Carbachol modulates voltage sensitivity of calcium channels in bronchial smooth muscle of rats

1992 ◽  
Vol 263 (1) ◽  
pp. C69-C77 ◽  
Author(s):  
T. Kamishima ◽  
M. T. Nelson ◽  
J. B. Patlak
Keyword(s):  
Smooth Muscle ◽  
Channel Blocker ◽  
Single Channel ◽  
Membrane Depolarization ◽  
Bay K 8644 ◽  
Voltage Sensitivity ◽  
Ca Channel ◽  
Ca Channels ◽  
Open Probability ◽  
Voltage Dependent

The role of voltage-dependent Ca channels in carbachol (CCh)-induced contraction of rat bronchus was investigated. Membrane depolarization and BAY K 8644, a Ca channel opener, significantly enhanced CCh-induced contractions. Nisoldipine, an organic Ca channel blocker, significantly inhibited the contractions. Cadmium, an inorganic Ca channel blocker, completely inhibited maintained contractions caused by CCh. These results suggested that the voltage-dependent Ca channels play an important role in sustained cholinergic contractions. This hypothesis was tested further by investigating the properties of single Ca channels of rat bronchus smooth muscle cells. We used 10 mM Ba as the charge carrier and BAY K 8644 to increase open times. The single-channel conductance was 16.8 pS. Steady-state open probability (NP(o)) increased steeply with membrane depolarization (e-fold for 4 mV). The primary effect of CCh (10 microM) on Ca channels was to shift the membrane potential at which NP(o) was half maximal from -34 to -43 mV without changing the steepness factor or maximal NP(o). This CCh-induced increase in NP(o) was not caused by depolarization, because the single-channel current amplitude was unchanged by CCh. We conclude that one of the mechanisms by which CCh opens Ca channels of rat bronchus smooth muscle is by shifting the activation curve in the hyperpolarized direction.

Single calcium channel currents of arterial smooth muscle at physiological calcium concentrations

1992 ◽  
Vol 263 (5) ◽  
pp. C948-C952 ◽  
Author(s):  
M. Gollasch ◽  
J. Hescheler ◽  
J. M. Quayle ◽  
J. B. Patlak ◽  
M. T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Single Channel ◽  
Membrane Potentials ◽  
Ca Channel ◽  
Ca Channels ◽  
Arterial Smooth Muscle ◽  
Voltage Dependent ◽  
High Concentrations ◽  
Single Channel Currents ◽  
Channel Currents

Entry of Ca through voltage-dependent Ca channels is an important regulator of the function of smooth muscle, cardiac muscle, and neurons. Although Ca channels have been extensively studied since the first descriptions of Ca action potentials (P. Fatt and B. Katz. J. Physiol. Lond. 120: 171-204, 1953), the permeation rate of Ca through single Ca channels has not been measured directly under physiological conditions. Instead, single Ca channels have typically been examined using high concentrations (80-110 mM) of another divalent charge carrier, Ba, so as to maximize the amplitude of the single-channel currents. Calculations of unitary currents at 2 mM Ca indicated that the single-channel currents would be immeasurably small (i.e., < 0.1 pA). We provide here the first direct measurements of single Ca channel currents at a physiological Ca concentration. Contrary to earlier estimates, we have found that currents through single Ca channels in arterial smooth muscle are 0.1-0.3 pA at 2 mM Ca and physiological membrane potentials. These relatively large unitary currents permit direct measurement of Ca channel properties under conditions that do not distort their function. Our data also indicate that Ca permeates these channels at relatively high rates in physiological Ca concentrations and membrane potentials.

