scholarly journals Intra- and Intersubunit Cooperativity in Activation of BK Channels by Ca2+

2006 ◽  
Vol 128 (4) ◽  
pp. 389-404 ◽  
Author(s):  
Xiang Qian ◽  
Xiaowei Niu ◽  
Karl L. Magleby
Keyword(s):  
Bk Channels ◽  
Open Probability ◽  
High Affinity ◽  
Cooperative Activation ◽  
Allosteric Model

The activation of BK channels by Ca2+ is highly cooperative, with small changes in intracellular Ca2+ concentration having large effects on open probability (Po). Here we examine the mechanism of cooperative activation of BK channels by Ca2+. Each of the four subunits of BK channels has a large intracellular COOH terminus with two different high-affinity Ca2+ sensors: an RCK1 sensor (D362/D367) located on the RCK1 (regulator of conductance of K+) domain and a Ca-bowl sensor located on or after the RCK2 domain. To determine interactions among these Ca2+ sensors, we examine channels with eight different configurations of functional high-affinity Ca2+ sensors on the four subunits. We find that the RCK1 sensor and Ca bowl contribute about equally to Ca2+ activation of the channel when there is only one high-affinity Ca2+ sensor per subunit. We also find that an RCK1 sensor and a Ca bowl on the same subunit are much more effective in increasing Po than when they are on different subunits, indicating positive intrasubunit cooperativity. If it is assumed that BK channels have a gating ring similar to MthK channels with alternating RCK1 and RCK2 domains and that the Ca2+ sensors act at the flexible (rather than fixed) interfaces between RCK domains, then a comparison of the distribution of Ca2+ sensors with the observed responses suggest that the interface between RCK1 and RCK2 domains on the same subunit is flexible. On this basis, intrasubunit cooperativity arises because two high-affinity Ca2+ sensors acting across a flexible interface are more effective in opening the channel than when acting at separate interfaces. An allosteric model incorporating intrasubunit cooperativity nested within intersubunit cooperativity could approximate the Po vs. Ca2+ response for eight possible subunit configurations of the high-affinity Ca2+ sensors as well as for three additional configurations from a previous study.

scholarly journals Effects of Multiple Metal Binding Sites on Calcium and Magnesium-dependent Activation of BK Channels

2005 ◽  
Vol 127 (1) ◽  
pp. 35-50 ◽  
Author(s):  
Lei Hu ◽  
Huanghe Yang ◽  
Jingyi Shi ◽  
Jianmin Cui
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Quantitative Estimation ◽  
Bk Channels ◽  
Channel Activation ◽  
Metal Binding Sites ◽  
High Affinity ◽  
A Site ◽  
Metal Sensing ◽  
Allosteric Model

BK channels are activated by physiological concentrations of intracellular Ca2+ and Mg2+ in a variety of cells. Previous studies have identified two sites important for high-affinity Ca2+ sensing between [Ca2+]i of 0.1–100 μM and a site important for Mg2+ sensing between [Mg2+]i of 0.1–10 mM. BK channels can be also activated by Ca2+ and Mg2+ at concentrations >10 mM so that the steady-state conductance and voltage (G-V) relation continuously shifts to more negative voltage ranges when [Mg2+]i increases from 0.1–100 mM. We demonstrate that a novel site is responsible for metal sensing at concentrations ≥10 mM, and all four sites affect channel activation independently. As a result, the contributions of these sites to channel activation are complex, depending on the combination of Ca2+ and Mg2+ concentrations. Here we examined the effects of each of these sites on Ca2+ and Mg2+-dependent activation and the data are consistent with the suggestion that these sites are responsible for metal binding. We provide an allosteric model for quantitative estimation of the contributions that each of these putative binding sites makes to channel activation at any [Ca2+]i and [Mg2+]i.

scholarly journals Mg2+ binding to open and closed states can activate BK channels provided that the voltage sensors are elevated

2011 ◽  
Vol 138 (6) ◽  
pp. 593-607 ◽  
Author(s):  
Ren-Shiang Chen ◽  
Yanyan Geng ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Closed Interval ◽  
Open Interval ◽  
Bk Channels ◽  
Voltage Sensor ◽  
Interval Duration ◽  
Open Probability ◽  
Voltage Sensors ◽  
High Affinity ◽  
The Mean

