scholarly journals Deletion of cytosolic gating ring decreases gate and voltage sensor coupling in BK channels

2017 ◽  
Vol 149 (3) ◽  
pp. 373-387 ◽  
Author(s):  
Guohui Zhang ◽  
Yanyan Geng ◽  
Yakang Jin ◽  
Jingyi Shi ◽  
Kelli McFarland ◽  
...  
Keyword(s):  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Voltage Sensitivity ◽  
Gating Currents ◽  
Voltage Sensors ◽  
Transmembrane Voltage ◽  
Wide Range ◽  
Activation Gate ◽  
Sensor Activation

Large conductance Ca2+-activated K+ channels (BK channels) gate open in response to both membrane voltage and intracellular Ca2+. The channel is formed by a central pore-gate domain (PGD), which spans the membrane, plus transmembrane voltage sensors and a cytoplasmic gating ring that acts as a Ca2+ sensor. How these voltage and Ca2+ sensors influence the common activation gate, and interact with each other, is unclear. A previous study showed that a BK channel core lacking the entire cytoplasmic gating ring (Core-MT) was devoid of Ca2+ activation but retained voltage sensitivity (Budelli et al. 2013. Proc. Natl. Acad. Sci. USA. http://dx.doi.org/10.1073/pnas.1313433110). In this study, we measure voltage sensor activation and pore opening in this Core-MT channel over a wide range of voltages. We record gating currents and find that voltage sensor activation in this truncated channel is similar to WT but that the coupling between voltage sensor activation and gating of the pore is reduced. These results suggest that the gating ring, in addition to being the Ca2+ sensor, enhances the effective coupling between voltage sensors and the PGD. We also find that removal of the gating ring alters modulation of the channels by the BK channel’s β1 and β2 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 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 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.

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

2002 ◽  
Vol 120 (3) ◽  
pp. 267-305 ◽  
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.

scholarly journals Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels

2015 ◽  
Vol 112 (15) ◽  
pp. 4809-4814 ◽  
Author(s):  
Karen Castillo ◽  
Gustavo F. Contreras ◽  
Amaury Pupo ◽  
Yolima P. Torres ◽  
Alan Neely ◽  
...  
Keyword(s):  
Bk Channels ◽  
Bk Channel ◽  
Tissue Type ◽  
Charge Movement ◽  
Structural Determinants ◽  
Gating Currents ◽  
Wide Range ◽  
Lysine Residues ◽  
Regulatory Effects ◽  
Cellular Types

Being activated by depolarizing voltages and increases in cytoplasmic Ca2+, voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.

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 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 Differential Effects of β1 and β2 Subunits on BK Channel Activity

2005 ◽  
Vol 125 (4) ◽  
pp. 395-411 ◽  
Author(s):  
Patricio Orio ◽  
Ramon Latorre
Keyword(s):  
Steady State ◽  
Calcium Binding ◽  
Voltage Dependence ◽  
Calcium Sensitivity ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensitivity ◽  
Channel Activation ◽  
Α Subunit ◽  
Voltage Sensors

High conductance, calcium- and voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (β) subunits. β1 and β2 subunits increase apparent channel calcium sensitivity. The β1 subunit also decreases the voltage sensitivity of the channel and the β2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the β1 and β2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage (Horrigan and Aldrich, 2002). In this study, we used a β2 subunit without its N-type inactivation domain (β2IR). The results indicate that the β2IR subunit, like the β1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the β1 subunit, the β2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied β subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the β1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both β subunits reduce the intrinsic equilibrium constant for channel opening (L0). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the β1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the α subunit is coexpressed with the β2IR subunit.

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.

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