Differential responses of Ca-activated K channels to bradykinin in sensory neurons and F-11 cells

1992 ◽  
Vol 262 (2) ◽  
pp. C453-C460 ◽  
Author(s):  
K. Naruse ◽  
D. S. McGehee ◽  
G. S. Oxford
Keyword(s):  
Intracellular Signaling ◽  
Single Channel ◽  
Outward Current ◽  
Transient Outward Current ◽  
Ca Channel ◽  
Ca Channels ◽  
Drg Neurons ◽  
Open Probability ◽  
K Channels ◽  
Drg Neuron

The nonapeptide bradykinin (BK) excites a subset of dorsal root ganglion (DRG) neurons with putative nociceptive functions by stimulating an inward cation current. In addition, BK stimulates various intracellular signaling pathways including an elevation of intracellular Ca2+. In a DRG neuron x neuroblastoma hybrid cell (F-11), BK stimulates similar increases in intracellular [Ca2+] and inward current but also elicits a large transient outward current through Ca(2+)-activated K channels. We have investigated the mechanisms underlying differential expression of outward current responses in the two cell types at the single channel level. Although K(Ca) channel activity appears in inside-out patches from both cells exposed to Ca2+, BK applied to the extrapatch membrane of cell-attached patches activates K(Ca) channels in F-11 but not DRG neurons. Whereas single K(Ca) channels are quantitatively similar in terms of conductance, voltage-dependence, and sensitivity to tetraethylammonium, they differ in sensitivity to intracellular Ca2+. Channel activation in both cells requires at least four Ca2+ ions, but half-maximal activation occurs at slightly higher [Ca2+] for DRG neurons. The shift in the Ca2+ dose-response curve combined with the steep [Ca2+] dependence of channel open probability makes it less likely that a BK-induced rise in internal [Ca2+] induced will trigger a transient outward current and resultant hyperpolarization in a DRG neuron.

scholarly journals Cardiac calcium channels in planar lipid bilayers. L-type channels and calcium-permeable channels open at negative membrane potentials.

1988 ◽  
Vol 92 (1) ◽  
pp. 27-54 ◽  
Author(s):  
R L Rosenberg ◽  
P Hess ◽  
R W Tsien
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Detailed Comparison ◽  
Membrane Potentials ◽  
Bay K 8644 ◽  
Ca Channel ◽  
Ca Channels ◽  
Channel Activity ◽  
Intact Cells ◽  
Free System

Planar lipid bilayer recordings were used to study Ca channels from bovine cardiac sarcolemmal membranes. Ca channel activity was recorded in the absence of nucleotides or soluble enzymes, over a range of membrane potentials and ionic conditions that cannot be achieved in intact cells. The dihydropyridine-sensitive L-type Ca channel, studied in the presence of Bay K 8644, was identified by a detailed comparison of its properties in artificial membranes and in intact cells. L-type Ca channels in bilayers showed voltage dependence of channel activation and inactivation, open and closed times, and single-channel conductances in Ba2+ and Ca2+ very similar to those found in cell-attached patch recordings. Open channels were blocked by micromolar concentrations of external Cd2+. In this cell-free system, channel activity tended to decrease during the course of an experiment, reminiscent of Ca2+ channel "rundown" in whole-cell and excised-patch recordings. A purely voltage-dependent component of inactivation was observed in the absence of Ca2+ stores or changes in intracellular Ca2+. Millimolar internal Ca2+ reduced unitary Ba2+ influx but did not greatly increase the rate or extent of inactivation or the rate of channel rundown. In symmetrical Ba2+ solutions, unitary conductance saturated as the Ba2+ concentration was increased up to 500 mM. The bilayer recordings also revealed activity of a novel Ca2+-permeable channel, termed "B-type" because it may contribute a steady background current at negative membrane potentials, which is distinct from L-type or T-type Ca channels previously reported. Unlike L-type channels, B-type channels have a small unitary Ba2+ conductance (7 pS), but do not discriminate between Ba2+ and Ca2+, show no obvious sensitivity to Bay K 8644, and do not run down. Unlike either L- or T-type channels, B-type channels did not require a depolarization for activation and displayed mean open times of greater than 100 ms.

scholarly journals Hydrogen Sulfide Relaxes Human Uterine Artery via Activating Smooth Muscle BKCa Channels

