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 IKs ion-channel pore conductance can result from individual voltage sensor movements

2019 ◽  
Vol 116 (16) ◽  
pp. 7879-7888 ◽  
Author(s):  
Maartje Westhoff ◽  
Jodene Eldstrom ◽  
Christopher I. Murray ◽  
Emely Thompson ◽  
David Fedida
Keyword(s):  
Single Channel ◽  
Voltage Sensor ◽  
Open Probability ◽  
Voltage Sensors ◽  
Repolarization Reserve ◽  
Accessory Subunits ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Open States ◽  
Channel Currents

The IKs current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the IKs complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of IKs channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in IKs current kinetics, including the nonlinear depolarization of the conductance–voltage (G–V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.

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 The BK channel gating ring is strongly coupled to the voltage sensor

2018 ◽  
Author(s):  
Pablo Miranda ◽  
Miguel Holmgren ◽  
Teresa Giraldez
Keyword(s):  
Conformational Changes ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Voltage Sensitivity ◽  
Divalent Ions ◽  
Gating Currents ◽  
Open Probability ◽  
Voltage Dependent ◽  
Tightly Coupled

ABSTRACTThe open probability of large conductance voltage- and calcium-dependent potassium (BK) channels is regulated allosterically by changes in the transmembrane voltage and intracellular concentration of divalent ions (Ca2+ and Mg2+). The divalent cation sensors reside within the gating ring formed by eight Regulator of Conductance of Potassium (RCK) domains, two from each of the four channel subunits. Overall, the gating ring contains 12 sites that can bind Ca2+ with different affinities. Using patch-clamp fluorometry, we have shown robust changes in FRET signals within the gating ring in response to divalent ions and voltage, which do not directly track open probability. Only the conformational changes triggered through the RCK1 binding site are voltage-dependent in presence of Ca2+. Because the gating ring is outside the electric field, it must gain voltage sensitivity from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of voltage sensor dynamics known to shift gating currents produce a cognate shift in the gating ring voltage dependence, whereas changing BK channels’ relative probability of opening had little effect. These results strongly suggest that the conformational changes of the RCK1 domain of the gating ring are tightly coupled to the voltage sensor function, and this interaction is central to the allosteric modulation of BK channels.

scholarly journals Tamoxifen inhibits BK channels in chick cochlea without alterations in voltage-dependent activation

2009 ◽  
Vol 297 (1) ◽  
pp. C75-C85 ◽  
Author(s):  
Mingjie Tong ◽  
R. Keith Duncan
Keyword(s):  
Hair Cells ◽  
Molecular Mechanisms ◽  
Single Channel ◽  
Frequency Tuning ◽  
Low Frequency ◽  
Bk Channels ◽  
Bk Channel ◽  
Basilar Papilla ◽  
Channel Kinetics ◽  
Open Probability

Large-conductance, Ca2+-activated, and voltage-gated potassium channels (BK, BKCa, or Maxi-K) play an important role in electrical tuning in nonmammalian vertebrate hair cells. Systematic changes in tuning frequency along the tonotopic axis largely result from variations in BK channel kinetics, but the molecular changes underpinning these functional variations remain unknown. Auxiliary β1 have been implicated in low-frequency tuning at the cochlear apex because these subunits dramatically slow channel kinetics. Tamoxifen (Tx), a (xeno)estrogen compound known to activate BK channels through the β-subunit, was used to test for the functional presence of β1. The hypotheses were that Tx would activate the majority of BK channels in hair cells from the cochlear apex due to the presence of β1 and that the level of activation would exhibit a tonotopic gradient following the expression profile of β1. Outside-out patches of BK channels were excised from tall hair cells along the apical half of the chicken basilar papilla. In low-density patches, single-channel conductance was reduced and the averaged open probability was unaffected by Tx. In high-density patches, the amplitude of ensemble-averaged BK current was inhibited, whereas half-activation potential and activation kinetics were unaffected by Tx. In both cases, no tonotopic Tx-dependent activation of channel activity was observed. Therefore, contrary to the hypotheses, electrophysiological assessment suggests that molecular mechanisms other than auxiliary β-subunits are involved in generating a tonotopic distribution of BK channel kinetics and electric tuning in chick basilar papilla.

scholarly journals Bidirectional control of BK channel open probability by CAMKII and PKC in medial vestibular nucleus neurons

2011 ◽  
Vol 105 (4) ◽  
pp. 1651-1659 ◽  
Author(s):  
Ingrid van Welie ◽  
Sascha du Lac
Keyword(s):  
Single Channel ◽  
Vestibular Nucleus ◽  
Critical Role ◽  
Bk Channels ◽  
Bk Channel ◽  
Medial Vestibular Nucleus ◽  
Intrinsic Excitability ◽  
Action Potential Firing ◽  
Open Probability ◽  
Single Channel Currents

Large conductance K+ (BK) channels are a key determinant of neuronal excitability. Medial vestibular nucleus (MVN) neurons regulate eye movements to ensure image stabilization during head movement, and changes in their intrinsic excitability may play a critical role in plasticity of the vestibulo-ocular reflex. Plasticity of intrinsic excitability in MVN neurons is mediated by kinases, and BK channels influence excitability, but whether endogenous BK channels are directly modulated by kinases is unknown. Double somatic patch-clamp recordings from MVN neurons revealed large conductance potassium channel openings during spontaneous action potential firing. These channels displayed Ca2+ and voltage dependence in excised patches, identifying them as BK channels. Recording isolated single channel currents at physiological temperature revealed a novel kinase-mediated bidirectional control in the range of voltages over which BK channels are activated. Application of activated Ca2+/calmodulin-dependent kinase II (CAMKII) increased BK channel open probability by shifting the voltage activation range towards more hyperpolarized potentials. An opposite shift in BK channel open probability was revealed by inhibition of phosphatases and was occluded by blockade of protein kinase C (PKC), suggesting that active PKC associated with BK channel complexes in patches was responsible for this effect. Accordingly, direct activation of endogenous PKC by PMA induced a decrease in BK open probability. BK channel activity affects excitability in MVN neurons and bidirectional control of BK channels by CAMKII, and PKC suggests that cellular signaling cascades engaged during plasticity may dynamically control excitability by regulating BK channel open probability.

