Channels In Skeletal Muscle Research Articles

Testing for microscopic reversibility in the gating of maxi K+ channels using two-dimensional dwell-time distributions

An assumption usually made when developing kinetic models for the gating of ion channels is that the transitions among the various states involved in the gating obey microscopic reversibility. If this assumption is incorrect, then the models and estimated rate constants made with the assumption would be in error. This paper examines whether the gating of a large conductance Ca-activated K+ channel in skeletal muscle is consistent with microscopic reversibility. If microscopic reversibility is obeyed, then the number of forward and backward transitions per unit time for each individual reaction step will, on average, be identical and, consequently, the gating must show time reversibility. To look for time reversibility, two-dimensional dwell-time distributions of the durations of open and closed intervals were obtained from single-channel current records analyzed in the forward and in the backward directions. Two-dimensional dwell-time distributions of pairs of open intervals and of pairs of closed intervals were also analyzed to extend the resolution of the method to special circumstances in which intervals from different closed (or open) states might have similar durations. No significant differences were observed between the forward and backward analysis of the two-dimensional dwell-time distributions, suggesting time reversibility. Thus, we find no evidence to indicate that the gating of the maxi K+ channel violates microscopic reversibility.

Modulation of skeletal muscle sodium channels in a satellite cell line by protein kinase C

Adult vertebrate skeletal muscle sodium channels are responsible for the spread of excitation from the end-plate through the muscle membrane and transverse tubular system that ultimately leads to contraction. These channels can be distinguished from other sodium channels by their sensitivity to both mu-conotoxin and TTX. The mouse satellite muscle cell line MM14 expresses only TTX- and mu-conotoxin-sensitive sodium channels having the physiological characteristics of adult skeletal muscle channels in both undifferentiated myoblasts and differentiated myotubes. Using undifferentiated and differentiated MM14 cells as well as primary cultures of rat skeletal muscle, we have examined modulation of adult skeletal muscle sodium channels by activators of protein kinase C (PKC). Stimulation of PKC by 1-oleoyl-2-acetyl-sn-glycerol (OAG) slows sodium current macroscopic inactivation rate by up to 70% and reduces the peak sodium current as much as 88%. Single-channel analysis reveals prolonged single channel openings and greatly increased probability of multiple channel openings during sustained depolarizations. These effects are due to PKC activation since they are blocked by a specific peptide inhibitor of PKC. The two effects of OAG are sequential. Low OAG concentrations can cause slowed macroscopic sodium current inactivation in the absence of peak current reduction, and intermediate concentrations of OAG cause slowing of inactivation followed by reduction of peak current. The separation of these two effects indicates that PKC modulation of the skeletal muscle sodium channel may occur by phosphorylation at two independent sites. PKC modulation of muscle sodium channels is expected to have important effects on muscle excitability and resultant contractile activity. Detection of adult skeletal muscle ion channels in replicating MM14 cells suggest that satellite cells may express a distinct subset of muscle-specific genes prior to activation of the terminal differentiation program.

Calcium-dependent block of ryanodine receptor channel of swine skeletal muscle by direct binding of calmodulin

The interaction of the Ca 2+ binding protein calmodulin (CaM) with the ryanodine receptor of the sarcoplasmic reticulum (SR) of pig skeletal muscle was investigated by [ 3H]-ryanodine binding, planar bilayer recordings, and rapid filtration of 45Ca 2+-loaded SR. Inhibition of [ 3H]-ryanodine binding by CaM was phosphorylation-independent, had an IC 50 of approximately 0.1 μM and was optimal at 10 μM Ca 2+. CaM also inhibited [ 3H]-ryanodine binding to CHAPS-solubilized and purified ryanodine receptors, suggesting a direct CaM-ryanodine receptor interaction. In single channel recordings, CaM blocked Ca 2+ release channels in a Ca 2+-dependent manner by decreasing the number of open events per unit time without affecting the mean open time or unitary channel conductance. Rapid filtration of 45Ca 2+ passively loaded into SR vesicles showed that CaM blocked Ca 2+ release within milliseconds of exposure of SR to a Ca 2+ release medium containing 10 μM CaM. In controls, an increase in extravesicular Ca 2+ from 7 nM to 10 μM resulted in a release of 47 ± 10% of the 45Ca 2+ in 20 ms. CaM reduced the release to 23 ± 12% in the same period. These results are compatible with a direct mechanism of Ca 2+ release channel blockade by CaM and suggest that CaM could play a significant role in the inactivation of SR Ca 2+ release during excitation-contraction coupling.

