Two Types of ACh Receptors Contribute to Fast Channel Gating on Mouse Skeletal Muscle

Shepherd, Dawn and Paul Brehm. Two types of ACh receptors contribute to fast channel gating on mouse skeletal muscle. J. Neurophysiol. 78: 2966–2974, 1997. Single-channel recordings from mouse C2 myotubes indicate that maturation of skeletal muscle is accompanied by the appearance of two types of fast acetylcholine (ACh) receptor channels that are each functionally distinct from the embryonic receptor type present at early stages of differentiation. The embryonic receptor type has a low conductance (45 pS) and long channel open time, rendering slowly decaying synaptic currents. One fast channel type that appears during muscle maturation is distinguished from the embryonic receptor type on the basis of both higher conductance (65 pS) and shorter open time. However, single-channel recordings from differentiated mouse skeletal muscle cell line (C2) point to the existence of a second fast receptor type, which has a conductance similar to the embryonic receptor type (45 pS), yet significantly reduced mean channel open time. Analyses of individual channel function at high ACh concentrations directly demonstrate the coexistence of two kinetically distinct types of 45 pS ACh receptors. Openings by fast type and slow embryonic type of 45 pS receptors occurred in bursts, allowing distinction on the basis of both mean open time and open probability for individual receptors. The embryonic type of 45 pS receptor has an open time approximately twofold longer than the fast-receptor counterpart. Additional differences were reflected in the open probability distributions for fast and slow 45 pS receptor types. Both types of 45 pS receptor were kinetically distinguishable from the 65 pS receptor. We found no support for the idea that the slow and fast 45 pS receptor types result from the interconversion of dual gating modes involving the same receptor protein. Our results are consistent with the idea that the acquisition of fast synaptic current decay, required at mature neuromuscular synapses, is the result of the up-regulation of two distinct fast types of nicotinic ACh receptors during skeletal muscle development.

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

AJP Cell Physiology ◽

10.1152/ajpcell.2000.278.3.c601 ◽

2000 ◽

Vol 278 (3) ◽

pp. C601-C611 ◽

Cited By ~ 17

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.

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Serine protease activation of near-silent epithelial Na+ channels

AJP Cell Physiology ◽

10.1152/ajpcell.00342.2003 ◽

2004 ◽

Vol 286 (1) ◽

pp. C190-C194 ◽

Cited By ~ 150

Author(s):

Ray A. Caldwell ◽

Richard C. Boucher ◽

M. Jackson Stutts

Keyword(s):

Plasma Membrane ◽

Serine Protease ◽

Serine Proteases ◽

Single Channel ◽

Open Probability ◽

Open Time ◽

Protease Activation ◽

Salt And Water Balance ◽

Channel Open Time ◽

Nih 3T3

The regulation of epithelial Na+ channel (ENaC) function is critical for normal salt and water balance. This regulation is achieved through cell surface insertion/retrieval of channels, by changes in channel open probability ( Po), or through a combination of these processes. Epithelium-derived serine proteases, including channel activating protease (CAP) and prostasin, regulate epithelial Na+ transport, but the molecular mechanism is unknown. We tested the hypothesis that extracellular serine proteases activate a near-silent ENaC population resident in the plasma membrane. Single-channel events were recorded in outside-out patches from fibroblasts (NIH/3T3) stably expressing rat α-, β-, and γ-subunits (rENaC), before and during exposure to trypsin, a serine protease homologous to CAP and prostasin. Under baseline conditions, near-silent patches were defined as having rENaC activity ( NPo) < 0.03, where N is the number of channels. Within 1–5 min of 3 μg/ml bath trypsin superfusion, NPo increased ∼66-fold ( n = 7). In patches observed to contain a single functional channel, trypsin increased Po from 0.02 ± 0.01 to 0.57 ± 0.03 ( n = 3, mean ± SE), resulting from the combination of an increased channel open time and decreased channel closed time. Catalytic activity was required for activation of near-silent ENaC. Channel conductance and the Na+/Li+ current ratio with trypsin were similar to control values. Modulation of ENaC Po by endogenous epithelial serine proteases is a potentially important regulator of epithelial Na+ transport, distinct from the regulation achieved by hormone-induced plasma membrane insertion of channels.

