Modes of Operation of the BKCa Channel β2 Subunit

The β2 subunit of the large conductance Ca2+- and voltage-activated K+ channel (BKCa) modulates a number of channel functions, such as the apparent Ca2+/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the β2 subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BKCa channels (hSlo), we have investigated the mechanisms of operation of the β2 subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with β2 subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the β2 subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BKCa channels coexpressed with the β2 subunit. The experimental observations are consistent with the view that the β2 subunit of BKCa channels facilitates channel activation by changing the voltage sensor equilibrium and that the β2-induced inactivation process does not follow a typical N-type mechanism.

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The β1 Subunit Enhances Oxidative Regulation of Large-Conductance Calcium-activated K+ Channels

The Journal of General Physiology ◽

10.1085/jgp.200409144 ◽

2004 ◽

Vol 124 (4) ◽

pp. 357-370 ◽

Cited By ~ 17

Author(s):

Lindsey Ciali Santarelli ◽

Jianguo Chen ◽

Stefan H. Heinemann ◽

Toshinori Hoshi

Keyword(s):

Bkca Channel ◽

Open Probability ◽

Bkca Channels ◽

Additional Increase ◽

Channel Activation ◽

Α Subunit ◽

Voltage Range ◽

Virtual Absence ◽

Methionine Residues ◽

Oxidative Regulation

Oxidative stress may alter the functions of many proteins including the Slo1 large conductance calcium-activated potassium channel (BKCa). Previous results demonstrated that in the virtual absence of Ca2+, the oxidant chloramine-T (Ch-T), without the involvement of cysteine oxidation, increases the open probability and slows the deactivation of BKCa channels formed by human Slo1 (hSlo1) α subunits alone. Because native BKCa channel complexes may include the auxiliary subunit β1, we investigated whether β1 influences the oxidative regulation of hSlo1. Oxidation by Ch-T with β1 present shifted the half-activation voltage much further in the hyperpolarizing direction (−75 mV) as compared with that with α alone (−30 mV). This shift was eliminated in the presence of high [Ca2+]i, but the increase in open probability in the virtual absence of Ca2+ remained significant at physiologically relevant voltages. Furthermore, the slowing of channel deactivation after oxidation was even more dramatic in the presence of β1. Oxidation of cysteine and methionine residues within β1 was not involved in these potentiated effects because expression of mutant β1 subunits lacking cysteine or methionine residues produced results similar to those with wild-type β1. Unlike the results with α alone, oxidation by Ch-T caused a significant acceleration of channel activation only when β1 was present. The β1 M177 mutation disrupted normal channel activation and prevented the Ch-T–induced acceleration of activation. Overall, the functional effects of oxidation of the hSlo1 pore-forming α subunit are greatly amplified by the presence of β1, which leads to the additional increase in channel open probability and the slowing of deactivation. Furthermore, M177 within β1 is a critical structural determinant of channel activation and oxidative sensitivity. Together, the oxidized BKCa channel complex with β1 has a considerable chance of being open within the physiological voltage range even at low [Ca2+]i.

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BKCa channel activation increases cardiac contractile recovery following hypothermic ischemia/reperfusion

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00818.2014 ◽

2015 ◽

Vol 309 (4) ◽

pp. H625-H633 ◽

Cited By ~ 7

Author(s):

