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 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.

Mechanisms of Pi regulation of the skeletal muscle SR Ca2+ release channel

2000 ◽  
Vol 278 (3) ◽  
pp. C601-C611 ◽  
Author(s):  
Edward M. Balog ◽  
Bradley R. Fruen ◽  
Patricia K. Kane ◽  
Charles F. Louis
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Adenine Nucleotides ◽  
Muscle Fibers ◽  
Open Probability ◽  
Release Channel ◽  
High Concentrations ◽  
Channel Open Time ◽  
Stimulation Of

Inorganic phosphate (Pi) accumulates in the fibers of actively working muscle where it acts at various sites to modulate contraction. To characterize the role of Pi as a regulator of the sarcoplasmic reticulum (SR) calcium (Ca2+) release channel, we examined the action of Pi on purified SR Ca2+ release channels, isolated SR vesicles, and skinned skeletal muscle fibers. In single channel studies, addition of Pi to the cis chamber increased single channel open probability ( P o; 0.079 ± 0.020 in 0 Pi, 0.157 ± 0.034 in 20 mM Pi) by decreasing mean channel closed time; mean channel open times were unaffected. In contrast, the ATP analog, β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP), enhanced P o by increasing single channel open time and decreasing channel closed time. Pi stimulation of [3H]ryanodine binding by SR vesicles was similar at all concentrations of AMP-PCP, suggesting Pi and adenine nucleotides act via independent sites. In skinned muscle fibers, 40 mM Pi enhanced Ca2+-induced Ca2+ release, suggesting an in situ stimulation of the release channel by high concentrations of Pi. Our results support the hypothesis that Pi may be an important endogenous modulator of the skeletal muscle SR Ca2+ release channel under fatiguing conditions in vivo, acting via a mechanism distinct from adenine nucleotides.

scholarly journals Spermine Block of the Strong Inward Rectifier Potassium Channel Kir2.1

2002 ◽  
Vol 120 (1) ◽  
pp. 53-66 ◽  
Author(s):  
Lai-Hua Xie ◽  
Scott A. John ◽  
James N. Weiss
Keyword(s):  
Single Channel ◽  
Quantitative Model ◽  
Inward Rectification ◽  
Channel Blockers ◽  
Surface Charges ◽  
Open Probability ◽  
Dependent Component ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Inside Out

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg2+. Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.

Blockade of Adenosine Triphosphate–sensitive Potassium Channels by Thiamylal in Rat Ventricular Myocytes

2000 ◽  
Vol 92 (4) ◽  
pp. 1154-1159 ◽  
Author(s):  
Yasuo Tsutsumi ◽  
Shuzo Oshita ◽  
Hiroshi Kitahata ◽  
Yasuhiro Kuroda ◽  
Takashi Kawano ◽  
...  
Keyword(s):  
Adenosine Triphosphate ◽  
Katp Channel ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Katp Channels ◽  
Dependent Manner ◽  
Open Probability ◽  
Rat Ventricular Myocytes ◽  
Inside Out ◽  
Cell Attached Configuration

Background The adenosine triphosphate (ATP)-sensitive potassium (KATP) channels protect myocytes during ischemia and reperfusion. This study investigated the effects of thiamylal on the activities of KATP channels in isolated rat ventricular myocytes during simulated ischemia. Methods Male Wistar rats were anesthetized with ether. Single, quiescent ventricular myocytes were dispersed enzymatically. Membrane currents were recorded using patch-clamp techniques. In the cell-attached configuration, KATP channel currents were assessed before and during activation of these channels by 2,4-dinitrophenol and after administration of 25, 50, and 100 mg/l thiamylal. The open probability was determined from current-amplitude histograms. In the inside-out configuration, the current-voltage relation was obtained before and after the application of thiamylal (50 mg/1). Results In the cell-attached configuration, 2,4-dinitrophenol caused frequent channel opening. 2,4-Dinitrophenol-induced channel activities were reduced significantly by glibenclamide, suggesting that the channels studied were KATP channels. Open probability of KATP channels was reduced by thiamylal in a concentration-dependent manner. KATP channels could be activated in the inside-out configuration because of the absence of ATP. Thiamylal inhibited KATP channel activity without changing the single-channel conductance. Conclusions The results obtained in this study indicate that thiamylal inhibits KATP channel activities in cell-attached and inside-out patches, suggesting a direct action of this drug on these channels.