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Single channel recordings of Nt- and L-type Ca2+ currents in rat neurohypophysial terminals

Journal of Neurophysiology ◽

10.1152/jn.1993.70.4.1617 ◽

1993 ◽

Vol 70 (4) ◽

pp. 1617-1628 ◽

Cited By ~ 26

Author(s):

X. Wang ◽

S. N. Treistman ◽

J. R. Lemos

Keyword(s):

Time Constant ◽

Voltage Pulse ◽

Single Channel ◽

Single Channels ◽

Open Probability ◽

Time Constants ◽

Channel Current ◽

Open Time ◽

The Mean ◽

Channel Open Time

1. Ca2+ currents through single channels in acutely dissociated nerve terminals from rat neurohypophyses were recorded using cell-attached patch recordings with 110 mM Ba2+ as the charge carrier. 2. One type (Nt, where the t denotes terminal) of single Ca2+ channel current was evoked only by depolarizing steps from holding potentials less negative than -50 mV. Because this channel opened primarily at the beginning of a 180-ms-long voltage pulse, the averaged ensemble current decayed rapidly (approximately 30 ms). Infrequently, the channel opened throughout such a long pulse, resulting in a long-lasting averaged ensemble current. The averaged channel open time constant (tau) was 0.34 ms and the two averaged closed time constants were 1.78 (tau 1) and 86.57 (tau 2) ms. The mean unitary slope conductance for this channel was 11 pS and its threshold for activation was approximately -10 mV. 3. The other type (L) of single Ca2+ channel current could be evoked in isolation by depolarizations from holding potentials more positive than or equal to -50 mV. This channel opened throughout an entire 180-ms-long voltage pulse. The averaged ensemble current, therefore, showed little inactivation. The averaged channel open-time constant was 0.49 ms and the two average closed time constants were 2.02 (tau 1) and 79.91 (tau 2) ms. The mean unitary slope conductance for this channel was 25 pS. 4. Bay K 8644 (5 microM), a dihydropyridine (DHP) Ca2+ channel agonist, increased the open probability of the larger-conductance L-type Ca2+ channel by prolonging the average duration (to 2.79 ms) of channel openings, but did not alter the single channel slope conductance. In contrast, the same concentration of Bay K 8644 did not affect the smaller-conductance Nt-type Ca2+ channel. The DHP Ca2+ channel antagonist nicardipine (5 microM), but not nifedipine (5 microM), reduced the open probability of the large-conductance L-type Ca2+ channel by shortening the duration (to 0.36 ms) of channel openings. 5. The voltage- and time-dependent properties of these two types of single Ca2+ channel currents are in close agreement with those of the two components of macroscopic Ca2+ currents previously reported using the "whole-terminal" recording method. Therefore these two types of single channels appear to underlie the macroscopic current. 6. Our studies suggest that the terminal Nt-type Ca2+ channel differs from the conventional somatic N- and T-type Ca2+ channels in some respects, and that the terminal L-type Ca2+ channel is similar to the conventional somatic L-type Ca2+ channel.(ABSTRACT TRUNCATED AT 400 WORDS)

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Mechanisms of GABAB receptor enhancement of extrasynaptic GABAA receptor currents in cerebellar granule cells

Scientific Reports ◽

10.1038/s41598-019-53087-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Shailesh N. Khatri ◽

Wan-Chen Wu ◽

Ying Yang ◽

Jason R. Pugh

Keyword(s):

Granule Cells ◽

Single Channel ◽

Cerebellar Granule Cells ◽

Gabab Receptor ◽

Open Probability ◽

Cerebellar Granule ◽

Channel Current ◽

Open Time ◽

Patch Clamp Electrophysiology ◽

Channel Open Time

Abstract Many neurons, including cerebellar granule cells, exhibit a tonic GABA current mediated by extrasynaptic GABAA receptors. This current is a critical regulator of firing and the target of many clinically relevant compounds. Using a combination of patch clamp electrophysiology and photolytic uncaging of RuBi-GABA we show that GABAB receptors are tonically active and enhance extrasynaptic GABAA receptor currents in cerebellar granule cells. This enhancement is not associated with meaningful changes in GABAA receptor potency, mean channel open-time, open probability, or single-channel current. However, there was a significant (~40%) decrease in the number of channels participating in the GABA uncaging current and an increase in receptor desensitization. Furthermore, we find that adenylate cyclase, PKA, CaMKII, and release of Ca2+ from intracellular stores are necessary for modulation of GABAA receptors. Overall, this work reveals crosstalk between postsynaptic GABAA and GABAB receptors and identifies the signaling pathways and mechanisms involved.