scholarly journals Highly Specific Alternative Splicing of Transcripts Encoding BK Channels in the Chicken's Cochlea Is a Minor Determinant of the Tonotopic Gradient

2010 ◽  
Vol 30 (14) ◽  
pp. 3646-3660 ◽  
Author(s):  
Soledad Miranda-Rottmann ◽  
Andrei S. Kozlov ◽  
A. J. Hudspeth
Keyword(s):  
Cellular Response ◽  
Single Channel ◽  
Electrical Potential ◽  
Sensory Epithelium ◽  
Bk Channels ◽  
Bk Channel ◽  
Kinetic Properties ◽  
Auditory Hair Cells ◽  
Specific Alternative ◽  
Single Channel Currents

ABSTRACT The frequency sensitivity of auditory hair cells in the inner ear varies with their longitudinal position in the sensory epithelium. Among the factors that determine the differential cellular response to sound is the resonance of a hair cell's transmembrane electrical potential, whose frequency correlates with the kinetic properties of the high-conductance Ca2+-activated K+ (BK) channels encoded by a Slo (kcnma1) gene. It has been proposed that the inclusion of specific alternative axons in the Slo transcripts along the cochlea underlies the gradient of BK-channel kinetics. By analyzing the complete sequences of chicken Slo gene (cSlo) cDNAs from the chicken's cochlea, we show that most transcripts lack alternative exons. Transcripts with more than one alternative exon constitute only 10% of the total. Although the fraction of transcripts containing alternative exons increases from the cochlear base to the apex, the combination of alternative exons is not regulated. There is also a clear increase in the expression of BK transcripts with long carboxyl termini toward the apex. When long and short BK transcripts are expressed in HEK-293 cells, the kinetics of single-channel currents differ only slightly, but they are substantially slowed when the channels are coexpressed with the auxiliary β subunit that occurs more widely at the apex. These results argue that the tonotopic gradient is not established by the selective inclusion of highly specific cSlo exons. Instead, a gradient in the expression of β subunits slows BK channels toward the low-frequency apex of the cochlea.

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 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 Voltage-dependent dynamics of the BK channel cytosolic gating ring are coupled to the membrane-embedded voltage sensor

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Pablo Miranda ◽  
Miguel Holmgren ◽  
Teresa Giraldez
Keyword(s):  
Conformational Changes ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Cation Binding ◽  
Voltage Sensitivity ◽  
Open Probability ◽  
Calcium Dependent ◽  
Transmembrane Voltage ◽  
Voltage Dependent

In humans, large conductance voltage- and calcium-dependent potassium (BK) channels are regulated allosterically by transmembrane voltage and intracellular Ca2+. Divalent cation binding sites reside within the gating ring formed by two Regulator of Conductance of Potassium (RCK) domains per subunit. Using patch-clamp fluorometry, we show that Ca2+ binding to the RCK1 domain triggers gating ring rearrangements that depend on transmembrane voltage. Because the gating ring is outside the electric field, this voltage sensitivity must originate from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of the voltage sensor, either by mutagenesis or regulation by auxiliary subunits, are paralleled by changes in the voltage dependence of the gating ring movements, whereas modifications of the relative open probability are not. These results strongly suggest that conformational changes of RCK1 domains are specifically coupled to the voltage sensor function during allosteric modulation of BK channels.

scholarly journals Coupling of Ca2+ and voltage activation in BK channels through the αB helix/voltage sensor interface

2020 ◽  
Author(s):  
Yanyan Geng ◽  
Zengqin Deng ◽  
Guohui Zhang ◽  
Gonzalo Budelli ◽  
Alice Butler ◽  
...  
Keyword(s):  
Modular Design ◽  
Cell Types ◽  
Cytoplasmic Domain ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Membrane Excitability ◽  
Gating Currents ◽  
Voltage Sensors ◽  
Voltage Activation

AbstractLarge conductance Ca2+ and voltage activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is comprised of a voltage sensor domain (VSD), a central pore gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We now explore mechanism by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the HCA model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high affinity Ca2+ sites transduce their effect, at least in part, through the αB helix. Mg2+ activation was also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to WT, without other notable differences in the CTD, indicating structural change from the mutation was confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+ binding and voltage depolarization to pore opening, and that shared Ca2+ and voltage transduction pathways involving the αB helix may be involved.SignificanceLarge conductance BK (Slo1) K+ channels are activated by voltage, Ca2+, and Mg2+ to modulate membrane excitability in neurons, muscle, and other cells. BK channels are of modular design, with pore-gate and voltage sensors as transmembrane domains and a large cytoplasmic domain CTD containing the Ca2+ sensors. Previous observations suggest that voltage and Ca2+ sensors interact, but less is known about this interaction and its involvement in the gating process. We show that a previously identified structural interface between the CTD and voltage sensors is required for effective activation by both voltage and Ca2+, suggesting that these processes may share common allosteric activation pathways. Such knowledge should help explain disease processes associated with BK channel dysfunction.

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