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

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.

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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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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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Cysteine Modification Alters Voltage- and Ca2+-dependent Gating of Large Conductance (BK) Potassium Channels

The Journal of General Physiology ◽

10.1085/jgp.200409149 ◽

2005 ◽

Vol 125 (2) ◽

pp. 213-236 ◽

Cited By ~ 27

Author(s):

Guangping Zhang ◽

Frank T. Horrigan

Keyword(s):

Steady State ◽

Bk Channels ◽

Bk Channel ◽

Tail Domain ◽

Cysteine Modification ◽

Α Subunit ◽

Channel Opening ◽

Rck Domain ◽

Sensor Activation ◽

And Shifts

The Ca2+-activated K+ (BK) channel α-subunit contains many cysteine residues within its large COOH-terminal tail domain. To probe the function of this domain, we examined effects of cysteine-modifying reagents on channel gating. Application of MTSET, MTSES, or NEM to mSlo1 or hSlo1 channels changed the voltage and Ca2+ dependence of steady-state activation. These reagents appear to modify the same cysteines but have different effects on function. MTSET increases IK and shifts the GK–V relation to more negative voltages, whereas MTSES and NEM shift the GK–V in the opposite direction. Steady-state activation was altered in the presence or absence of Ca2+ and at negative potentials where voltage sensors are not activated. Combinations of [Ca2+] and voltage were also identified where Po is not changed by cysteine modification. Interpretation of our results in terms of an allosteric model indicate that cysteine modification alters Ca2+ binding and the relative stability of closed and open conformations as well as the coupling of voltage sensor activation and Ca2+ binding and to channel opening. To identify modification-sensitive residues, we examined effects of MTS reagents on mutant channels lacking one or more cysteines. Surprisingly, the effects of MTSES on both voltage- and Ca2+-dependent gating were abolished by replacing a single cysteine (C430) with alanine. C430 lies in the RCK1 (regulator of K+ conductance) domain within a series of eight residues that is unique to BK channels. Deletion of these residues shifted the GK–V relation by >−80 mV. Thus we have identified a region that appears to strongly influence RCK domain function, but is absent from RCK domains of known structure. C430A did not eliminate effects of MTSET on apparent Ca2+ affinity. However an additional mutation, C615S, in the Haem binding site reduced the effects of MTSET, consistent with a role for this region in Ca2+ binding.

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An S6 Mutation in BK Channels Reveals β1 Subunit Effects on Intrinsic and Voltage-dependent Gating

The Journal of General Physiology ◽

10.1085/jgp.200609596 ◽

2006 ◽

Vol 128 (6) ◽

pp. 731-744 ◽

Cited By ~ 33

Author(s):

Bin Wang ◽

Robert Brenner

Keyword(s):

Smooth Muscle ◽

Steady State ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

Open Probability ◽

Α Subunit ◽

Channel Opening ◽

Sensor Activation ◽

Intracellular Domains

Large conductance, Ca2+- and voltage-activated K+ (BK) channels are exquisitely regulated to suit their diverse roles in a large variety of physiological processes. BK channels are composed of pore-forming α subunits and a family of tissue-specific accessory β subunits. The smooth muscle–specific β1 subunit has an essential role in regulating smooth muscle contraction and modulates BK channel steady-state open probability and gating kinetics. Effects of β1 on channel's gating energetics are not completely understood. One of the difficulties is that it has not yet been possible to measure the effects of β1 on channel's intrinsic closed-to-open transition (in the absence of voltage sensor activation and Ca2+ binding) due to the very low open probability in the presence of β1. In this study, we used a mutation of the α subunit (F315Y) that increases channel openings by greater than four orders of magnitude to directly compare channels' intrinsic open probabilities in the presence and absence of the β1 subunit. Effects of β1 on steady-state open probabilities of both wild-type α and the F315Y mutation were analyzed using the dual allosteric HA model. We found that mouse β1 has two major effects on channel's gating energetics. β1 reduces the intrinsic closed-to-open equilibrium that underlies the inhibition of BK channel opening seen in submicromolar Ca2+. Further, PO measurements at limiting slope allow us to infer that β1 shifts open channel voltage sensor activation to negative membrane potentials, which contributes to enhanced channel opening seen at micromolar Ca2+ concentrations. Using the F315Y α subunit with deletion mutants of β1, we also demonstrate that the small N- and C-terminal intracellular domains of β1 play important roles in altering channel's intrinsic opening and voltage sensor activation. In summary, these results demonstrate that β1 has distinct effects on BK channel intrinsic gating and voltage sensor activation that can be functionally uncoupled by mutations in the intracellular domains.

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

The Journal of General Physiology ◽

10.1085/jgp.117.6.607 ◽

2001 ◽

Vol 117 (6) ◽

pp. 607-628 ◽

Cited By ~ 28

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.

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Alcohol modulation of BK channel gating depends on β subunit composition

The Journal of General Physiology ◽

10.1085/jgp.201611594 ◽

2016 ◽

Vol 148 (5) ◽

pp. 419-440 ◽

Cited By ~ 6

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.