Inactivating 'ball' peptide from Shaker B blocks Ca(2+)-activated but not ATP-dependent K+ channels of rat skeletal muscle.

1. Shaker B inactivating 'ball' peptide is shown to produce no detectable block of ATP-dependent K+ channels of rat skeletal muscle fibres at concentrations up to 300 microM or membrane potentials up to +30 mV. 2. The peptide does produce a voltage-dependent block of large-conductance Ca(2+)-activated K+ channels at lower concentrations (K1 = 55 microM at 0 mV). An appropriate point mutation (L7E) abolishes block. 3. Mean open times, corrected for missed closures, are little affected by the blocking peptide, but burst durations are substantially reduced. 4. The inactivating peptide increases occupancy of substates, whose amplitudes are 0.27 and 0.64 of fully open.

Blockade by MPTP of the nicotinic acetylcholine receptor Channels in embryonic Xenopus muscle cells

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on nicotinic acetylcholine (ACh) receptor channels were studied in cultured myocytes of 1-day-old Xenopus embryos. The amplitude and decay time of iontophoretic ACh-induced currents were reduced by MPTP in a concentration-dependent manner. The inhibitory action of MPTP was frequency-dependent, being enhanced by higher frequencies of stimulation at 7 and 30 Hz. Neither pargyline nor tranylcypromine affected the inhibitory effects of MPTP on ACh-induced currents. Single channel recordings showed that MPTP decreased the opening frequency, mean open time as well as the conductance of both low- and high-conductance ACh channels. These results suggest that the inhibitory actions of MPTP on ACh receptor channels in skeletal muscle may contribute to the depression of the nerve-evoked contraction of the mouse diaphragm as previously reported.

Dual actions of procainamide on batrachotoxin-activated sodium channels: open channel block and prevention of inactivation

We have investigated the action of procainamide on batrachotoxin (BTX)-activated sodium channels from bovine heart and rat skeletal muscle. When applied to the intracellular side, procainamide induced rapid, open-channel block. We estimated rate constants using amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157). Membrane depolarization increased the blocking rate and slowed unblock. The rate constants were similar in both magnitude and voltage dependence for cardiac and skeletal muscle channels. Qualitatively, this block resembled the fast open-channel block by lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:80), but procainamide was about sevenfold less potent. Molecular modeling suggests that the difference in potency between procainamide and lidocaine might arise from the relative orientation of their aromatic rings, or from differences in the structure of the aryl-amine link. For the cardiac channels, procainamide reduced the frequency of transitions to a long-lived closed state which shows features characteristic of inactivation (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys J. 65:91). Mean durations of kinetically identified closed states were not affected. The degree of fast block and of inhibition of the slow closures were correlated. Internally applied QX-314, a lidocaine derivative and also a fast blocker, produced a similar effect. Thus, drug binding to the fast blocking site appears to inhibit inactivation in BTX-activated cardiac channels.

Dissecting lidocaine action: diethylamide and phenol mimic separate modes of lidocaine block of sodium channels from heart and skeletal muscle

We have investigated block of sodium channels by diethylamide and phenol, which resemble the hydrophilic tertiary amine head and the hydrophobic aromatic tail of the lidocaine molecule, respectively. Diethylamide and phenol separately mimicked the fast and slow modes of block caused by lidocaine. Experiments were performed using single batrachotoxin-activated bovine cardiac and rat skeletal muscle sodium channels incorporated into neutral planar lipid bilayers. Diethylamide, only from the intracellular side, caused a voltage-dependent reduction in apparent single channel amplitude ('fast' block). Block was similar for cardiac and skeletal muscle channels, and increased in potency when extracellular sodium was replaced by N-methylglucamine, consistent with an intrapore blocking site. Thus, although occurring at 15-fold higher concentrations, block by diethylamide closely resembles the fast mode of block by lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:80-90). For cardiac sodium channels, phenol bound to a closed state causing the appearance of long blocked events whose duration increased with phenol concentration. This slow block depended neither on voltage nor on the side of application, and disappeared upon treatment of the channel with trypsin. For skeletal muscle channels, slow phenol block occurred with only very low probability. Thus, phenol block resembles the slow mode of block observed for lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:91-100). Our data suggest that there are separate sites for fast lidocaine block of the open channel and slow block of the "inactivated" channel. Fast block by diethylamide inhibited the long, spontaneous, trypsin-sensitive (inactivation-like) closures of cardiac channels, and hence secondarily antagonized slow block by phenol or lidocaine. This antagonism would potentiate shifts in the balance between the two modes of action of a tertiary amine drug caused by changes in the relative concentrations of the charged (fast blocking) and neutral (slow blocking) forms of the drug.

