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.

Calcium-dependent halothane activation of sarcoplasmic reticulum calcium channels from frog skeletal muscle

1994 ◽  
Vol 266 (2) ◽  
pp. C391-C396 ◽  
Author(s):  
R. Bull ◽  
J. J. Marengo
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Calcium Channels ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Free Calcium ◽  
Frog Skeletal Muscle ◽  
Time Constants ◽  
Calcium Dependent ◽  
Open Time

The effect of halothane on calcium channels present in sarcoplasmic reticulum membranes isolated from frog skeletal muscle was studied at the single channel level after fusing the isolated vesicles into planar lipid bilayers. Addition of 91 microM halothane to the cytosolic compartment containing 1 microM free calcium activated the channel by increasing fractional open time from 0.11 to 0.59, without changing the channel conductance. The activation of the channels by halothane was calcium dependent. At resting calcium concentrations in the cytosolic compartment, halothane failed to activate the channel, whereas maximal activation was found at 10 microM calcium. The free energy of halothane binding to the channel decreased from -5.8 kcal/mol at 1 microM calcium to -6.6 kcal/mol at 10 microM calcium. Halothane increased the open time constants and decreased the closed time constants, indicating that it binds to both the open and the closed configurations of the channel.

scholarly journals Reduction in mdx mouse muscle degeneration by low-intensity endurance exercise: a proteomic analysis in quadriceps muscle of exercised compared with sedentary mdx mice

2015 ◽  
Vol 35 (3) ◽  
Author(s):  
Simona Fontana ◽  
Odessa Schillaci ◽  
Monica Frinchi ◽  
Marco Giallombardo ◽  
Giuseppe Morici ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Endurance Exercise ◽  
Proteomic Analysis ◽  
Quadriceps Muscle ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Muscle Degeneration ◽  
Wild Type ◽  
Mouse Muscle ◽  
Low Intensity

By proteomic analysis we found an up-regulation of four carbonic anhydrase-3 (CA3) isoforms and a down-regulation of superoxide dismutase [Cu-Zn] (SODC) in quadriceps of sedentary X-linked muscular dystrophy (mdx) mice as compared with wild–type (WT) mice and the levels were significantly restored to WT values following low-intensity endurance exercise.

scholarly journals Reversible Block of the Calcium Release Channel/Ryanodine Receptor by Protamine, a Heparin Antidote

2000 ◽  
Vol 11 (7) ◽  
pp. 2213-2219 ◽  
Author(s):  
Peter Koulen ◽  
Barbara E. Ehrlich
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Calcium Release ◽  
Single Channel ◽  
Reversible Inhibitor ◽  
Channel Activity ◽  
Calcium Release Channel ◽  
Release Channel ◽  
Mouse Muscle ◽  
Cytoplasmic Side

Channel activity of the calcium release channel from skeletal muscle, ryanodine receptor type 1, was measured in the presence and absence of protamine sulfate on the cytoplasmic side of the channel. Single-channel activity was measured after incorporating channels into planar lipid bilayers. Optimally and suboptimally calcium-activated calcium release channels were inactivated by the application of protamine to the cytoplasmic side of the channel. Recovery of channel activity was not observed while protamine was present. The addition of protamine bound to agarose beads did not change channel activity, implying that the mechanism of action involves an interaction with the ryanodine receptor rather than changes in the bulk calcium concentration of the medium. The block of channel activity by protamine could be reversed either by removal by perfusion with buffer or by the addition of heparin to the cytoplasmic side of the channel. Microinjection of protamine into differentiated C2C12 mouse muscle cells prevented caffeine-induced intracellular calcium release. The results suggest that protamine acts on the ryanodine receptor in a similar but opposite manner from heparin and that protamine can be used as a potent, reversible inhibitor of ryanodine receptor activity.

