the data. These observations show that the voltage-dependent single-channel kinetics of BKB channels can be approximated by highly idealized allosteric models in which voltage sensor movement increases Po mainly through an increase in channel opening rates, with limited effects on closing rates.

scholarly journals Voltage and Ca2+ Activation of Single Large-Conductance Ca2+-Activated K+ Channels Described by a Two-Tiered Allosteric Gating Mechanism

2000 ◽  
Vol 116 (1) ◽  
pp. 75-100 ◽  
Author(s):  
Brad S. Rothberg ◽  
Karl L. Magleby
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Open Probability ◽  
Gating Charge ◽  
Gating Mechanism ◽  
Single Channel Analysis ◽  
Voltage Dependent ◽  
The Mean

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, qeff, of 2.3 ± 0.6 eo). Estimates of qeff were little changed for intracellular Ca2+ (Ca2+i) ranging from 0.0003 to 1,024 μM. Increasing Ca2+i from 0.03 to 1,024 μM shifted the voltage for half maximal activation (V1/2) 175 mV in the hyperpolarizing direction. V1/2 was independent of Ca2+i for Ca2+i ≤ 0.03 μM, indicating that the channel can be activated in the absence of Ca2+i. Open and closed dwell-time distributions for data obtained at different Ca2+i and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (qeff = −0.5 eo) and an increase in the mean opening rate (qeff = 1.8 eo), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+i (∼0 through 1,024 μM), voltage (+80 to −80 mV), and Po (10−4 to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.

scholarly journals Allosteric Gating of a Large Conductance Ca-activated K+ Channel

1997 ◽  
Vol 110 (3) ◽  
pp. 257-281 ◽  
Author(s):  
D.H. Cox ◽  
J. Cui ◽  
R.W. Aldrich
Keyword(s):  
Steady State ◽  
General Scheme ◽  
Kinetic Scheme ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Kinetic Properties ◽  
Allosteric Proteins ◽  
Channel Opening ◽  
Voltage Dependent

Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca2+. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca2+ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca2+ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca2+ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca2+ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations.

scholarly journals Gating Properties Conferred on Bk Channels by the β3b Auxiliary Subunit in the Absence of Its Nh2- and Cooh Termini

2001 ◽  
Vol 117 (6) ◽  
pp. 607-628 ◽  
Author(s):  
Xu-Hui Zeng ◽  
J.-P. Ding ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Current ◽  
Leftward Shift ◽  
Auxiliary Subunit ◽  
Voltage Dependent ◽  
Primary Determinant

Both β1 and β2 auxiliary subunits of the BK-type K+ channel family profoundly regulate the apparent Ca2+ sensitivity of BK-type Ca2+-activated K+ channels. Each produces a pronounced leftward shift in the voltage of half-activation (V0.5) at a given Ca2+ concentration, particularly at Ca2+ above 1 μM. In contrast, the rapidly inactivating β3b auxiliary produces a leftward shift in activation at Ca2+ below 1 μM. In the companion work (Lingle, C.J., X.-H. Zeng, J.-P. Ding, and X.-M. Xia. 2001. J. Gen. Physiol. 117:583–605, this issue), we have shown that some of the apparent β3b-mediated shift in activation at low Ca2+ arises from rapid unblocking of inactivated channels, unlike the actions of the β1 and β2 subunits. Here, we compare effects of the β3b subunit that arise from inactivation, per se, versus those that may arise from other functional effects of the subunit. In particular, we examine gating properties of the β3b subunit and compare it to β3b constructs lacking either the NH2- or COOH terminus or both. The results demonstrate that, although the NH2 terminus appears to be the primary determinant of the β3b-mediated shift in V0.5 at low Ca2+, removal of the NH2 terminus reveals two other interesting aspects of the action of the β3b subunit. First, the conductance-voltage curves for activation of channels containing the β3b subunit are best described by a double Boltzmann shape, which is proposed to arise from two independent voltage-dependent activation steps. Second, the presence of the β3b subunit results in channels that exhibit an anomalous instantaneous outward current rectification that is correlated with a voltage dependence in the time-averaged single-channel current. The two effects appear to be unrelated, but indicative of the variety of ways that interactions between β and α subunits can affect BK channel function. The COOH terminus of the β3b subunit produces no discernible functional effects.

scholarly journals Regulatory γ1 subunits defy symmetry in functional modulation of BK channels

2018 ◽  
Vol 115 (40) ◽  
pp. 9923-9928 ◽  
Author(s):  
Vivian Gonzalez-Perez ◽  
Manu Ben Johny ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Single Channel ◽  
Regulatory Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bk Channels ◽  
Bk Channel ◽  
Single Type ◽  
Regulatory Subunits ◽  
And Function

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, β and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four β subunits per channel, with each β subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+channel.

Sign in / Sign up

Export Citation Format

Share Document