Blockers and activators for stretch-activated ion channels of chick skeletal muscle.

Annals of the New York Academy of SciencesVolume 707, Issue 1 p. 417-420 Blockers and Activators for Stretch-Activated Ion Channels of Chick Skeletal Musclea MASAHIRO SOKABE, MASAHIRO SOKABE Department of Physiology Nagoya University School of Medicine 65 Tsurumai Nagoya 466, JapanSearch for more papers by this authorNOBORU HASEGAWA, NOBORU HASEGAWA Department of Physiology Nagoya University School of Medicine 65 Tsurumai Nagoya 466, JapanSearch for more papers by this authorKIMIKO YAMAMORI, KIMIKO YAMAMORI Department of Physiology Nagoya University School of Medicine 65 Tsurumai Nagoya 466, JapanSearch for more papers by this author MASAHIRO SOKABE, MASAHIRO SOKABE Department of Physiology Nagoya University School of Medicine 65 Tsurumai Nagoya 466, JapanSearch for more papers by this authorNOBORU HASEGAWA, NOBORU HASEGAWA Department of Physiology Nagoya University School of Medicine 65 Tsurumai Nagoya 466, JapanSearch for more papers by this authorKIMIKO YAMAMORI, KIMIKO YAMAMORI Department of Physiology Nagoya University School of Medicine 65 Tsurumai Nagoya 466, JapanSearch for more papers by this author First published: December 1993 https://doi.org/10.1111/j.1749-6632.1993.tb38086.xCitations: 17 a Supported by grants from the Ministry of Education, Science and Culture of Japan (0344073, 03454558, 04263291). AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume707, Issue1Molecular Basis of Ion Channels and Receptors Involved in Nerve Excitation, Synaptic Transmission, and Muscle ContractionDecember 1993Pages 417-420 RelatedInformation

Block by ruthenium red of the ryanodine-activated calcium release channel of skeletal muscle.

The effects of ruthenium red and the related compounds tetraamine palladium (4APd) and tetraamine platinum (4APt) were studied on the ryanodine activated Ca2+ release channel reconstituted in planar bilayers with the immunoaffinity purified ryanodine receptor. Ruthenium red, applied at submicromolar concentrations to the myoplasmic side (cis), induced an all-or-none flickery block of the ryanodine activated channel. The blocking effect was strongly voltage dependent, as large positive potentials that favored the movement of ruthenium red into the channel conduction pore produced stronger block. The half dissociation constants (Kd) for ruthenium red block of the 500 pS channel were 0.22, 0.38, and 0.62 microM, at +100, +80, and +60 mV, respectively. Multiple ruthenium red molecules seemed to be involved in the inhibition, because a Hill coefficient of close to 2 was obtained from the dose response curve. The half dissociation constant of ruthenium red block of the lower conductance state of the ryanodine activated channel (250 pS) was higher (Kd = 0.82 microM at +100 mV), while the Hill coefficient remained approximately the same (nH = 2.7). Ruthenium red block of the channel was highly asymmetric, as trans ruthenium red produced a different blocking effect. The blocking and unblocking events (induced by cis ruthenium red) can be resolved at the single channel level at a cutoff frequency of 2 kHz. The closing rate of the channel in the presence of ruthenium red increased linearly with ruthenium red concentration, and the unblocking rate of the channel was independent of ruthenium red concentrations. This suggests that ruthenium red block of the channel occurred via a simple blocking mechanism. The on-rate of ruthenium red binding to the channel was 1.32 x 10(9) M-1 s-1, and the off-rate of ruthenium red binding was 0.75 x 10(3) s-1 at +60 mV, in the presence of 200 nM ryanodine. The two related compounds, 4APd and 4APt, blocked the channel in a similar way to that of ruthenium red. These compounds inhibited the open channel with lower affinities (Kd = 170 microM, 4APd; Kd = 656 microM, 4APt), and had Hill coefficients of close to 1. The results suggest that ruthenium red block of the ryanodine receptor is due to binding to multiple sites located in the conduction pore of the channel.