Brenda Cordeiro ◽

Dmitry Terentyev ◽

Richard T. Clements

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Left Ventricular ◽

Bkca Channel ◽

Ros Generation ◽

H9c2 Cells ◽

Bkca Channels ◽

Channel Activation

Mitochondrial Ca2+-activated large-conductance K+ (BKCa) channels are thought to provide protection during ischemic insults in the heart. Rottlerin (mallotoxin) has been implicated as a potent BKCa activator. The purpose of this study was twofold: 1) to investigate the efficacy of BKCa channel activation as a cardioprotective strategy during ischemic cardioplegic arrest and reperfusion (CP/R) and 2) to assess the specificity of rottlerin for BKCa channels. Wild-type (WT) and BKCa knockout (KO) mice were subjected to an isolated heart model of ischemic CP/R. A mechanism of rottlerin-induced cardioprotection was also investigated using H9c2 cells subjected to in vitro CP/reoxygenation and assessed for mitochondrial membrane potential and reactive oxygen species (ROS) production. CP/R decreased left ventricular developed pressure, positive and negative first derivatives of left ventricular pressure, and coronary flow (CF) in WT mice. Rottlerin dose dependently increased the recovery of left ventricular function and CF to near baseline levels. BKCa KO hearts treated with or without 500 nM rottlerin were similar to WT CP hearts. H9c2 cells subjected to in vitro CP/R displayed reduced mitochondrial membrane potential and increased ROS generation, both of which were significantly normalized by rottlerin. We conclude that activation of BKCa channels rescues ischemic damage associated with CP/R, likely via effects on improved mitochondrial membrane potential and reduced ROS generation.

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Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

The Journal of General Physiology ◽

10.1085/jgp.201010453 ◽

2010 ◽

Vol 136 (3) ◽

pp. 283-291 ◽

Cited By ~ 51

Author(s):

Guiling Zhao ◽

Zachary P. Neeb ◽

M. Dennis Leo ◽

Judith Pachuau ◽

Adebowale Adebiyi ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Bkca Channel ◽

Ip3 Receptors ◽

Arterial Smooth Muscle ◽

Bkca Channels ◽

Channel Activation ◽

Arterial Smooth Muscle Cells

Plasma membrane large-conductance Ca2+-activated K+ (BKCa) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BKCa channel regulation by IP3 and IP3Rs in rat and mouse cerebral artery smooth muscle cells. IP3 activated BKCa channels both in intact cells and in excised inside-out membrane patches. IP3 caused concentration-dependent BKCa channel activation with an apparent dissociation constant (Kd) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca2+ concentration ([Ca2+]i; 10 µM). IP3 also caused a leftward-shift in BKCa channel apparent Ca2+ sensitivity and reduced the Kd for free [Ca2+]i from ∼20 to 12 µM, but did not alter the slope or maximal Po. BAPTA, a fast Ca2+ buffer, or an elevation in extracellular Ca2+ concentration did not alter IP3-induced BKCa channel activation. Heparin, an IP3R inhibitor, and a monoclonal type 1 IP3R (IP3R1) antibody blocked IP3-induced BKCa channel activation. Adenophostin A, an IP3R agonist, also activated BKCa channels. IP3 activated BKCa channels in inside-out patches from wild-type (IP3R1+/+) mouse arterial smooth muscle cells, but had no effect on BKCa channels of IP3R1-deficient (IP3R1−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP3R1 is located in close spatial proximity to BKCa α subunits. The IP3R1 monoclonal antibody coimmunoprecipitated IP3R1 and BKCa channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP3R1 activation elevates BKCa channel apparent Ca2+ sensitivity through local molecular coupling in arterial smooth muscle cells.

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Nitrovasodilators relax mesenteric microvessels by cGMP-induced stimulation of Ca-activated K channels

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1997.273.1.h76 ◽

1997 ◽

Vol 273 (1) ◽

pp. H76-H84 ◽

Cited By ~ 35

Author(s):

G. O. Carrier ◽

L. C. Fuchs ◽

A. P. Winecoff ◽

A. D. Giulumian ◽

R. E. White

Keyword(s):

Molecular Mechanisms ◽

Single Channel ◽

Peripheral Resistance ◽

K Channel ◽

Bkca Channel ◽

Bkca Channels ◽

K Channels ◽

Arterial Blood ◽

Rat Mesentery ◽

Stimulation Of

Nitric oxide (NO) released from endothelial cells or exogenous nitrates is a potent dilator of arterial smooth muscle; however, the molecular mechanisms mediating relaxation to NO in the microcirculation have not been characterized. The present study investigated the relaxant effect of nitrovasodilators on microvessels obtained from the rat mesentery and also employed whole cell and single-channel patch-clamp techniques to identify the molecular target of NO action in myocytes from these vessels. Both sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) relaxed phenylephrine-induced contractions by approximately 80% but were significantly less effective in relaxing contractions induced by 40 mM KCl. Relaxation to SNP was also inhibited by the K(+)-channel blocker tetraethylammonium or by inhibition of the activity of the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG). These results suggest that SNP stimulated K+ efflux by opening K+ channels via PKG-mediated phosphorylation. Perforated-patch experiments revealed that both SNP and SNAP increased outward currents in microvascular myocytes, and single-channel studies identified the high-conductance Ca(2+)- and voltage-activated K+ (BKCa) channel as the target of nitrovasodilator action. The effects of nitrovasodilators on BKCa channels were mimicked by cGMP and inhibited by blocking the activity of PKG. We conclude that stimulation of BKCa-channel activity via cGMP-dependent phosphorylation contributes to the vasodilatory effect of NO on microvessels and that a direct effect of NO on BKCa channels does not play a major role in this process. We propose that this mechanism is important for the therapeutic effect of nitrovasodilators on peripheral resistance and arterial blood pressure.

