scholarly journals An extracellular domain of the accessory β1 subunit is required for modulating BK channel voltage sensor and gate

2011 ◽  
Vol 139 (1) ◽  
pp. 57-67 ◽  
Author(s):  
Aleksandra Gruslova ◽  
Iurii Semenov ◽  
Bin Wang
Keyword(s):  
Bk Channel ◽  
Free Energy Calculations ◽  
Voltage Sensor ◽  
Functional Domain ◽  
Β Subunit ◽  
Gating Model ◽  
Channel Opening ◽  
Subunit Function ◽  
Sensor Activation ◽  
Open States

A family of tissue-specific auxiliary β subunits modulates large conductance voltage- and calcium-activated potassium (BK) channel gating properties to suit their diverse functions. Paradoxically, β subunits both promote BK channel activation through a stabilization of voltage sensor activation and reduce BK channel openings through an increased energetic barrier of the closed-to-open transition. The molecular determinants underlying β subunit function, including the dual gating effects, remain unknown. In this study, we report the first identification of a β1 functional domain consisting of Y74, S104, Y105, and I106 residues located in the extracellular loop of β1. These amino acids reside within two regions of highest conservation among related β1, β2, and β4 subunits. Analysis in the context of the Horrigan-Aldrich gating model revealed that this domain functions to both promote voltage sensor activation and also reduce intrinsic gating. Free energy calculations suggest that the dual effects of the β1 Y74 and S104–I106 domains can be largely accounted for by a relative destabilization of channels in open states that have few voltage sensors activated. These results suggest a unique and novel mechanism for β subunit modulation of voltage-gated potassium channels wherein interactions between extracellular β subunit residues with the external portions of the gate and voltage sensor regulate channel opening.

scholarly journals Hydrophobic interaction between contiguous residues in the S6 transmembrane segment acts as a stimuli integration node in the BK channel

2014 ◽  
Vol 145 (1) ◽  
pp. 61-74 ◽  
Author(s):  
Willy Carrasquel-Ursulaez ◽  
Gustavo F. Contreras ◽  
Romina V. Sepúlveda ◽  
Daniel Aguayo ◽  
Fernando González-Nilo ◽  
...  
Keyword(s):  
Transmembrane Helix ◽  
Energy Difference ◽  
Bk Channel ◽  
Voltage Sensor ◽  
K Channel ◽  
Open Probability ◽  
Channel Opening ◽  
Integration Node ◽  
Sensor Activation ◽  
Dynamics Simulations

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.

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.

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

2006 ◽  
Vol 128 (6) ◽  
pp. 731-744 ◽  
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.

scholarly journals cAMP-dependent regulation of IKs single-channel kinetics

2017 ◽  
Vol 149 (8) ◽  
pp. 781-798 ◽  
Author(s):  
Emely Thompson ◽  
Jodene Eldstrom ◽  
Maartje Westhoff ◽  
Donald McAfee ◽  
Elise Balse ◽  
...  
Keyword(s):  
Action Potential ◽  
Single Channel ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Surface Expression ◽  
Voltage Sensor ◽  
Single Channels ◽  
Adrenergic Stimulation ◽  
Channel Opening ◽  
Sensor Activation

The delayed potassium rectifier current, IKs, is composed of KCNQ1 and KCNE1 subunits and plays an important role in cardiac action potential repolarization. During β-adrenergic stimulation, 3′-5′-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylates KCNQ1, producing an increase in IKs current and a shortening of the action potential. Here, using cell-attached macropatches and single-channel recordings, we investigate the microscopic mechanisms underlying the cAMP-dependent increase in IKs current. A membrane-permeable cAMP analog, 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), causes a marked leftward shift of the conductance–voltage relation in macropatches, with or without an increase in current size. Single channels exhibit fewer silent sweeps, reduced first latency to opening (control, 1.61 ± 0.13 s; cAMP, 1.06 ± 0.11 s), and increased higher-subconductance-level occupancy in the presence of cAMP. The E160R/R237E and S209F KCNQ1 mutants, which show fixed and enhanced voltage sensor activation, respectively, largely abolish the effect of cAMP. The phosphomimetic KCNQ1 mutations, S27D and S27D/S92D, are much less and not at all responsive, respectively, to the effects of PKA phosphorylation (first latency of S27D + KCNE1 channels: control, 1.81 ± 0.1 s; 8-CPT-cAMP, 1.44 ± 0.1 s, P < 0.05; latency of S27D/S92D + KCNE1: control, 1.62 ± 0.1 s; cAMP, 1.43 ± 0.1 s, nonsignificant). Using total internal reflection fluorescence microscopy, we find no overall increase in surface expression of the channel during exposure to 8-CPT-cAMP. Our data suggest that the cAMP-dependent increase in IKs current is caused by an increase in the likelihood of channel opening, combined with faster openings and greater occupancy of higher subconductance levels, and is mediated by enhanced voltage sensor activation.