Genotype-phenotype correlations in human skeletal muscle sodium channel diseases.

Over the past 3 years, the genetics of the myotonic diseases have been substantially elaborated. Three genetically different groups of myotonic disease can be discerned: (1) the chloride channel myotonias, (2) the adynamia-paramyotonia complex, and (3) myotonic dystrophy. Electrophysiology has suggested and molecular biology has proven that the diseases belonging to the adynamia-paramyotonia complex, ie, paramyotonia congenita, hyperkalemic and normokalemic periodic paralysis, and some rare forms of myotonic disease, are caused by point mutations in the gene encoding the alpha subunit of the sodium channel in adult human skeletal muscle, located on chromosome 17q23. Thirteen different mutations have been described by various groups in the United States and Germany. The various mutations causing a particular form of the complex are not located in the gene in a predictable or easily understandable regular manner. Further study of the genotype-phenotype correlations should not only increase our understanding of the variability of signs in this group of diseases, it could also provide us with a deeper insight in the function of the various regions of the sodium channel protein.

A possible role of sarcoplasmic Ca2+ release in modulating the slow Ca2+ current of skeletal muscle.

Ca2+ channels are regulated in a variety of different ways, one of which is modulation by the Ca2+ ion itself. In skeletal muscle, Ca2+ release sites are presumably located in the vicinity of the dihydropyridine-sensitive Ca2+ channel. In this study, we have tried to investigate the effects of Ca2+ release from the sarcoplasmic reticulum on the L-type Ca2+ channel in frog skeletal muscle, using the double Vaseline gap technique. We found an increase in Ca2+ current amplitude on application of caffeine, a well-known potentiator of Ca2+ release. Addition of the fast Ca2+ buffer BAPTA to the intracellular solution led to a gradual decline in Ca2+ current amplitude and eventually caused complete inhibition. Similar observations were made when the muscle fibre was perfused internally with the Ca2+ release channel blocker ruthenium red. The time course of Ca2+ current decline followed closely the increase in ruthenium red concentration. This suggests that Ca2+ release from the sarcoplasmic reticulum is involved in the regulation of L-type Ca2+ channels in frog skeletal muscle.

Characterization of [ 3H]Ryanodine Binding Sites in Smooth Muscle of Dog Mesentery Artery

The ryanodine receptor has been widely demonstrated to be a Ca 2+ release channel in both skeletal and cardiac muscle. Ryanodine also functionally affects Ca 2+ release from an intracellular pool of smooth muscle. In the present study, both high and low affinity ryanodine binding sites are identified in dog mesenteric artery (with Kd 5 nM and 269 nM, respectively). [ 3H]ryanodine binding is not correlated with the density of innervation in different smooth muscles. This study provides direct evidence of the presence of ryanodine binding sites in dog mesentery artery. These ryanodine binding sites are of smooth muscle origin.

Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel, although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles

Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs(+)-conducting medium. We found that the pharmacology, Cs+ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10(-7) to 10(-3) M Ca2+, but the proportion of closed and open states shifted to shorter closings and openings, respectively. However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca2+, respectively, the cardiac SR channel remained activated in the presence of high [Ca2+]. In correlation to these studies, [3H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca2+] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [3H]ryanodine binding of cardiac SR at millimolar Ca2+ in the absence of adenine nucleotides. When Ca(2+)-induced Ca2+ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca(2+)-induced inactivation at high [Ca2+] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca2+ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca(2+)-inactivated site.

Molecular properties of calcium channels in skeletal muscle and neurons.

Molecular properties of calcium channels in skeletal muscle and neurons.