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Evidence that BKCa channel activation contributes to K+ channel opener induced relaxation of the porcine coronary artery

Naunyn-Schmiedeberg s Archives of Pharmacology ◽

10.1007/bf00176777 ◽

1995 ◽

Vol 352 (2) ◽

Cited By ~ 11

Author(s):

JosephL. Balwierczak ◽

ChristineM. Krulan ◽

HelenS. Kim ◽

Dominick DelGrande ◽

GeorgeB. Weiss ◽

...

Keyword(s):

Coronary Artery ◽

K Channel ◽

Bkca Channel ◽

Channel Activation ◽

Porcine Coronary Artery ◽

Channel Opener ◽

K Channel Opener

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The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating

The Journal of General Physiology ◽

10.1085/jgp.200509321 ◽

2005 ◽

Vol 126 (3) ◽

pp. 227-241 ◽

Cited By ~ 23

Author(s):

Gayathri Krishnamoorthy ◽

Jingyi Shi ◽

David Sept ◽

Jianmin Cui

Keyword(s):

Conformational Changes ◽

K Channel ◽

Bkca Channel ◽

Bkca Channels ◽

Allosteric Coupling ◽

Channel Opening ◽

Activation Gate ◽

Rck Domain ◽

Dual Activation ◽

Α Subunits

Large conductance, voltage- and Ca2+-activated K+ (BKCa) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four α subunits of BKCa may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BKCa gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BKCa activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BKCa channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.

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Similar Enhancement of BKCa Channel Function Despite Different Aerobic Exercise Frequency in Aging Cerebrovascular Myocytes

Physiological Research ◽

10.33549/physiolres.933111 ◽

2016 ◽

pp. 447-459 ◽

Cited By ~ 1

Author(s):

N. LI ◽

B. LIU ◽

S. XIANG ◽

L. SHI

Keyword(s):

Aerobic Exercise ◽

Aerobic Exercise Training ◽

Bkca Channel ◽

Voltage Sensitivity ◽

Open Probability ◽

Exercise Frequency ◽

Bkca Channels ◽

Functional Changes ◽

Young Rats ◽

Training Frequency

Aerobic exercise showed beneficial influence on cardiovascular systems in aging, and mechanisms underlying vascular adaption remain unclear. Large-conductance Ca2+-activated K+ (BKCa) channels play critical roles in regulating cellular excitability and vascular tone. This study determined the effects of aerobic exercise on aging-associated functional changes in BKCa channels in cerebrovascular myocytes, Male Wistar rats aged 20-22 months were randomly assigned to sedentary (O-SED), low training frequency (O-EXL), and high training frequency group (O-EXH). Young rats were used as control. Compared to young rats, whole-cell BKCa current was decreased, and amplitude of spontaneous transient outward currents were reduced. The open probability and Ca2+/voltage sensitivity of single BKCa channel were declined in O-SED, accompanied with a reduction of tamoxifen-induced BKCa activation; the mean open time of BKCa channels was shortened whereas close time was prolonged. Aerobic exercise training markedly alleviated the aging-associated decline independent of training frequency. Exercise three times rather than five times weekly may be a time and cost-saving training volume required to offer beneficial effects to offset the functional declines of BKCa during aging.