BK channels are activated by intracellular Ca2+ and Mg2+ as well as by depolarization. Such activation is possible because each of the four subunits has two high-affinity Ca2+ sites, one low-affinity Mg2+ site, and a voltage sensor. This study further investigates the mechanism of Mg2+ activation by using single-channel recording to determine separately the action of Mg2+ on the open and closed states of the channel. To limit Mg2+ action to the Mg2+ sites, the two high-affinity Ca2+ sites are disabled by mutation. When the voltage is stepped from negative holding potentials to +100 mV, we find that 10 mM Mg2+ decreases the mean closed latency to the first channel opening 2.1-fold, decreases the mean closed interval duration 8.7-fold, increases mean burst duration 10.1-fold, increases the number of openings per burst 4.4-fold, and increases mean open interval duration 2.3-fold. Hence, Mg2+ can bind to closed BK channels, increasing their opening rates, and to open BK channels, decreasing their closing rates. To explore the relationship between Mg2+ action and voltage sensor activation, we record single-channel activity in macropatches containing hundreds of channels. Open probability (Po) is dramatically increased by 10 mM Mg2+ when voltage sensors are activated with either depolarization or the mutation R210C. The increased Po arises from large decreases in mean closed interval durations and moderate increases in mean open interval durations. In contrast, 10 mM Mg2+ has no detectable effects on Po or interval durations when voltage sensors are deactivated with very negative potentials or the mutation R167E. These observations are consistent with a model in which Mg2+ can bind to and alter the gating of both closed and open states to increase Po, provided that one or more voltage sensors are activated.

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

2014 ◽  
Vol 306 (5) ◽  
pp. C460-C470 ◽  
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.

scholarly journals Interactions of a Reversible Ryanoid (21-Amino-9α-Hydroxy-Ryanodine) with Single Sheep Cardiac Ryanodine Receptor Channels

1998 ◽  
Vol 112 (1) ◽  
pp. 55-69 ◽  
Author(s):  
Bhavna Tanna ◽  
William Welch ◽  
Luc Ruest ◽  
John L. Sutko ◽  
Alan J. Williams
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Single Channel ◽  
Single Channels ◽  
Open Probability ◽  
Release Channel ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Channel Protein ◽  
High Affinity ◽  
Conductance State

The binding of ryanodine to a high affinity site on the sarcoplasmic reticulum Ca2+-release channel results in a dramatic alteration in both gating and ion handling; the channel enters a high open probability, reduced-conductance state. Once bound, ryanodine does not dissociate from its site within the time frame of a single channel experiment. In this report, we describe the interactions of a synthetic ryanoid, 21-amino-9α-hydroxy-ryanodine, with the high affinity ryanodine binding site on the sheep cardiac sarcoplasmic reticulum Ca2+-release channel. The interaction of 21-amino-9α-hydroxy-ryanodine with the channel induces the occurrence of a characteristic high open probability, reduced-conductance state; however, in contrast to ryanodine, the interaction of this ryanoid with the channel is reversible under steady state conditions, with dwell times in the modified state lasting seconds. By monitoring the reversible interaction of this ryanoid with single channels under voltage clamp conditions, we have established a number of novel features of the ryanoid binding reaction. (a) Modification of channel function occurs when a single molecule of ryanoid binds to the channel protein. (b) The ryanoid has access to its binding site only from the cytosolic side of the channel and the site is available only when the channel is open. (c) The interaction of 21-amino-9α-hydroxy-ryanodine with its binding site is influenced strongly by transmembrane voltage. We suggest that this voltage dependence is derived from a voltage-driven conformational alteration of the channel protein that changes the affinity of the binding site, rather than the translocation of the ryanoid into the voltage drop across the channel.

Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

2001 ◽  
Vol 281 (6) ◽  
pp. C1769-C1775 ◽  
Author(s):  
Guillermo J. Pérez ◽  
Adrian D. Bonev ◽  
Mark T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Laser Scanning ◽  
Cerebral Arteries ◽  
Bk Channels ◽  
Bk Channel ◽  
Hill Coefficient ◽  
Channel Activity ◽  
Acetoxymethyl Ester ◽  
Open Probability ◽  
Apparent Dissociation Constant

The goal of the present study was to test the hypothesis that local Ca2+ release events (Ca2+ sparks) deliver high local Ca2+concentration to activate nearby Ca2+-sensitive K+ (BK) channels in the cell membrane of arterial smooth muscle cells. Ca2+ sparks and BK channels were examined in isolated myocytes from rat cerebral arteries with laser scanning confocal microscopy and patch-clamp techniques. BK channels had an apparent dissociation constant for Ca2+ of 19 μM and a Hill coefficient of 2.9 at −40 mV. At near-physiological intracellular Ca2+ concentration ([Ca2+]i; 100 nM) and membrane potential (−40 mV), the open probability of a single BK channel was low (1.2 × 10−6). A Ca2+spark increased BK channel activity to 18. Assuming that 1–100% of the BK channels are activated by a single Ca2+ spark, BK channel activity increases 6 × 105-fold to 6 × 103-fold, which corresponds to ∼30 μM to 4 μM spark Ca2+ concentration. 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid acetoxymethyl ester caused the disappearance of all Ca2+sparks while leaving the transient BK currents unchanged. Our results support the idea that Ca2+ spark sites are in close proximity to the BK channels and that local [Ca2+]i reaches micromolar levels to activate BK channels.