Pharmacological properties of ATP-sensitive K + channels in mammalian skeletal muscle cells

The patch-clamp technique (single-channel recordings) was used to study the effects of glibenclamide and some channel openers on the K ATP channel in mouse skeletal muscle. In outside/out membrane patches, glibenclamide reversibly inhibited K ATP channel activity in a dose-dependent manner with an apparent K i of 190 nM. In inside/out membrane patches, RP 61419 increased K ATP channel activity both in the absence and in the presence of internal ATP while other K + channel openers such as nicorandil and cromakalim required the presence of internal ATP to evoke channel activation. The half-maximal activity effect for cromakalim, with 0.5 mM ATP at the cytoplasmic face, was observed at about 220 μM. Pinacidil was unable to activate the K ATP channel in the absence of internal ATP and could even reduce channel opening in situations where activity was high in the control. In the presence of internal Mg 2+, activation by pinacidil occurred when ATP or low and weakly activating concentrations of ADP were present at the cytoplasmic side. Pinacidil activation could also be observed in the presence of ATP or ADP when Mg 2+ was absent from the internal solution. The mechanism of action of pinacidil is discussed in terms of interactions between the different nucleotide regulatory sites and the K + channel opener binding site of the K ATP channel. Half-maximum activation of the K ATP channel in the presence of 0.5 mM ATP at the cytoplasmic face was observed at 125 μM pinacidil.

A brain-specific transcript from the 3′-terminal region of the skeletal muscle ryanodine receptor gene

We have shown previously that the skeletal muscle ryanodine receptor mRNA of ∼16,000 nucleotides codes 5,037 amino acid residues constituting the calcium release channel in skeletal muscle. In this study, RNA blot hybridization analysis shows that the brain contains an RNA species with an estimated size of ∼2,400 nucleotides hybridizable with the 3′-terminal region of the skeletal muscle ryanodine receptor cDNA. cDNA cloning and genome analysis indicated that two transcripts differing in their start sites are produced from the skeletal muscle ryanodine receptor gene in a tissue-specific fashion, and that the mRNA in brain may code the carboxyl-terminal region of the ryanodine receptor molecule. cDNA expression experiments suggested that the ATG triplet encoding Met 4382 of the skeletal muscle ryanodine receptor can function as a translation initiation codon, and that the expressed protein composed of the carboxy terminal 656 amino acid residues of the receptor is located on the endoplasmic reticulum membrane.

ATP-sensitive potassium channels in smooth muscle cells from guinea pig urinary bladder.

We explored the possibility that ATP-sensitive potassium (KATP) channels exist in urinary bladder smooth muscle, since synthetic openers (e.g., lemakalim) of KATP channels in other tissues relax bladder smooth muscle. Unitary currents through single potassium channels and whole cell potassium currents were measured in smooth muscle cells isolated from the detrusor muscle of the guinea pig bladder. Lemakalim (10 microM) increased whole cell K+ currents by 50 pA at -80 mV with 60 mM external K+ when the cells were dialyzed with 0.1 mM ATP and 140 mM K+. Glibenclamide (10 microM), a sulfonylurea blocker of KATP channels in other tissues, inhibited the entire lemakalim-stimulated current as well as 19 pA of the steady-state K+ current. Glibenclamide-sensitive K+ currents were not dependent on voltage. Increasing intracellular ATP from 0.1 to 3.0 mM reduced the glibenclamide-sensitive K+ current in both the presence and absence of lemakalim by about fourfold. External barium (100 microM) which blocks KATP channels in skeletal muscle reduced KATP channel currents in bladder smooth muscle by 50% at -80 mV. Lemakalim (10 microM) increased the open-state probability of single K+ channels in outside-out patches (with 0.1 mM internal ATP) by sixfold. The single-channel conductance was approximately 7 pS at 0 mV with a physiological K+ gradient. This single-channel conductance was in accord with estimates of conductance made from noise analysis of the lemakalim-induced whole cell current. Glibenclamide inhibited these channels. The number of channels per cell was estimated to be approximately 425. We conclude that urinary bladder smooth muscle has KATP channels and that these channels can be opened by the K+ channel opening drug, lemakalim, and blocked by external glibenclamide and barium. We propose that modulation of these channels may regulate bladder contractility.

