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1.
J Physiol ; 535(Pt 1): 83-93, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11507159

ABSTRACT

1. In the present work we investigated the dependence on temperature of the ionic conductance and gating of human muscle ClC-1 chloride channels, transiently expressed in human embryonic kidney (HEK 293) cells. 2. At normal pH, ClC-1 currents deactivated at negative potentials with a double-exponential time course. The time constants of the exponential components, corresponding to the relaxations of the fast and slow gates, were temperature dependent with Q(10) values of approximately 3 and approximately 4, respectively. Current amplitude increased with increasing temperature with a Q(10) of approximately 1.6. 3. The voltage dependence of the two gating processes was shifted towards more positive potentials with increasing temperature. The half-saturation voltage (V(1/2)) of the steady-state open probability (P(o)) was shifted by approximately 23 and approximately 34 mV per 10 degrees C increase in temperature, for the fast and slow gate, respectively. 4. At low pH, the voltage dependence of ClC-1 was reversed and currents were activated by hyperpolarisation with a single-exponential time course. This type of gating in ClC-1 resembled the slow gating of the Torpedo ClC-0 homologue, but differed with respect to its kinetics and temperature dependence, with a Q(10) of gating relaxations at negative potentials of approximately 5. The Arrhenius plot of ClC-1 conductance at low pH had a clear break point at approximately 25 degrees C, with higher Q(10) values at lower temperatures. 5. The temperature sensitivity of relaxation and open probability of the slow gate, which in both ClC-0 and ClC-1 controls two pores simultaneously, implies that the slow gating of ClC-1 is mechanistically different from that of ClC-0.


Subject(s)
Chloride Channels/physiology , Temperature , Cell Line , Electric Conductivity , Humans , Hydrogen-Ion Concentration , Ion Channel Gating , Time Factors
2.
Mol Pharmacol ; 60(1): 200-8, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11408615

ABSTRACT

Our knowledge about ClC-1 muscle chloride channel gating, previously gained from single-channel recording and noise analysis, provides a theoretical basis for further analysis of macroscopic currents. In the present study, we propose a simple method of calculation of open probabilities (P(o)) of fast and slow gates from the relative amplitudes of ClC-1 inward current components. With this method, we investigated the effects of 2-(4-chlorophenoxy) propionic acid (CPP), a drug known to produce myotonia in animals, and dominant negative myotonic mutations, F307S and A313T, on fast and slow gating of ClC-1. We have shown that these mutations affected the P(o) of the slow gate, as expected from their mode of inheritance, and that CPP predominantly affected the fast gating process. CPP's action on the fast gating of mutant channels was similar to its effect in wild-type channels. Comparison of the effects of CPP and the mutations on fast and slow gating with the effects produced by reduction of external Cl(-) concentration suggested that CPP and mutations exert their action by affecting the transition of the channel from its closed to open state after Cl(-) binding to the gating site.


Subject(s)
2-Methyl-4-chlorophenoxyacetic Acid/pharmacology , Chloride Channels/metabolism , 2-Methyl-4-chlorophenoxyacetic Acid/analogs & derivatives , Binding, Competitive , Cells, Cultured , Chloride Channels/drug effects , Chloride Channels/genetics , Chloride Channels/physiology , Electrophysiology , Humans , Kinetics , Mutagenesis, Site-Directed
3.
J Physiol ; 530(Pt 3): 379-93, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11158270

ABSTRACT

Permeation of a range of hydrophobic anions through the rat skeletal muscle chloride channel, rClC-1, expressed in Sf-9 (a Spodoptera frugiperda insect cell line) cells has been studied using the whole-cell patch-clamp technique. Bi-ionic reversal potentials measured with external application of foreign anions gave the following permeability sequence: Cl- (1) > benzoate (0.15) > hexanoate (0.12) > butyrate (0.09) > propionate (0.047) approximately formate (0.046). Anions with larger hydrophobic moieties were more permeant, which suggested that ClC-1 selectivity for hydrophobic anions is dominated by their interaction with a hydrophobic region in the external mouth of the pore. All anions studied when applied from outside show an apparently paradoxical voltage-dependent block of inward currents; this voltage-dependent block could be qualitatively described by a discrete-state permeation model with two binding sites and three barriers. Effects of the external anions with aliphatic side-chains on the apparent open probability (Po) suggested that they are unable to gate the channel, but can modulate ClC-1 gating, probably, by changing Cl- affinity to the gating site. Effects of internal application of benzoate, hexanoate or propionate mimicked those of increasing internal pH, and similarly depended on the channel protonation from the external side. Results for internal benzoate support the concept of a negatively charged cytoplasmic particle being involved in the ClC-1 gating mechanism sensitive to the internal pH.