Comparison of large-conductance Ca(2+)-activated K+ channels in artificial bilayer and patch-clamp experiments

1994 ◽  
Vol 266 (3) ◽  
pp. C601-C610 ◽  
Author(s):  
C. L. Kapicka ◽  
A. Carl ◽  
M. L. Hall ◽  
A. L. Percival ◽  
B. W. Frey ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Apparent Distance ◽  
Bk Channels ◽  
Open Probability ◽  
K Channels ◽  
Artificial Lipid Bilayers ◽  
Excised Patches ◽  
Lipid Environment ◽  
Artificial Bilayers ◽  
Channel Properties

We compared the gating, ion conduction, and pharmacology of large-conductance Ca(2+)-activated K+ channels (BK channels) from canine colon in artificial lipid bilayers and in excised patches. Both protocols identified 270-pS K(+)-selective channels activated by depolarization and Ca2+ (approximately 130-mV shift of half-activation voltage per 10-fold change in Ca2+) that were inhibited by extracellular tetraethylammonium (TEA) and charybdotoxin. These similarities suggest that the same BK channels are studied in the two techniques. However, we found three quantitative differences between channels in artificial bilayers and patches. 1) Channels in artificial bilayers required fivefold higher free Ca2+ or 80-mV stronger depolarization for activation. 2) The voltage dependence of TEA block was smaller for channels in artificial bilayers. The apparent distance across the membrane field for the TEA binding site was 0.031 for channels in artificial bilayers and 0.23 for channels in patches. 3) ATP (2 mM) decreased open probability (Po) of channels in artificial bilayers, whereas channels in patches were unaffected. Neither GTP nor UTP reduced Po of channels in artificial bilayers. It is possible that these differences may be due to a lack of molecular identity between the channels studied in the two protocols. Alternatively, they may be attributed to alterations in channel properties during reconstitution or to influences of the artificial lipid environment.

Fast BK-Type Channel Mediates the Ca2+-Activated K+ Current in Crayfish Muscle

1999 ◽  
Vol 82 (4) ◽  
pp. 1655-1661 ◽  
Author(s):  
Alfonso Araque ◽  
Washington Buño
Keyword(s):  
Electrical Activity ◽  
Single Channel ◽  
Muscle Fibers ◽  
Ionic Channels ◽  
Bk Channels ◽  
Intrinsic Properties ◽  
Open Probability ◽  
Crayfish Muscle ◽  
Fast Activation ◽  
Inside Out

The role of the Ca2+-activated K+ current ( I K(Ca)) in crayfish opener muscle fibers is functionally important because it regulates the graded electrical activity that is characteristic of these fibers. Using the cell-attached and inside-out configurations of the patch-clamp technique, we found three different classes of channels with properties that matched those expected of the three different ionic channels mediating the depolarization-activated macroscopic currents previously described (Ca2+, K+, and Ca2+-dependent K+ currents). We investigated the properties of the ionic channels mediating the extremely fast activating and persistent I K(Ca). These voltage- and Ca2+-activated channels had a mean single-channel conductance of ∼ 70 pS and showed a very fast activation. Both the single-channel open probability and the speed of activation increased with depolarization. Both parameters also increased in inside-out patches, i.e., in high Ca2+concentration. Intracellular loading with the Ca2+ chelator bis(2-aminophenoxy) ethane- N, N,N′,N′-tetraacetic acid gradually reduced and eventually prevented channel openings. The channels opened at very brief delays after the pulse depolarization onset (<5 ms), and the time-dependent open probability was constant during sustained depolarization (≤560 ms), matching both the extremely fast activation kinetics and the persistent nature of the macroscopic I K(Ca). However, the intrinsic properties of these single channels do not account for the partial apparent inactivation of the macroscopic I K(Ca), which probably reflects temporal Ca2+ variations in the whole muscle fiber. We conclude that the channels mediating I K(Ca) in crayfish muscle are voltage- and Ca2+-gated BK channels with relatively small conductance. The intrinsic properties of these channels allow them to act as precise Ca2+ sensors that supply the exact feedback current needed to control the graded electrical activity and therefore the contraction of opener muscle fibers.

scholarly journals Functional Coupling of the β1 Subunit to the Large Conductance Ca2+-Activated K+ Channel in the Absence of Ca2+

2000 ◽  
Vol 115 (6) ◽  
pp. 719-736 ◽  
Author(s):  
Crina M. Nimigean ◽  
Karl L. Magleby
Keyword(s):  
Fold Increase ◽  
Bk Channels ◽  
Burst Duration ◽  
K Channel ◽  
Functional Coupling ◽  
Open Probability ◽  
Α Subunit ◽  
Gating Kinetics ◽  
Leftward Shift ◽  
Wide Range