Antioxidants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1127
Author(s):  
Yan Li ◽  
Jin Bai ◽  
Yi-hua Yang ◽  
Naoto Hoshi ◽  
Dong-bao Chen
Keyword(s):  
Smooth Muscle ◽  
Hydrogen Sulfide ◽  
Uterine Artery ◽  
Plasma Membranes ◽  
Single Channel ◽  
Patch Clamps ◽  
Open Probability ◽  
Bkca Channels ◽  
Voltage Dependent

Opening of large conductance calcium-activated and voltage-dependent potassium (BKCa) channels hyperpolarizes plasma membranes of smooth muscle (SM) to cause vasodilation, underling a key mechanism for mediating uterine artery (UA) dilation in pregnancy. Hydrogen sulfide (H2S) has been recently identified as a new UA vasodilator, yet the mechanism underlying H2S-induced UA dilation is unknown. Here, we tested whether H2S activated BKCa channels in human UA smooth muscle cells (hUASMC) to mediate UA relaxation. Multiple BKCa subunits were found in human UA in vitro and hUASMC in vitro, and high β1 and γ1 proteins were localized in SM cells in human UA. Baseline outward currents, recorded by whole-cell and single-channel patch clamps, were significantly inhibited by specific BKCa blockers iberiotoxin (IBTX) or tetraethylammonium, showing specific BKCa activity in hUASMC. H2S dose (NaHS, 1–1000 µM)-dependently potentiated BKCa currents and open probability. Co-incubation with a Ca2+ blocker nifedipine (5 µM) or a chelator (ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 5 mM) did not alter H2S-potentiated BKCa currents and open probability. NaHS also dose-dependently relaxed phenylephrine pre-constricted freshly prepared human UA rings, which was inhibited by IBTX. Thus, H2S stimulated human UA relaxation at least partially via activating SM BKCa channels independent of extracellular Ca2+.

Prepulse-induced mode 2 gating behavior with and without β-adrenergic stimulation in cardiac L-type Ca channels

1999 ◽  
Vol 276 (6) ◽  
pp. C1338-C1345 ◽  
Author(s):  
Yuji Hirano ◽  
Takashi Yoshinaga ◽  
Mitsushige Murata ◽  
Masayasu Hiraoka
Keyword(s):  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Ca Channel ◽  
Ca Channels ◽  
Adrenergic Stimulation ◽  
Open Probability ◽  
Mode 2 ◽  
Voltage Dependent ◽  
Phosphorylation Mechanism ◽  
Prepulse Facilitation

Mode 2 gating of L-type Ca channels is characterized by high channel open probability ( NP o) and long openings. In cardiac myocytes, this mode is evoked physiologically in two apparently different circumstances: membrane depolarization (prepulse facilitation) and activation of protein kinase A. To examine whether the phosphorylation mechanism is involved during prepulse-induced facilitation of cardiac L-type Ca channels, we used isolated guinea pig ventricular myocytes to analyze depolarization-induced modal gating behavior under different basal levels of phosphorylation. In control, NP o measured at 0 mV was augmented as the duration of prepulse to +100 mV was prolonged from 50 to 400 ms. This was due to the induction of mode 2 gating behavior clustered at the beginning of test pulses. Analysis of open time distribution revealed that the prepulse evoked an extra component, the time constant of which is not dependent on prepulse duration. When isoproterenol (1 μM) was applied to keep Ca channels at an enhanced level of phosphorylation, basal NP o without prepulse was increased by a factor of 3.6 ± 2.2 ( n = 6). Under these conditions, prepulse further increased NP o by promoting long openings with the same kinetics of transition to mode 2 gating (τ ≅ 200 ms at +100 mV). Likewise, recovery from mode 2 gating, as estimated by the decay of averaged unitary current, was not affected after β-stimulation (τ ≅ 25 ms at 0 mV). The kinetic behavior independent from the basal level of phosphorylation or activity of cAMP-dependent protein kinase suggests that prepulse facilitation of the cardiac Ca channel involves a mechanism directly related to voltage-dependent conformational change rather than voltage-dependent phosphorylation.

scholarly journals The dihydropyridine-sensitive calcium channel subtype in cone photoreceptors.