Subject(s)
Chloride Channels/physiology , Muscle, Skeletal/physiology , Animals , Anions/metabolism , Benzoates/pharmacology , Cell Line , Chloride Channels/drug effects , Chloride Channels/genetics , Hydrogen-Ion Concentration , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Kinetics , Membrane Potentials/physiology , Patch-Clamp Techniques , Rats , Spodoptera , Structure-Activity Relationship , Transfection
4.
Br J Pharmacol ; 126(6): 1375-82, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10217531

ABSTRACT

1. Using whole-cell patch-clamping and Sf-9 cells expressing the rat skeletal muscle chloride channel, rCIC-1, the cellular mechanism responsible for the myotonic side effects of clofibrate derivatives was examined. 2. RS-(+/-) 2-(4-chlorophenoxy)propionic acid (RS-(+/-) CPP) and its S-(-) enantiomer produced pronounced effects on CIC-1 gating. Both compounds caused the channels to deactivate more rapidly at hyperpolarizing potentials, which showed as a decrease in the time constants of both the fast and slow deactivating components of the whole cell currents. Both compounds also produced a concentration-dependent shift in the voltage dependence of channel apparent open probability to more depolarizing potentials, with an EC50 of 0.79 and 0.21 mM for the racemate and S-(-) enantiomer respectively. R-(+) CPP at similar concentrations had no effect on gating. RS-(+/-) CPP did not block the passage of Cl- through the pore of rCIC-1. 3. CIC-1 is gated by Cl- binding to a site within an access channel and S-(-) CPP alters gating of the channel by decreasing the affinity of this binding site for Cl-. Comparison of the EC50 for RS-(+/-) CPP and S-(-) CPP indicates that R-(+) CPP can compete with the S-(-) enantiomer for the site but that it is without biological activity. 4. RS-(+/-) CPP produced the same effect on rCIC-1 gating when added to the interior of the cell and in the extracellular solution. 5. S-(-) CPP modulates the gating of CIC-1 to decrease the membrane Cl- conductance (GCl), which would account for the myotonic side effects of clofibrate and its derivatives.


Subject(s)
2-Methyl-4-chlorophenoxyacetic Acid/analogs & derivatives , Chloride Channels/drug effects , Ion Channel Gating/drug effects , Muscle Proteins/drug effects , 2-Methyl-4-chlorophenoxyacetic Acid/chemistry , 2-Methyl-4-chlorophenoxyacetic Acid/pharmacology , Animals , Cell Line , Chloride Channels/physiology , Chlorides/pharmacology , Dose-Response Relationship, Drug , Electric Stimulation , Hydrogen-Ion Concentration , Membrane Potentials/drug effects , Muscle Proteins/physiology , Patch-Clamp Techniques , Rats , Stereoisomerism
5.
Hum Mol Genet ; 7(11): 1753-60, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9736777

ABSTRACT

Mutations in the ClC-1 muscle chloride channel cause either recessive or dominant myotonia congenita. Using a systematic screening procedure, we have now identified four novel missense mutations in dominant (V286A, F307S) and recessive myotonia (V236L, G285E), and have analysed the effect of these and other recently described mutations (A313T, I556N) on channel properties in the Xenopus oocyte expression system. Mutations V286A, F307S and A313T displayed a 'classical' dominant phenotype: their voltage dependence was shifted towards positive potentials and displayed a dominant-negative effect by significantly imparting a voltage shift on mutant-wild-type heteromeric channels as found in heterozygous patients. In contrast, the recessive mutation V236L also shifted the voltage dependence to positive values, but co-expression with wild-type ClC-1 gave almost wild-type currents. I556N, a mutation found in patients with benign dominant myotonia, drastically shifts the voltage dependence, but only a slight shift is seen when co-expressed with wild-type ClC-1. Thus, the voltage dependence of mutant heteromeric channels is not always intermediate between those of the constituent homomeric channel subunits, a conclusion further supported by mixing different ClC-1 mutants. These complex interactions correlate clinically with various inheritance patterns, ranging from autosomal dominant with various degrees of penetrance to autosomal recessive.