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Coupling between Voltage Sensor Activation, Ca2+ Binding and Channel Opening in Large Conductance (BK) Potassium Channels

The Journal of General Physiology ◽

10.1085/jgp.20028605 ◽

2002 ◽

Vol 120 (3) ◽

pp. 267-305 ◽

Cited By ~ 335

Author(s):

Frank T. Horrigan ◽

Richard W. Aldrich

Keyword(s):

Steady State ◽

Conformational Change ◽

Bk Channels ◽

Voltage Sensor ◽

Kinetic Properties ◽

Open Probability ◽

Voltage Sensors ◽

Channel Opening ◽

Wide Range ◽

Sensor Activation

To determine how intracellular Ca2+ and membrane voltage regulate the gating of large conductance Ca2+-activated K+ (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca2+ over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305–336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277–304). In 0 Ca2+, the steady-state gating charge-voltage (QSS-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (GK-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 μM Ca2+. This change reflects a differential effect of Ca2+ on voltage sensor activation and channel opening. Ca2+ has only a small effect on the fast component of ON gating current, indicating that Ca2+ binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than −80 mV) increases more than 1,000-fold in 70 μM Ca2+, demonstrating that Ca2+ increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca2+ binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca2+ sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca2+ sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic IK kinetics indicate that Ca2+ and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.

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The liverwort Marchantia polymorpha operates a depolarization-activated Slowpoke (SLO) K+ channel that recognises pH changes in the environment

10.1101/2021.06.01.446568 ◽

2021 ◽

Author(s):

Frances C. Sussmilch ◽

Jennifer Boehm ◽

Guido Gessner ◽

Tobias Maierhofer ◽

Thomas D. Mueller ◽

...

Keyword(s):

Critical Role ◽

Bk Channels ◽

Bk Channel ◽

K Channel ◽

Marchantia Polymorpha ◽

Terrestrial Environment ◽

Ph Changes ◽

Kv Channels ◽

Voltage Dependent ◽

Genes Encoding

Voltage-dependent ion channels are a prerequisite for cellular excitability and electrical communication - important traits for multicellular organisms to thrive in a changeable terrestrial environment. Based on their presence in extant embryophytes and closely-related green algae, the first plants to survive on land likely possessed genes encoding channels with homology to large-conductance calcium-activated K+ channels (BK channels from the Slo family) in addition to primary voltage-gated potassium channels from the plant VG-type family (Shaker or Kv channels). While the function and gating of Shaker channels has been characterised in flowering plants, so far knowledge of BK channels has been limited to animal models. In humans, BK-mediated K+ efflux has a critical role in sperm motility and membrane polarisation to enable fertilisation. In the liverwort Marchantia polymorpha, the MpBK2a channel gene is most highly expressed in male reproductive tissue, suggesting that these channels may function in sexual reproduction. We characterised MpBK2a channels and found them to be strongly K+-selective, outward-rectifying, 80-pS channels capable of repolarising the membrane after stimulus-dependent depolarisation. In contrast to its animal counterpart, MpBK2a is insensitive to cytoplasmic Ca2+ variations but effectively gated by pH changes. Given that this plant BK channel is active even in the presence of trace amounts of external K+ and at low pH, the liverwort channel could have stabilised the membrane potential under stressful pre-historic conditions including nutrient-depleted and acid environments as early plant pioneers conquered land.

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Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00325.2013 ◽

2014 ◽

Vol 306 (5) ◽

pp. C460-C470 ◽

Cited By ~ 18

Author(s):

Kiril L. Hristov ◽

Amy C. Smith ◽

Shankar P. Parajuli ◽

John Malysz ◽

Georgi V. Petkov

Keyword(s):

Smooth Muscle ◽

Membrane Potential ◽

Guinea Pig ◽

Bk Channels ◽

Bk Channel ◽

K Channel ◽

Channel Activity ◽

Open Probability ◽

Channel Regulation ◽

Pkc Activation

Large-conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility.

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BK channel inhibition by strong extracellular acidification

eLife ◽

10.7554/elife.38060 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 3

Author(s):

Yu Zhou ◽

Xiao-Ming Xia ◽

Christopher J Lingle

Keyword(s):

Bk Channels ◽

Bk Channel ◽

Extracellular Acidification ◽

Channel Inhibition ◽

Wide Range ◽

The Family ◽

Voltage Dependent ◽

Intracellular Organelles ◽

Extracellular Side ◽

Key Residues

Mammalian BK-type voltage- and Ca2+-dependent K+ channels are found in a wide range of cells and intracellular organelles. Among different loci, the composition of the extracellular microenvironment, including pH, may differ substantially. For example, it has been reported that BK channels are expressed in lysosomes with their extracellular side facing the strongly acidified lysosomal lumen (pH ~4.5). Here we show that BK activation is strongly and reversibly inhibited by extracellular H+, with its conductance-voltage relationship shifted by more than +100 mV at pHO 4. Our results reveal that this inhibition is mainly caused by H+ inhibition of BK voltage-sensor (VSD) activation through three acidic residues on the extracellular side of BK VSD. Given that these key residues (D133, D147, D153) are highly conserved among members in the voltage-dependent cation channel superfamily, the mechanism underlying BK inhibition by extracellular acidification might also be applicable to other members in the family.

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