Coexpression of the β1 subunit with the α subunit (mSlo) of BK channels increases the apparent Ca2+ sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca2+ sensitivity requires Ca2+, by comparing the gating in 0 Ca2+i of BK channels composed of α subunits to those composed of α+β1 subunits. The β1 subunit increased burst duration ∼20-fold and the duration of gaps between bursts ∼3-fold, giving an ∼10-fold increase in open probability (Po) in 0 Ca2+i. The effect of the β1 subunit on increasing burst duration was little changed over a wide range of Po achieved by varying either Ca2+i or depolarization. The effect of the β1 subunit on increasing the durations of the gaps between bursts in 0 Ca2+i was preserved over a range of voltage, but was switched off as Ca2+i was increased into the activation range. The Ca2+-independent, β1 subunit-induced increase in burst duration accounted for 80% of the leftward shift in the Po vs. Ca2+i curve that reflects the increased Ca2+ sensitivity induced by the β1 subunit. The Ca2+-dependent effect of the β1 subunit on the gaps between bursts accounted for the remaining 20% of the leftward shift. Our observation that the major effects of the β1 subunit are independent of Ca2+i suggests that the β1 subunit mainly alters the energy barriers of Ca2+-independent transitions. The changes in gating induced by the β1 subunit differ from those induced by depolarization, as increasing Po by depolarization or by the β1 subunit gave different gating kinetics. The complex gating kinetics for both α and α+β1 channels in 0 Ca2+i arise from transitions among two to three open and three to five closed states and are inconsistent with Monod-Wyman-Changeux type models, which predict gating among only one open and one closed state in 0 Ca2+i.

scholarly journals Transduction of Voltage and Ca2+ Signals by Slo1 BK Channels

Physiology ◽  
2013 ◽  
Vol 28 (3) ◽  
pp. 172-189 ◽  
Author(s):  
T. Hoshi ◽  
A. Pantazis ◽  
R. Olcese
Keyword(s):  
Gate Voltage ◽  
Bk Channels ◽  
Activation Mechanism ◽  
Channel Activation ◽  
Regulatory Factors ◽  
Voltage Sensors ◽  
Voltage Gated ◽  
Intracellular Ca ◽  
Biophysical Mechanism ◽  
Allosteric Model

Large-conductance Ca2+- and voltage-gated K+ channels are activated by an increase in intracellular Ca2+ concentration and/or depolarization. The channel activation mechanism is well described by an allosteric model encompassing the gate, voltage sensors, and Ca2+ sensors, and the model is an excellent framework to understand the influences of auxiliary β and γ subunits and regulatory factors such as Mg2+. Recent advances permit elucidation of structural correlates of the biophysical mechanism.

scholarly journals G protein pathway suppressor 2 enhanced the renal large-conductance Ca2+-activated potassium channel expression via inhibiting ERK1/2 signaling pathway

2018 ◽  
Vol 315 (3) ◽  
pp. F503-F511 ◽  
Author(s):  
Zhizhi Zhuang ◽  
Jia Xiao ◽  
Xinxin Chen ◽  
Xiaohan Hu ◽  
Ruidian Li ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
G Protein ◽  
Mapk Signaling ◽  
Bk Channels ◽  
Bk Channel ◽  
Mapk Signaling Pathway ◽  
Dependent Manner ◽  
Channel Activity ◽  
Open Probability

G protein pathway suppressor 2 (GPS2) is a multifunctional protein and transcriptional regulation factor that is involved in the G protein MAPK signaling pathway. It has been shown that the MAPK signaling pathway plays an important role in the regulation of renal large-conductance Ca2+-activated potassium (BK) channels. In this study, we investigated the effects of GPS2 on BK channel activity and protein expression. In human embryonic kidney (HEK) BK stably expressing cells transfected with either GPS2 or its vector control, a single-cell recording showed that GPS2 significantly increased BK channel activity ( NPo), increasing BK open probability ( Po), and channel number ( N) compared with the control. In Cos-7 cells and HEK 293 T cells, GPS2 overexpression significantly enhanced the total protein expression of BK in a dose-dependent manner. Knockdown of GPS2 expression significantly decreased BK protein expression, while increasing ERK1/2 phosphorylation. Knockdown of ERK1/2 expression reversed the GPS2 siRNA-mediated inhibition of BK protein expression in Cos-7 cells. Pretreatments of Cos-7 cells with either the lysosomal inhibitor bafilomycin A1 or the proteasomal inhibitor MG132 partially reversed the inhibitory effects of GPS2 siRNA on BK protein expression. In addition, feeding a high-potassium diet significantly increased both GPS2 and BK protein abundance in mice. These data suggest that GPS2 enhances BK channel activity and its protein expression by reducing ERK1/2 signaling-mediated degradation of the channel.

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