scholarly journals Single channel properties of newly synthesized acetylcholine receptors following denervation of mammalian skeletal muscle.

1987 ◽  
Vol 89 (6) ◽  
pp. 999-1014 ◽  
Author(s):  
L P Henderson ◽  
J D Lechleiter ◽  
P Brehm
Keyword(s):  
Single Channel ◽  
Synaptic Contact ◽  
Synaptic Membrane ◽  
Mammalian Skeletal Muscle ◽  
Denervated Muscle ◽  
Mouse Muscle ◽  
Alpha Bungarotoxin ◽  
Channel Properties ◽  
Ach Receptors

We have examined the single channel properties of newly synthesized acetylcholine (ACh) receptors in denervated adult mouse muscle. Patch-clamp recordings were made on freshly isolated fibers from flexor digitorum brevis (fdb) muscles that had been denervated in vivo for periods up to 3 wk. Muscles were treated with alpha-bungarotoxin (alpha-BTX), immediately before denervation, in order to block pre-existing receptors. Denervated fibers exhibited two types of ACh receptor channels, which differed in terms of single channel conductance (45 and 70 pS) and mean channel open time (approximately 7 and 2.5 ms, respectively). In contrast to innervated muscle, where only 3% of the total openings were contributed by the low-conductance channel type, greater than 80% of the openings in the nonsynaptic membrane of denervated muscle were of this type. Importantly, a similar increase in the proportion of low-conductance channels was observed for recordings from synaptic membrane after denervation. These data argue against the proposal that, in denervated muscle, the low-conductance channels undergo continued conversion to the high-conductance type focally at the site of former synaptic contact. Rather, our findings provide additional support for the idea that the functional properties of ACh receptors are governed uniformly by the state of innervation of the fiber and not by proximity to the site of synaptic contact.

Altering creatine kinase isoenzymes in transgenic mouse muscle by overexpression of the B subunit

1993 ◽  
Vol 264 (1) ◽  
pp. C151-C160 ◽  
Author(s):  
M. J. Brosnan ◽  
S. P. Raman ◽  
L. Chen ◽  
A. P. Koretsky
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Transgenic Mice ◽  
Mammalian Skeletal Muscle ◽  
Maximal Velocity ◽  
Coding Region ◽  
Mouse Muscle ◽  
Control Muscle ◽  
B Subunit

To change the levels of expression and isoenzyme distribution of creatine kinase (CK) in muscle, transgenic technology was used to express the B subunit of CK in mouse muscle. Normally, mammalian skeletal muscle contains the MM dimer of CK. The BB dimer and MB heterodimer of CK can be found in brain and heart, respectively. Heterologous genes consisting of skeletal and cardiac muscle-specific actin promoters fused to the genomic coding region of the B form of CK were used to create transgenic mice. Lines were established from the three highest expressing founders. Analysis of skeletal muscle extracts revealed that all three lines had an increase in total CK activity measured under maximal velocity conditions. The highest expressing line, 7001, had a CK activity 150% that of control muscle. Nuclear magnetic resonance saturation transfer was used to measure the in vivo rate of the CK reaction. In 7001 hindlimb muscles, the CK catalyzed reaction was 200% that of control muscle. The elevation in CK activity in transgenic muscle was accompanied by significant changes in the composition of the cytosolic isoenzyme ratio of CK. In control, 100% of CK was MM, whereas 7001 had 60 +/- 18% MM, 32 +/- 10% MB, and 8 +/- 2% BB. There were no changes in ATP, phosphocreatine, Pi, or creatine levels in transgenic muscle compared with control. Immunofluorescence of myofibrils isolated from control and transgenic muscle revealed specific association of CK to the M line. Small amounts of MB CK were detected on myofibrils from transgenic mice. Transgenic mice expressing the B subunit of CK in muscle represent a first step toward altering CK isoforms so as to elucidate the specific roles of these isoforms in energy metabolism.

scholarly journals Gating Dynamics of the Acetylcholine Receptor Extracellular Domain

2004 ◽  
Vol 123 (4) ◽  
pp. 341-356 ◽  
Author(s):  
Sudha Chakrapani ◽  
Timothy D. Bailey ◽  
Anthony Auerbach
Keyword(s):  
Conformational Change ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Extracellular Domain ◽  
Mouse Muscle ◽  
Α Subunit ◽  
Acetylcholine Binding Protein ◽  
Almost All ◽  
Loop 2 ◽  
Sandwich Core