Quinidine blocks adenosine 5'-triphosphate-sensitive potassium channels in heart

1990 ◽  
Vol 259 (5) ◽  
pp. H1609-H1612 ◽  
Author(s):  
A. I. Undrovinas ◽  
N. Burnashev ◽  
D. Eroshenko ◽  
I. Fleidervish ◽  
C. F. Starmer ◽  
...  
Keyword(s):  
Single Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Pipette Solution ◽  
Channel Current ◽  
Closed Intervals ◽  
Ventricular Cells ◽  
Voltage Dependent ◽  
Inside Out ◽  
Ischemic Arrhythmias

The ATP-sensitive potassium channel current (IK-ATP) was studied in excised inside-out patches from rat ventricular cells at 20-23 degrees C. The bath solution contained 140 mM KF, and the pipette solution contained 140 mM KCl and 1.2 mM MgCl2. ATP (0.5 mM) in the bath inhibited IK-ATP. In the absence of ATP, 10 microM quinidine decreased open probability 67 +/- 1% (n = 6) at -50 mV and 28 +/- 12% at -130 mV (n = 5) without affecting single channel conductance (48-52 pS). The block increased with 25 and 50 microM quinidine and could be reversed on washing quinidine for several minutes. Interburst (closed) intervals were increased by quinidine, whereas open and closed time distributions within bursts were not changed. We conclude that quinidine blocks IK-ATP in a "slow" and voltage-dependent manner in clinically relevant concentrations. Because of the postulated role for IK-ATP in cardiac ischemia, quinidine block of this channel may play a role in ischemic arrhythmias.

IP3-activated Ca2+ channels in the plasma membrane of cultured vascular endothelial cells

1995 ◽  
Vol 269 (3) ◽  
pp. C733-C738 ◽  
Author(s):  
L. Vaca ◽  
D. L. Kunze
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Single Channel ◽  
Vascular Endothelial Cells ◽  
Channel Activity ◽  
Open Probability ◽  
Aortic Endothelial Cells ◽  
Control Value ◽  
Selective Channel ◽  
Inside Out

Although it is clear that D-myo-inositol 1,4,5-trisphosphate (IP3) plays an important role in the activation of Ca2+ influx, the mechanisms by which this occurs remain controversial. In an attempt to determine the role of IP3 in the activation of Ca2+ influx, patch-clamp single-channel experiments in the cell-attached, inside-out, and outside-out configurations were performed on cultured bovine aortic endothelial cells (BAEC). The results presented indicate that both IP3 and intracellular Ca2+ can modulate the activity of a Ca(2+)-selective channel found in the plasma membrane of these cells. Addition of 10 microM IP3 increased channel open probability (P(o)) from a control value of 0.12 +/- 0.05 to 0.7 +/- 0.13 at a constant intracellular Ca2+ of 1 nM in excised inside-out patches. D-Myo-inositol 1,3,4,5-tetrakisphosphate at 50 microM was ineffective in altering channel P(o). Channel activity declined after approximately 2 min in the continuous presence of IP3. Three to four minutes after addition of IP3, channel P(o) was reduced from 0.7 +/- 0.2 to 0.2 +/- 0.1, indicating that an additional regulator might be required to maintain channel activity in excised patches. The channel was reversibly blocked by application of 1 microgram/ml heparin to the intracellular side of inside-out patches. This Ca(2+)-selective channel is indistinguishable from the depletion-activated Ca2+ channel we have previously described in BAEC.