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Some properties of acetylcholine receptors in human cultured myotubes

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1985.0028 ◽

1985 ◽

Vol 224 (1235) ◽

pp. 183-196 ◽

Cited By ~ 3

Keyword(s):

Acetylcholine Receptors ◽

Single Channel ◽

Fold Increase ◽

Resting Potential ◽

Patch Clamp Technique ◽

Membrane Hyperpolarization ◽

Human Myotubes ◽

Cultured Myotubes ◽

Channel Open Time ◽

Ach Receptors

The distribution and single channel properties of acetylcholine (ACh) receptors in human myotubes grown in tissue culture have been examined. Radioautography of myotubes labelled with [ 125 I]α-bungarotoxin showed that ACh receptors are distributed uniformly over the myotube surface at a density of 3.9 ± 0.5 receptors per square micrometre. Accumulations of ACh receptors (hot spots) were found rarely. The conductance and kinetics of ACh-activated channels were investigated with the patch-clamp technique. Cell-attached membrane patches were used in all experiments. A single channel conductance in the range 40–45 pS was calculated. No sublevels of conductance (substates) of the activated channel were observed. The distribution of channel open-times varied with ACh concentration. With 100 nM ACh, the distribution was best fitted by the sum of two exponentials, whereas with 1 μM ACh a single exponential could be fitted. The mean channel open-time at the myotube resting potential (ca. — 70 mV, 22°C) was 8.2 ms. The distribution of channel closed-times was complex at all concentrations of ACh studied (100 nM to 10 μm). With desensitizing doses of ACh (10 μM), channel openings occurred in obvious bursts; each burst usually appeared as part of a ‘cluster’ of bursts. Both burst duration and mean interval between bursts increased with membrane hyperpolarization. Individual channel open-times and burst durations showed similar voltage dependence (e-fold increase per 80 mV hyperpolarization), whereas both the channel closed-times within a burst and the number of openings per burst were independent of membrane potential.

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Ryanodine receptor dysfunction in porcine stunned myocardium

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1997.273.2.h796 ◽

1997 ◽

Vol 273 (2) ◽

pp. H796-H804 ◽

Cited By ~ 3

Author(s):

C. Valdivia ◽

J. O. Hegge ◽

R. D. Lasley ◽

H. H. Valdivia ◽

R. Mentzer

Keyword(s):

Coronary Artery ◽

Lipid Bilayers ◽

Ryanodine Receptor ◽

Myocardial Stunning ◽

Single Channel ◽

Stunned Myocardium ◽

Wall Thickening ◽

Planar Lipid Bilayers ◽

Open Probability ◽

Single Channel Recordings

We investigated the effects of myocardial stunning on the function of the two main Ca2+ transport proteins of the sarcoplasmic reticulum (SR), the Ca(2+)-adenosinetriphosphatase and the Ca(2+)-release channel or ryanodine receptor. Regional myocardial stunning was induced in open-chest pigs (n = 6) by a 10-min occlusion of the left anterior descending coronary artery (LAD) and 2 h reperfusion. SR vesicles isolated from the LAD-perfused region (stunned) and the normal left circumflex coronary artery (LC)-perfused region were used to assess the oxalate-supported 45Ca2+ uptake, [3H]ryanodine binding, and single-channel recordings of ryanodine-sensitive Ca(2+)-release channels in planar lipid bilayers. Myocardial stunning decreased LAD systolic wall thickening to 20% of preischemic values. The rate of SR 45Ca2+ uptake in the stunned LAD bed was reduced by 37% compared with that of the normal LC bed (P < 0.05). Stunning was also associated with a 38% reduction in the maximal density of high-affinity [3H]ryanodine binding sites (P < 0.05 vs. normal LC) but had no effect on the dissociation constant. The open probability of ryanodine-sensitive Ca(2+)-release channels determined by single channel recordings in planar lipid bilayers was 26 +/- 2% for control SR (n = 33 channels from 3 animals) and 14 +/- 2% for stunned SR (n = 21 channels; P < 0.05). This depressed activity of SR function observed in postischemic myocardium could be one of the mechanisms underlying myocardial stunning.