Ammonium ion enhances the calcium-dependent gating of a mammalian large conductance, calcium-sensitive K+ channel

2001 ◽  
Vol 79 (11) ◽  
pp. 919-923 ◽  
Author(s):  
Andrew P Braun
Keyword(s):  
Single Channel ◽  
Ammonium Ion ◽  
K Channel ◽  
Free Calcium ◽  
Dependent Manner ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Hek 293 ◽  
Calcium Dependent ◽  
Single Channel Currents

We observed that the current amplitude and activation of expressed, mouse brain large conductance, calcium-sensitive K+ channels (BKCa channels) may be reversibly enhanced following addition of low concentrations of the weakly permeant cation NH4+ to the cytoplasmic face of the channel in excised, inside-out membrane patches from HEK 293 cells. Conductance-voltage relations were left-shifted along the voltage axis by addition of NH4Cl in a concentration-dependent manner, with an EC50 of 18.5 mM. Furthermore, this effect was observed in the presence of cytosolic free calcium (~1 µM), but was absent in a cytosolic bath solution containing nominally zero free calcium (e.g., 5 mM EGTA only), a condition under which these channels undergo largely voltage-dependent gating. Recordings of single BKCa channel events indicated that NH4+ increased the channel open probability of single channel activity ~3-fold, but did not alter the amplitude of single channel currents. These findings suggest that the calcium-sensitive gating of mammalian BKCa channels may be modified by other ions present in cytosolic solution.Key words: potassium channel, calcium, modulation, electrophysiology.

scholarly journals Membrane-delimited Inhibition of Maxi-K Channel Activity by the Intermediate Conductance Ca2+-activated K Channel

2006 ◽  
Vol 127 (2) ◽  
pp. 159-169 ◽  
Author(s):  
Jill Thompson ◽  
Ted Begenisich
Keyword(s):  
Single Channel ◽  
Expression System ◽  
Chemical Activation ◽  
K Channel ◽  
Channel Activity ◽  
Single Family ◽  
Open Probability ◽  
K Channels ◽  
Channel Proteins ◽  
K Activity

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.

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.

scholarly journals ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

1989 ◽  
Vol 94 (4) ◽  
pp. 693-717 ◽  
Author(s):  
B Ribalet ◽  
S Ciani ◽  
G T Eddlestone
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Insulin Secreting Cells ◽  
Dependent Protein ◽  
Inside Out

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.

scholarly journals TOK Homologue in Neurospora crassa: First Cloning and Functional Characterization of an Ion Channel in a Filamentous Fungus

2003 ◽  
Vol 2 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Stephen K. Roberts
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Neurospora Crassa ◽  
Filamentous Fungus ◽  
Single Channel ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Channel Activity ◽  
Biophysical Properties ◽  
Patch Clamp Technique

ABSTRACT In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K+ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K+ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K+ efflux. NcTOKA channel gating was sensitive to extracellular K+ such that channel activation was dependent on the reversal potential for K+. However, expression of NcTOKA was able to overcome the K+ auxotrophy of a yeast mutant missing the K+ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K+ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K+ currents at potentials negative of EK. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca2+, verapamil, quinine, and TEA+ but was insensitive to Cs+, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.

Beneficial effects of voluntary wheel running on the properties of dystrophic mouse muscle

1996 ◽  
Vol 80 (2) ◽  
pp. 670-679 ◽  
Author(s):  
A. Hayes ◽  
D. A. Williams
Keyword(s):  
Skeletal Muscles ◽  
Wheel Running ◽  
Voluntary Exercise ◽  
Mdx Mice ◽  
Free Access ◽  
Type I ◽  
Fiber Types ◽  
Force Output ◽  
Mouse Muscle ◽  
Beneficial Effects

Effects of voluntary exercise on the isometric contractile, fatigue, and histochemical properties of hindlimb dystrophic (mdx and 129ReJ dy/dy) skeletal muscles were investigated. Mice were allowed free access to a voluntary running wheel at 4 wk of age for a duration of 16 (mdx) or 5 (dy/dy) wk. Running performance of mdx mice (approximately 4 km/day at 1.6 km/h) was inferior to normal mice (approximately 6.5 km/day at 2.1 km/h). However, exercise improved the force output (approximately 15%) and the fatigue resistance of both C57BL/10 and mdx soleus muscles. These changes coincided with increased proportions of smaller type I fibers and decreased proportions of larger type IIa fibers in the mdx soleus. The extensor digitorum longus of mdx, but not of normal, mice also exhibited improved resistance to fatigue and conversion towards oxidative fiber types. The dy/dy animals were capable of exercising, yet ran significantly less than normal animals (approximately 0.5 km/day). Despite this, running increased the force output of the plantaris muscle (approximately 50%). Taken together, the results showed that exercise can have beneficial effects on dystrophic skeletal muscles.

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