We used single-channel recording and model-based kinetic analyses to quantify the effects of mutations in the extracellular domain (ECD) of the α-subunit of mouse muscle–type acetylcholine receptors (AChRs). The crystal structure of an acetylcholine binding protein (AChBP) suggests that the ECD is comprised of a β-sandwich core that is surrounded by loops. Here we focus on loops 2 and 7, which lie at the interface of the AChR extracellular and transmembrane domains. Side chain substitutions in these loops primarily affect channel gating by either decreasing or increasing the gating equilibrium constant. Many of the mutations to the β-core prevent the expression of functional AChRs, but of the mutants that did express almost all had wild-type behavior. Rate-equilibrium free energy relationship analyses reveal the presence of two contiguous, distinct synchronously-gating domains in the α-subunit ECD that move sequentially during the AChR gating reaction. The transmitter-binding site/loop 5 domain moves first (Φ = 0.93) and is followed by the loop 2/loop 7 domain (Φ = 0.80). These movements precede that of the extracellular linker (Φ = 0.69). We hypothesize that AChR gating occurs as the stepwise movements of such domains that link the low-to-high affinity conformational change in the TBS with the low-to-high conductance conformational change in the pore.

Some properties of acetylcholine receptors in human cultured myotubes

1985 ◽  
Vol 224 (1235) ◽  
pp. 183-196 ◽  
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.

scholarly journals Myotubes from transgenic mdx mice expressing full-length dystrophin show normal calcium regulation.

1994 ◽  
Vol 5 (10) ◽  
pp. 1159-1167 ◽  
Author(s):  
W F Denetclaw ◽  
F W Hopf ◽  
G A Cox ◽  
J S Chamberlain ◽  
R A Steinhardt
Keyword(s):  
Single Channel ◽  
Calcium Regulation ◽  
Full Length ◽  
Mdx Mice ◽  
Free Calcium ◽  
Muscle Degeneration ◽  
Channel Activity ◽  
Mouse Muscle ◽  
Calcium Dependent ◽  
Normal Calcium

A lack of dystrophin results in muscle degeneration in Duchenne muscular dystrophy. Dystrophin-deficient human and mouse muscle cells have higher resting levels of intracellular free calcium ([Ca2+]i) and show a related increase in single-channel open probabilities of calcium leak channels. Elevated [Ca2+]i results in high levels of calcium-dependent proteolysis, which in turn increases calcium leak channel activity. This process could initiate muscle degeneration by further increasing [Ca2+]i and proteolysis in a positive feedback loop. Here, we tested the direct effect of restoration of dystrophin on [Ca2+]i and channel activity in primary myotubes from mdx mice made transgenic for full-length dystrophin. Transgenic mdx mice have been previously shown to have normal dystrophin localization and no muscle degeneration. Fura-2 calcium measurements and single-channel patch recordings showed that resting [Ca2+]i levels and open probabilities of calcium leak channels of transgenic mdx myotubes were similar to normal levels and significantly lower than mdx littermate controls (mdx) that lack dystrophin. Thus, restoration of normal calcium regulation in transgenic mdx mice may underlie the resulting absence of degeneration.

scholarly journals Effects of Strontium on the Permeation and Gating Phenotype of Calcium Channels in Hair Cells

2008 ◽  
Vol 100 (4) ◽  
pp. 2115-2124 ◽  
Author(s):  
Adrian Rodriguez-Contreras ◽  
Ping Lv ◽  
Jun Zhu ◽  
Hyo Jeong Kim ◽  
Ebenezer N. Yamoah
Keyword(s):  
Hair Cell ◽  
Single Channel ◽  
Single Channels ◽  
Physiological Concentration ◽  
Closed State ◽  
Dependent Inactivation ◽  
Intracellular Ca ◽  
High Concentrations ◽  
Latency Distributions ◽  
Channel Properties

To minimize the effects of Ca2+ buffering and signaling, this study sought to examine single Ca2+ channel properties using Sr2+ ions, which substitute well for Ca2+ but bind weakly to intracellular Ca2+ buffers. Two single-channel fluctuations were distinguished by their sensitivity to dihydropyridine agonist (L-type) and insensitivity toward dihydropyridine antagonist (non-L-type). The L- and non-L-type single channels were observed with single-channel conductances of 16 and 19 pS at 70 mM Sr2+ and 11 and 13 pS at 5 mM Sr2+, respectively. We obtained KD estimates of 5.2 and 1.9 mM for Sr2+ for L- and non-L-type channels, respectively. At Ca2+ concentration of ∼2 mM, the single-channel conductances of Sr2+ for the L-type channel was ∼1.5 and 4.0 pS for the non-L-type channels. Thus the limits of single-channel microdomain at the membrane potential of a hair cell (e.g., −65 mV) for Sr2+ ranges from 800 to 2,000 ion/ms, assuming an ECa of 100 mV. The channels are ≥4-fold more sensitive at the physiological concentration ranges than at concentrations >10 mM. Additionally, the channels have the propensity to dwell in the closed state at high concentrations of Sr2+, which is reflected in the time constant of the first latency distributions. It is concluded that the concentration of the permeant ion modulates the gating of hair cell Ca2+ channels. Finally, the closed state/s that is/are altered by high concentrations of Sr2+ may represent divalent ion-dependent inactivation of the L-type channel.

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