1996 ◽  
Vol 107 (5) ◽  
pp. 621-630 ◽  
Author(s):  
M F Wilkinson ◽  
S Barnes
Keyword(s):  
Channel Blocker ◽  
Channel Blockers ◽  
Ca Channel ◽  
Ca Channels ◽  
Cone Photoreceptors ◽  
Channel Current ◽  
Ca Channel Blockers ◽  
Voltage Dependent ◽  
P Type ◽  
Ca Channel Current

High-voltage activated Ca channels in tiger salamander cone photoreceptors were studied with nystatin-permeabilized patch recordings in 3 mM Ca2+ and 10 mM Ba2+. The majority of Ca channel current was dihydropyridine sensitive, suggesting a preponderance of L-type Ca channels. However, voltage-dependent, incomplete block (maximum 60%) by nifedipine (0.1-100 microM) was evident in recordings of cones in tissue slice. In isolated cones, where the block was more potent, nifedipine (0.1-10 microM) or nisoldipine (0.5-5 microM) still failed to eliminate completely the Ca channel current. Nisoldipine was equally effective in blocking Ca channel current elicited in the presence of 10 mM Ba2+ (76% block) or 3 mM Ca2+ (88% block). 15% of the Ba2+ current was reversibly blocked by omega-conotoxin GVIA (1 microM). After enhancement with 1 microM Bay K 8644, omega-conotoxin GVIA blocked a greater proportion (22%) of Ba2+ current than in control. After achieving partial block of the Ba2+ current with nifedipine, concomitant application of omega-conotoxin GVIA produced no further block. The P-type Ca channel blocker, omega-agatoxin IVA (200 nM), had variable and insignificant effects. The current persisting in the presence of these blockers could be eliminated with Cd2+ (100 microM). These results indicate that photoreceptors express an L-type Ca channel having a distinguishing pharmacological profile similar to the alpha 1D Ca channel subtype. The presence of additional Ca channel subtypes, resistant to the widely used L-, N-, and P-type Ca channel blockers, cannot, however, be ruled out.

Differential Ca2+ sensitivities of BK(Ca) isochannels in bovine mesenteric vascular smooth muscle

1994 ◽  
Vol 266 (5) ◽  
pp. C1182-C1189 ◽  
Author(s):  
S. C. Sansom ◽  
J. D. Stockand
Keyword(s):  
Smooth Muscle ◽  
Single Channel ◽  
Mesenteric Arteries ◽  
Ca Channels ◽  
Planar Bilayer ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Gating Charge ◽  
Gating Charges

The planar bilayer method was used to characterize the properties of large Ca(2+)-activated K+ [BK(Ca)] channels of smooth muscle from bovine mesenteric arteries. We found two isochannels of BK(Ca), differing in sensitivity to Ca2+ on the intracellular side of the channel. The first isochannel, Kc1, had a single-channel conductance of 287 +/- 8 pS and required a potential of -33 mV to activate to an open probability (Po) of 0.5 with 1 microM Ca2+. The single-channel conductance of the second isochannel, Kc2 (282 +/- 8 pS), was not statistically different from that of Kc1 but required a potential of 41 mV to activate to a Po of 0.5 with 1.0 microM Ca2+. At a channel voltage of 0 mV, the Ca2+ concentrations for activating Po to 0.5 were 0.2 and 10 microM for Kc1 and Kc2, respectively. The equivalent gating charges, estimated from the Boltzmann equation, were 2.4 and 2.2 for Kc1 and Kc2, respectively. The K/Cl selectivity of Kc1 was > 40 and not significantly different from Kc2. The Po of either isochannel did not change when protein kinase A or alkaline phosphatase was added to the intracellular side. We conclude that bovine mesenteric arteries contain two distinct isochannels of BK(Ca) that differ in Ca2+ sensitivity but are identical with respect to single-channel conductance, equivalent gating charge, and K+/Cl- selectivities.

