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.

Peroxynitrite reversibly inhibits Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

2000 ◽  
Vol 278 (6) ◽  
pp. H1883-H1890 ◽  
Author(s):  
Anna K. Brzezinska ◽  
Debebe Gebremedhin ◽  
William M. Chilian ◽  
Balaraman Kalyanaraman ◽  
Stephen J. Elliott
Keyword(s):  
Single Channel ◽  
Cerebral Arteries ◽  
Ionic Current ◽  
Inhibitory Effect ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Cell Current

Peroxynitrite (ONOO−) is a contractile agonist of rat middle cerebral arteries. To determine the mechanism responsible for this component of ONOO−bioactivity, the present study examined the effect of ONOO− on ionic current and channel activity in rat cerebral arteries. Whole cell recordings of voltage-clamped cells were made under conditions designed to optimize K+ current. The effects of iberiotoxin, a selective inhibitor of large-conductance Ca2+-activated K+ (BK) channels, and ONOO− (10–100 μM) were determined. At a pipette potential of +50 mV, ONOO− inhibited 39% of iberiotoxin-sensitive current. ONOO− was selective for iberiotoxin-sensitive current, whereas decomposed ONOO− had no effect. In excised, inside-out membrane patches, channel activity was recorded using symmetrical K+solutions. Unitary currents were sensitive to increases in internal Ca2+ concentration, consistent with activity due to BK channels. Internal ONOO− dose dependently inhibited channel activity by decreasing open probability and mean open times. The inhibitory effect of ONOO− could be overcome by reduced glutathione. Glutathione, added after ONOO−, restored whole cell current amplitude to control levels and reverted single-channel gating to control behavior. The inhibitory effect of ONOO− on membrane K+ current is consistent with its contractile effects in isolated cerebral arteries and single myocytes. Taken together, our data suggest that ONOO− has the potential to alter cerebral vascular tone by inhibiting BK channel activity.

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 The β Subunit Increases the Ca2+ Sensitivity of Large Conductance Ca2+-activated Potassium Channels by Retaining the Gating in the Bursting States

1999 ◽  
Vol 113 (3) ◽  
pp. 425-440 ◽  
Author(s):  
Crina M. Nimigean ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Β Subunit ◽  
Open Probability ◽  
Α Subunit ◽  
Closed Intervals ◽  
Single Channel Analysis ◽  
Channel Analysis ◽  
The Mean

Coexpression of the β subunit (KV,Caβ) with the α subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the β subunit increased open probability (Po) by increasing burst duration 20–100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the β subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the β subunit does not act by increasing all the Ca2+ binding rates proportionally. The β subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the β subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the β subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.

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 Testosterone decreases urinary bladder smooth muscle excitability via novel signaling mechanism involving direct activation of the BK channels

2016 ◽  
Vol 311 (6) ◽  
pp. F1253-F1259 ◽  
Author(s):  
Kiril L. Hristov ◽  
Shankar P. Parajuli ◽  
Aaron Provence ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Bladder Pressure ◽  
Resting Membrane Potential ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
Inside Out

In addition to improving sexual function, testosterone has been reported to have beneficial effects in ameliorating lower urinary tract symptoms by increasing bladder capacity and compliance, while decreasing bladder pressure. However, the cellular mechanisms by which testosterone regulates detrusor smooth muscle (DSM) excitability have not been elucidated. Here, we used amphotericin-B perforated whole cell patch-clamp and single channel recordings on inside-out excised membrane patches to investigate the regulatory role of testosterone in guinea pig DSM excitability. Testosterone (100 nM) significantly increased the depolarization-induced whole cell outward currents in DSM cells. The selective pharmacological inhibition of the large-conductance voltage- and Ca2+-activated K+ (BK) channels with paxilline (1 μM) completely abolished this stimulatory effect of testosterone, suggesting a mechanism involving BK channels. At a holding potential of −20 mV, DSM cells exhibited transient BK currents (TBKCs). Testosterone (100 nM) significantly increased TBKC activity in DSM cells. In current-clamp mode, testosterone (100 nM) significantly hyperpolarized the DSM cell resting membrane potential and increased spontaneous transient hyperpolarizations. Testosterone (100 nM) rapidly increased the single BK channel open probability in inside-out excised membrane patches from DSM cells, clearly suggesting a direct BK channel activation via a nongenomic mechanism. Live-cell Ca2+ imaging showed that testosterone (100 nM) caused a decrease in global intracellular Ca2+ concentration, consistent with testosterone-induced membrane hyperpolarization. In conclusion, the data provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in DSM cells, independent from genomic testosterone receptors, and thus regulates DSM excitability.