scholarly journals Role of Charged Residues in the S1–S4 Voltage Sensor of BK Channels

2006 ◽  
Vol 127 (3) ◽  
pp. 309-328 ◽  
Author(s):  
Zhongming Ma ◽  
Xing Jian Lou ◽  
Frank T. Horrigan
Keyword(s):  
Voltage Dependence ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Voltage Gating ◽  
Voltage Sensing ◽  
Kv Channels ◽  
Gating Charge ◽  
Charged Residues ◽  
Sensor Activation

The activation of large conductance Ca2+-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K+ (KV) channels. Yet BK and KV channels share many conserved charged residues in transmembrane segments S1–S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (Po) and IK kinetics (τ(IK)) over an extended voltage range in 0–50 μM [Ca2+]i. mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of PO. The voltage dependence of PO and τ(IK) at extreme negative potentials was also reduced, implying that the closed–open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and KV channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to KV channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1–S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3–7 kcal mol−1, indicating a strong contribution of non–voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.

scholarly journals Voltage Sensor Deactivation Inhibits BK Channel Opening by Mg2+

2011 ◽  
Vol 100 (3) ◽  
pp. 582a
Author(s):  
Ren-Shiang Chen ◽  
Yanyan Geng ◽  
Karl L. Magleby
Keyword(s):  
Bk Channel ◽  
Voltage Sensor ◽  
Channel Opening

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 Uncoupling Charge Movement from Channel Opening in Voltage-gated Potassium Channels by Ruthenium Complexes

2011 ◽  
Vol 286 (18) ◽  
pp. 16414-16425 ◽  
Author(s):  
Andrés Jara-Oseguera ◽  
Itzel G. Ishida ◽  
Gisela E. Rangel-Yescas ◽  
Noel Espinosa-Jalapa ◽  
José A. Pérez-Guzmán ◽  
...  
Keyword(s):  
Electromechanical Coupling ◽  
Voltage Dependence ◽  
Voltage Sensor ◽  
Delayed Rectifier ◽  
Charge Movement ◽  
Β Cells ◽  
Delayed Rectifier Current ◽  
Mechanism Of Inhibition ◽  
Channel Opening ◽  
Sensor Activation

The Kv2.1 channel generates a delayed-rectifier current in neurons and is responsible for modulation of neuronal spike frequency and membrane repolarization in pancreatic β-cells and cardiomyocytes. As with other tetrameric voltage-activated K+-channels, it has been proposed that each of the four Kv2.1 voltage-sensing domains activates independently upon depolarization, leading to a final concerted transition that causes channel opening. The mechanism by which voltage-sensor activation is coupled to the gating of the pore is still not understood. Here we show that the carbon-monoxide releasing molecule 2 (CORM-2) is an allosteric inhibitor of the Kv2.1 channel and that its inhibitory properties derive from the CORM-2 ability to largely reduce the voltage dependence of the opening transition, uncoupling voltage-sensor activation from the concerted opening transition. We additionally demonstrate that CORM-2 modulates Shaker K+-channels in a similar manner. Our data suggest that the mechanism of inhibition by CORM-2 may be common to voltage-activated channels and that this compound should be a useful tool for understanding the mechanisms of electromechanical coupling.

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

2005 ◽  
Vol 125 (2) ◽  
pp. 213-236 ◽  
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.

Impaired calcium release during fatigue

2008 ◽  
Vol 104 (1) ◽  
pp. 296-305 ◽  
Author(s):  
D. G. Allen ◽  
G. D. Lamb ◽  
H. Westerblad
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Action Potential ◽  
Inorganic Phosphate ◽  
Calcium Release ◽  
Voltage Sensor ◽  
Functional Importance ◽  
Intracellular Atp ◽  
Channel Opening ◽  
Sensor Activation

Impaired calcium release from the sarcoplasmic reticulum (SR) has been identified as a contributor to fatigue in isolated skeletal muscle fibers. The functional importance of this phenomenon can be quantified by the use of agents, such as caffeine, which can increase SR Ca2+ release during fatigue. A number of possible mechanisms for impaired calcium release have been proposed. These include reduction in the amplitude of the action potential, potentially caused by extracellular K+ accumulation, which may reduce voltage sensor activation but is counteracted by a number of mechanisms in intact animals. Reduced effectiveness of SR Ca2+ channel opening is caused by the fall in intracellular ATP and the rise in Mg2+ concentrations that occur during fatigue. Reduced Ca2+ available for release within the SR can occur if inorganic phosphate enters the SR and precipitates with Ca2+. Further progress requires the development of methods that can identify impaired SR Ca2+ release in intact, blood-perfused muscles and that can distinguish between the various mechanisms proposed.

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