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Gating of Single N-type Calcium Channels Recorded from Bullfrog Sympathetic Neurons

The Journal of General Physiology ◽

10.1085/jgp.113.1.111 ◽

1999 ◽

Vol 113 (1) ◽

pp. 111-124 ◽

Cited By ~ 23

Author(s):

Hye Kyung Lee ◽

Keith S. Elmslie

Keyword(s):

Calcium Channels ◽

Calcium Influx ◽

Sympathetic Neurons ◽

Channel Gating ◽

Open Probability ◽

Maximum Value ◽

Open Time ◽

Time Component ◽

Patch Technique ◽

Single Exponential

For many neurons, N-type calcium channels provide the primary pathway for calcium influx during an action potential. We investigated the gating properties of single N-type calcium channels using the cell-attached patch technique. With 100 mM Ba2+ in the pipet, mean N-channel open probability (Po, measured over 100 ms) increased with depolarization, but the range at a single voltage was large (e.g., Po at +40 mV ranged from 0.1 to 0.8). The open dwell time histograms were generally well fit by a single exponential with mean open time (τo) increasing from 0.7 ms at +10 mV to 3.1 ms at +40 mV. Shut time histograms were well fit by two exponentials. The brief shut time component (τsh1 = 0.3 ms) did not vary with the test potential, while the longer shut time component (τsh2) decreased with voltage from 18.9 ms at +10 mV to 2.3 ms at +40 mV. Although N-channel Po during individual sweeps at +40 mV was often high (∼0.8), mean Po was reduced by null sweeps, low Po gating, inactivation, and slow activation. The variability in mean Po across patches resulted from differences in the frequency these different gating processes were expressed by the channels. Runs analysis showed that null sweeps tended to be clustered in most patches, but that inactivating and slowly activating sweeps were generally distributed randomly. Low Po gating (Po = 0.2, τo = 1 ms at +40 mV) could be sustained for ∼1 min in some patches. The clustering of null sweeps and sweeps with low Po gating is consistent with the idea that they result from different modes of N-channel gating. While Po of the main N-channel gating state is high, the net Po is reduced to a maximum value of close to 0.5 by other gating processes.

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Interdomain Interactions within Ryanodine Receptors Regulate Ca2+ Spark Frequency in Skeletal Muscle

The Journal of General Physiology ◽

10.1085/jgp.119.1.15 ◽

2001 ◽

Vol 119 (1) ◽

pp. 15-32 ◽

Cited By ~ 40

Author(s):

Alexander Shtifman ◽

Christopher W. Ward ◽

Takeshi Yamamoto ◽

Jianli Wang ◽

Beth Olbinski ◽

...

Keyword(s):

Skeletal Muscle ◽

Lipid Bilayers ◽

Laser Scanning ◽

Muscle Fibers ◽

Mammalian Skeletal Muscle ◽

Open Probability ◽

Release Channel ◽

Pronounced Increase ◽

Open Time ◽

Interdomain Interactions

DP4 is a 36-residue synthetic peptide that corresponds to the Leu2442-Pro2477 region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca2+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618–11625). We have investigated the effects of DP4 on local SR Ca2+ release events (Ca2+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca2+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca2+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca2+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca2+ release channel(s) generating the sparks. DP4 also increased [3H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca2+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca2+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg17 to Cys17 replacement (DP4mut), which corresponds to the Arg2458-to-Cys2458 mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg2+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg2+-free RyR(s), thus promoting channel opening and production of Ca2+ sparks.

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