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 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 State-dependent inhibition of BK channels by the opioid agonist loperamide

2021 ◽  
Vol 153 (9) ◽  
Author(s):  
Alexandre G. Vouga ◽  
Michael E. Rockman ◽  
Jiusheng Yan ◽  
Marlene A. Jacobson ◽  
Brad S. Rothberg
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Open Probability ◽  
Opioid Agonist ◽  
State Dependent ◽  
Dependent Inhibition ◽  
Cytosolic Side ◽  
Relative Stabilization ◽  
Α Subunits

Large-conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K+ efflux through intestinal BK channels.

scholarly journals Gating and Conductance Properties of Bk Channels Are Modulated by the S9–S10 Tail Domain of the α Subunit

2001 ◽  
Vol 118 (6) ◽  
pp. 711-734 ◽  
Author(s):  
Brenda L. Moss ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Burst Duration ◽  
Tail Domain ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Time ◽  
The Mean ◽  
Single Channel Currents

The COOH-terminal S9–S10 tail domain of large conductance Ca2+-activated K+ (BK) channels is a major determinant of Ca2+ sensitivity (Schreiber, M., A. Wei, A. Yuan, J. Gaut, M. Saito, and L. Salkoff. 1999. Nat. Neurosci. 2:416–421). To investigate whether the tail domain also modulates Ca2+-independent properties of BK channels, we explored the functional differences between the BK channel mSlo1 and another member of the Slo family, mSlo3 (Schreiber, M., A. Yuan, and L. Salkoff. 1998. J. Biol. Chem. 273:3509–3516). Compared with mSlo1 channels, mSlo3 channels showed little Ca2+ sensitivity, and the mean open time, burst duration, gaps between bursts, and single-channel conductance of mSlo3 channels were only 32, 22, 41, and 37% of that for mSlo1 channels, respectively. To examine which channel properties arise from the tail domain, we coexpressed the core of mSlo1 with either the tail domain of mSlo1 or the tail domain of mSlo3 channels, and studied the single-channel currents. Replacing the mSlo1 tail with the mSlo3 tail resulted in the following: increased open probability in the absence of Ca2+; reduced the Ca2+ sensitivity greatly by allowing only partial activation by Ca2+ and by reducing the Hill coefficient for Ca2+ activation; decreased the voltage dependence ∼28%; decreased the mean open time two- to threefold; decreased the mean burst duration three- to ninefold; decreased the single-channel conductance ∼14%; decreased the Kd for block by TEAi ∼30%; did not change the minimal numbers of three to four open and five to seven closed states entered during gating; and did not change the major features of the dependency between adjacent interval durations. These observations support a modular construction of the BK channel in which the tail domain modulates the gating kinetics and conductance properties of the voltage-dependent core domain, in addition to determining most of the high affinity Ca2+ sensitivity.

scholarly journals Gating Kinetics of Single Large-Conductance Ca2+-Activated K+ Channels in High Ca2+ Suggest a Two-Tiered Allosteric Gating Mechanism✪

1999 ◽  
Vol 114 (1) ◽  
pp. 93-124 ◽  
Author(s):  
Brad S. Rothberg ◽  
Karl L. Magleby
Keyword(s):  
Dwell Time ◽  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Hill Coefficient ◽  
Channel Kinetics ◽  
Open Probability ◽  
Kinetic Schemes ◽  
Gating Mechanism ◽  
Open States

The Ca2+-dependent gating mechanism of large-conductance calcium-activated K+ (BK) channels from cultured rat skeletal muscle was examined from low (4 μM) to high (1,024 μM) intracellular concentrations of calcium (Ca2+i) using single-channel recording. Open probability (Po) increased with increasing Ca2+i (K0.5 11.2 ± 0.3 μM at +30 mV, Hill coefficient of 3.5 ± 0.3), reaching a maximum of ∼0.97 for Ca2+i ∼ 100 μM. Increasing Ca2+i further to 1,024 μM had little additional effect on either Po or the single-channel kinetics. The channels gated among at least three to four open and four to five closed states at high levels of Ca2+i (>100 μM), compared with three to four open and five to seven closed states at lower Ca2+i. The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of two-dimensional (2-D) dwell-time distributions obtained from low to high Ca2+i. Kinetic schemes drawn from the 10-state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca2+i. Kinetic schemes drawn from Eigen's general model for a ligand-activated tetrameric protein could approximate the dwell-time distributions but not the dependency (correlations) between adjacent intervals at high Ca2+i. However, models drawn from a general 50 state two-tiered scheme, in which there were 25 closed states on the upper tier and 25 open states on the lower tier, could approximate both the dwell-time distributions and the dependency from low to high Ca2+i. In the two-tiered model, the BK channel can open directly from each closed state, and a minimum of five open and five closed states are available for gating at any given Ca2+i. A model that assumed that the apparent Ca2+-binding steps can reach a maximum rate at high Ca2+i could also approximate the gating from low to high Ca2+i. The considered models can serve as working hypotheses for the gating of BK channels.

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