Isoflurane Reduces the Carbachol-evoked Ca2+Influx in Neuronal Cells

2004 ◽  
Vol 101 (4) ◽  
pp. 895-901 ◽  
Author(s):  
Alexandra Corrales ◽  
Fang Xu ◽  
Zayra V. Garavito-Aguilar ◽  
Thomas J. J. Blanck ◽  
Esperanza Recio-Pinto
Keyword(s):  
Plasma Membrane ◽  
Channel Blocker ◽  
Current Data ◽  
Ca Channel ◽  
Ca Channels ◽  
Human Neuroblastoma ◽  
Cationic Channel ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
A Site

Background The authors previously reported that the isoflurane-caused reduction of the carbachol-evoked cytoplasmic Ca transient increase ([Ca]cyt) was eliminated by K or caffeine-pretreatment. In this study the authors investigated whether the isoflurane-sensitive component of the carbachol-evoked [Ca]cyt transient involved Ca influx through the plasma membrane. Methods Perfused attached human neuroblastoma SH-SY5Y cells were exposed to carbachol (1 mm, 2 min) in the absence and presence of isoflurane (1 mm) and in the absence and presence of extracellular Ca (1.5 mm). The authors studied the effect of the nonspecific cationic channel blocker La (100 microm), of the L-type Ca channel blocker nitrendipine (10 microm), and of the N-type Ca channel blocker omega-conotoxin GVIA (0.1 microm) on isoflurane modulation of the carbachol-evoked [Ca]cyt transient. [Ca]cyt was detected with fura-2 and experiments were carried out at 37 degrees C. Results Isoflurane reduced the peak and area of the carbachol-evoked [Ca]cyt transient in the presence but not in the absence of extracellular Ca. La had a similar effect as the removal of extracellular Ca. Omega-conotoxin GVIA and nitrendipine did not affect the isoflurane sensitivity of the carbachol response although nitrendipine reduced the magnitude of the carbachol response. Conclusions The current data are consistent with previous observations in that the carbachol-evoked [Ca]cyt transient involves both Ca release from intracellular Ca stores and Ca entry through the plasma membrane. It was found that isoflurane attenuates the carbachol-evoked Ca entry. The isoflurane sensitive Ca entry involves a cationic channel different from the L- or N- type voltage-dependent Ca channels. These results indicate that isoflurane attenuates the carbachol-evoked [Ca]cyt transient at a site at the plasma membrane that is distal to the muscarinic receptor.

scholarly journals Calcium currents in the A7r5 smooth muscle-derived cell line. Increase in current and selective removal of voltage-dependent inactivation by intracellular trypsin.

1991 ◽  
Vol 98 (6) ◽  
pp. 1127-1140 ◽  
Author(s):  
C A Obejero-Paz ◽  
S W Jones ◽  
A Scarpa
Keyword(s):  
Smooth Muscle ◽  
Cell Line ◽  
Single Channel ◽  
Calcium Currents ◽  
Open Probability ◽  
Calcium Channel Inactivation ◽  
Voltage Curve ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Current Voltage Curve

We studied the effects of trypsin on L-type calcium current in the A7r5 smooth muscle cell line. Intracellular dialysis with trypsin increased the whole-cell current up to fivefold. The effect was concentration dependent, and was prevented by soybean trypsin inhibitor. Ensemble analysis indicated an increase in the number of functional channels, and possibly a smaller increase in the open probability, with no change in the single channel current. The shape of the current-voltage curve was unaffected. Trypsin also nearly eliminated inactivation of currents carried by Ba2+, but had little or no effect on the rapid inactivation process in Ca2+, This indicates that trypsin removes voltage-dependent but not Ca(2+)-dependent inactivation, suggesting the existence of distinct protein domains for these two mechanisms of calcium channel inactivation.

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