Single potassium channel currents activated by extracellular ATP in developing chick skeletal muscle: a role for second messengers

1. In developing chick skeletal muscle, extracellular ATP activates an early excitatory current and a delayed potassium current. Previous work had shown that the potassium current elicited by ATP is sensitive to temperature and activates with a delay of nearly 1 s, suggesting that a second messenger is involved. The existence of a second messenger was confirmed by the observation that single potassium channels were activated in cell-attached patches, when ATP was applied outside of the patch pipette. 2. Two classes of ATP-activated potassium-channel currents were observed in cell-attached patches: one had a slope conductance of 23 pS, whereas the other had a slope conductance of 51 pS. 3. Pharmacological manipulations suggested that activation of the whole-cell potassium current by ATP did not require cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), inositol 1,4,5-triphosphate (IP3), nitric oxide, or a rise in internal free calcium. Additional pharmacological experiments suggested that activation of the whole-cell potassium current might not require activation of a G protein and probably did not involve intracellular protein phosphorylation. 4. The ability of arachidonic acid and its metabolites to activate potassium channels in chick skeletal muscle was also tested. Arachidonic acid, several prostaglandins and several leukotrienes activated whole-cell potassium currents. However, results with several inhibitors suggested that arachidonic acid and its metabolites are not necessary for activation of the whole-cell potassium current by ATP. 5. In excised outside-out membrane patches, ATP activated a single class of potassium channels. The slope conductance of these channels indicated that they are likely to be identical to the smaller of the two classes of second messenger activated potassium channels observed in cell-attached patches. 6. The observation that the larger class of potassium channels observed in cell-attached patches was absent in excised patches suggests that activation of these channels by ATP requires a cytosolic factor that is easily dialyzed away. In contrast, the observation that the smaller class of potassium channels could still be activated by ATP in excised patches suggests that the two classes of potassium channels are activated by different mechanisms. These results also indicate that all the molecules involved in coupling ATP receptor activation to opening of the smaller class of potassium channels remain closely associated with an excised patch. One possible explanation is that there might be an intramembranous second messenger.

Characterization of a Cl−-Channel from Rabbit Transverse Tubules in the Planar Lipid Bilayer System

A Cl−-channel of the transverse tubule(TT) was incorporated into artificial planar lipid bilayers and its properties were investigated. The channel was found to have the following properties. (1) The single-channel conductance is 40 pS in choline-Cl solution. (2) The rate of gating of the channel does not depend on the membrane voltage. (3) The channel is blocked by stilbene derivatives (DIDS and SITS), which are known as inhibitors of voltage-dependent Cl−-channels of the Torpedo electric organ, from both sides of the membrane. (4) An inhibitor of voltage-dependent Cl−-channels of skeletal muscle, 9-anthracene carboxylic acid(9-AC), inhibits Cl−-current from the cis-side to which the TT vesicles were added.

Fab fragments from amyotrophic lateral sclerosis IgG affect calcium channels of skeletal muscle.

Amyotrophic lateral sclerosis (ALS) is a human disease involving upper and lower motoneurons. In this paper we studied the action of specific antigen-binding site (Fab) fragments of immunoglobulins from ALS patients on dihydropyridine (DHP)-sensitive Ca2+ channel function in situ. Ca2+ channels in single mammalian skeletal muscle fibers tested by the double Vaseline gap technique and single Ca2+ channels reconstituted into bilayer were tested in these experiments. Although the observed current-voltage relationship was not modified by the addition of Fab fragments (1.5 mg/ml), peak Ca2+ current (ICa) was significantly reduced. The effect of these Fab fragments on the peak ICa reached a stable value after 60 min of incubation. ALS Fab fragments also slowed the ICa rising phase and increased the rate of tail current deactivation. Studies with double pulses demonstrated that ICa inactivation time course, voltage dependence, and recovery were not modified by ALS Fab fragments. Fab fragments from normal subjects and heat-inactivated Fab fragments from ALS patients did not induce any modification on the charge movement and ICa. In single channel studies, ALS Fab fragments reduced channels amplitude. These data support the concept of an immunological interaction between the circulating antibodies from ALS patients and DHP-sensitive Ca2+ channels.