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Role of the β1 Subunit in Large-Conductance Ca2+-Activated K+ Channel Gating Energetics

The Journal of General Physiology ◽

10.1085/jgp.116.3.411 ◽

2000 ◽

Vol 116 (3) ◽

pp. 411-432 ◽

Cited By ~ 191

Author(s):

D.H. Cox ◽

R.W. Aldrich

Keyword(s):

Steady State ◽

Channel Gating ◽

K Channel ◽

Bkca Channel ◽

Voltage Sensitivity ◽

Specific Expression ◽

Α Subunit ◽

Gating Charge ◽

Channel Opening ◽

Wide Range

Over the past few years, it has become clear that an important mechanism by which large-conductance Ca2+-activated K+ channel (BKCa) activity is regulated is the tissue-specific expression of auxiliary β subunits. The first of these to be identified, β1, is expressed predominately in smooth muscle and causes dramatic effects, increasing the apparent affinity of the channel for Ca2+ 10-fold at 0 mV, and shifting the range of voltages over which the channel activates −80 mV at 9.1 μM Ca2+. With this study, we address the question: which aspects of BKCa gating are altered by β1 to bring about these effects: Ca2+ binding, voltage sensing, or the intrinsic energetics of channel opening? The approach we have taken is to express the β1 subunit together with the BKCa α subunit in Xenopus oocytes, and then to compare β1's steady state effects over a wide range of Ca2+ concentrations and membrane voltages to those predicted by allosteric models whose parameters have been altered to mimic changes in the aspects of gating listed above. The results of our analysis suggest that much of β1's steady state effects can be accounted for by a reduction in the intrinsic energy the channel must overcome to open and a decrease in its voltage sensitivity, with little change in the affinity of the channel for Ca2+ when it is either open or closed. Interestingly, however, the small changes in Ca2+ binding affinity suggested by our analysis (Kc 7.4 μM → 9.6 μM; Ko = 0.80 μM → 0.65 μM) do appear to be functionally important. We also show that β1 affects the mSlo conductance–voltage relation in the essential absence of Ca2+, shifting it +20 mV and reducing its apparent gating charge 38%, and we develop methods for distinguishing between alterations in Ca2+ binding and other aspects of BKCa channel gating that may be of general use.

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Effective Activation of BKCa Channels by QO-40 (5-(Chloromethyl)-3-(Naphthalen-1-yl)-2-(Trifluoromethyl)Pyrazolo [1,5-a]pyrimidin-7(4H)-one), Known to Be an Opener of KCNQ2/Q3 Channels

Pharmaceuticals ◽

10.3390/ph14050388 ◽

2021 ◽

Vol 14 (5) ◽

pp. 388

Author(s):

Wei-Ting Chang ◽

Sheng-Nan Wu

Keyword(s):

Single Channel ◽

Slow Component ◽

Ionic Channel ◽

Gh3 Cells ◽

Bkca Channel ◽

Bkca Channels ◽

Gating Charge ◽

Ramp Voltage ◽

K Current ◽

The Mean

QO-40 (5-(chloromethyl)-3-(naphthalene-1-yl)-2-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-7(4H)-one) is a novel and selective activator of KCNQ2/KCNQ3 K+ channels. However, it remains largely unknown whether this compound can modify any other type of plasmalemmal ionic channel. The effects of QO-40 on ion channels in pituitary GH3 lactotrophs were investigated in this study. QO-40 stimulated Ca2+-activated K+ current (IK(Ca)) with an EC50 value of 2.3 μM in these cells. QO-40-stimulated IK(Ca) was attenuated by the further addition of GAL-021 or paxilline but not by linopirdine or TRAM-34. In inside-out mode, this compound added to the intracellular leaflet of the detached patches stimulated large-conductance Ca2+-activated K+ (BKCa) channels with no change in single-channel conductance; however, there was a decrease in the slow component of the mean closed time of BKCa channels. The KD value required for the QO-40-mediated decrease in the slow component at the mean closure time was 1.96 μM. This compound shifted the steady-state activation curve of BKCa channels to a less positive voltage and decreased the gating charge of the channel. The application of QO-40 also increased the hysteretic strength of BKCa channels elicited by a long-lasting isosceles-triangular ramp voltage. In HEK293T cells expressing α-hSlo, QO-40 stimulated BKCa channel activity. Overall, these findings demonstrate that QO-40 can interact directly with the BKCa channel to increase the amplitude of IK(Ca) in GH3 cells.

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