Basolateral outward rectifier chloride channel in isolated crypts of mouse colon

2000 ◽  
Vol 279 (2) ◽  
pp. G277-G287 ◽  
Author(s):  
Olivier Mignen ◽  
Stéphane Egee ◽  
Martine Liberge ◽  
Brian J. Harvey
Keyword(s):  
Protein Kinase ◽  
Chloride Channels ◽  
Single Channel ◽  
Basolateral Membrane ◽  
Distal Colon ◽  
Open Probability ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Inside Out

Single channel patch-clamp techniques were used to demonstrate the presence of outwardly rectifying chloride channels in the basolateral membrane of crypt cells from mouse distal colon. These channels were rarely observed in the cell-attached mode and, in the inside-out configuration, only became active after a delay and depolarizing voltage steps. Single channel conductance was 23.4 pS between −100 and −40 mV and increased to 90.2 pS between 40 and 100 mV. The channel permeability sequence for anions was: I− > SCN− > Br−> Cl− > NO3 − > F−≫ SO4 2− ≈ gluconate. In inside-out patches, the channel open probability was voltage dependent but insensitive to intracellular Ca2+ concentration. In cell-attached mode, forskolin, histamine, carbachol, A-23187, and activators of protein kinase C all failed to activate the channel, and activity could not be evoked in inside-out patches by exposure to the purified catalytic subunit of cAMP-dependent protein kinase A. The channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, 9-anthracenecarboxylic acid, and DIDS. Stimulation of G proteins with guanosine 5′- O-(3-thiotriphosphate) decreased the channel open probability and conductance, whereas subsequent addition of guanosine 5′- O-(2-thiodiphosphate) reactivated the channel.

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 Basic properties and potential regulators of the apical K+ channel in macula densa cells.

1994 ◽  
Vol 103 (6) ◽  
pp. 1055-1070 ◽  
Author(s):  
A M Hurst ◽  
J Y Lapointe ◽  
A Laamarti ◽  
P D Bell
Keyword(s):  
Single Channel ◽  
Signal Transmission ◽  
Macula Densa ◽  
Tubuloglomerular Feedback ◽  
Rabbit Kidney ◽  
K Channel ◽  
Feedback Signal ◽  
Open Probability ◽  
Patch Pipette ◽  
Inside Out

These studies examine the properties of an apical potassium (K+) channel in macula densa cells, a specialized group of cells involved in tubuloglomerular feedback signal transmission. To this end, individual glomeruli with thick ascending limbs (TAL) and macula densa cells were dissected from rabbit kidney and the TAL covering macula densa cells was removed. Using patch clamp techniques, we found a high density (up to 54 channels per patch) of K+ channels in the apical membrane of macula densa cells. An inward conductance of 41.1 +/- 4.8 pS was obtained in cell-attached patches (patch pipette, 140 mM K+). In inside-out patches (patch pipette, 140 mM; bath, 5 mM K+), inward currents of 1.1 +/- 0.1 pA (n = 11) were observed at 0 mV and single channel current reversed at a pipette potential of -84 mV giving a permeability ratio (PK/PNa) of over 100. In cell-attached patches, mean channel open probability (N,Po, where N is number of channels in the patch and Po is single channel open probability) was unaffected by bumetanide, but was reduced from 11.3 +/- 2.7 to 1.6 +/- 1.3 (n = 5, p &lt; 0.02) by removal of bath sodium (Na+). Simultaneous removal of bath Na+ and calcium (Ca2+) prevented the Na(+)-induced decrease in N.Po indicating that the effect of Na+ removal on N.Po was probably mediated by stimulation of Ca2+ entry. This interpretation was supported by studies where ionomycin, which directly increases intracellular Ca2+, produced a fall in N.Po from 17.8 +/- 4.0 to 5.9 +/- 4.1 (n = 7, p &lt; 0.02). In inside-out patches, the apical K+ channel was not sensitive to ATP but was directly blocked by 2 mM Ca2+ and by lowering bath pH from 7.4 to 6.8. These studies constitute the first single channel observations on macula densa cells and establish some of the characteristics and regulators of this apical K+ channel. This channel is likely to be involved in macula densa transepithelial Cl- transport and perhaps in the tubuloglomerular feedback signaling process.

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.

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