Subject(s)
Chloride Channels/genetics , Chloride Channels/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , Mutation , Myotonia Congenita/genetics , Amino Acid Sequence , Animals , DNA Mutational Analysis/methods , Electrophysiology/methods , Female , Genes, Dominant , Genes, Recessive , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Oocytes/physiology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
6.
J Gen Physiol ; 111(5): 653-65, 1998 May.
Article in English | MEDLINE | ID: mdl-9565403

ABSTRACT

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl- as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO3-, BrO3-); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl- at the regulatory binding site but impair Cl- passage through the channel pore (Br-, NO3-, ClO3-, I-, ClO4-, SCN-). The permeability sequence for rClC-1, SCN- approximately ClO4- > Cl- > Br- > NO3- approximately ClO3- > I- >> BrO3- > HCO3- >> methanesulfonate approximately cyclamate approximately glutamate, was different from the sequence determined for blocking potency and ability to shift the Popen curve, SCN- approximately ClO4- > I- > NO3- approximately ClO3- approximately methanesulfonate > Br- > cyclamate > BrO3- > HCO3- > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be approximately 4.5 A. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl- and SCN- or ClO4- suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.


Subject(s)
Anions/metabolism , Chloride Channels/metabolism , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Animals , Binding, Competitive/physiology , Cell Line , Chloride Channels/physiology , Electric Conductivity , Humans , Insecta/cytology , Kinetics , Muscle Proteins/physiology , Permeability , Rats
7.
J Biol Chem ; 273(8): 4304-7, 1998 Feb 20.
Article in English | MEDLINE | ID: mdl-9468477

ABSTRACT

Although hydropathy analysis of the skeletal muscle chloride channel protein, ClC-1, initially predicted 13 potential membrane spanning domains (D1 to D13), later topological studies have suggested that domain D4 is extracellular and that D13, conserved in all eukaryotic ClC channels, is located within the extensive cytoplasmic tail that makes up the carboxyl terminus of the protein. We have examined the effect of deleting D13 (DeltaD13) and the function of the carboxyl tail by removing the final 72 (fs923X), 100 (fs895X), 125 (L869X), 398 (N596X), and 420 (Q574X) amino acids from rat ClC-1. Appropriate cDNA constructs were prepared and expressed using the baculovirus Sf9 insect cell system. Patch clamp analysis of chloride currents in Sf9 cells showed that only relatively insubstantial changes could be attributed to the expressed fs923X, fs895X, and DeltaD13 mutants compared with wild type rat ClC-1. For N596X and Q574X, however, adequate mRNA could be detected, but neither patch clamp nor polyacrylamide gel electrophoresis showed corresponding protein production. By contrast, expression of L869X was demonstrable by polyacrylamide gel electrophoresis, but no chloride conductance attributable to it could be detected. Overall, our results indicate that the domain D13 is dispensable, as are the final 100 amino acids, but not the final 125 amino acids or more, of the carboxyl tail. Some essential region of unknown significance, therefore, appears to reside in the 18 amino acids after D13, from Lys877 to Arg894.


Subject(s)
Chloride Channels/metabolism , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Amino Acid Sequence , Animals , Baculoviridae/genetics , Chloride Channels/chemistry , Chloride Channels/genetics , Kinetics , Molecular Sequence Data , Muscle Proteins/chemistry , Muscle Proteins/genetics , Mutagenesis , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Spodoptera
8.
J Physiol ; 501 ( Pt 2): 355-62, 1997 Jun 01.
Article in English | MEDLINE | ID: mdl-9192307

ABSTRACT

1. Gating of the skeletal muscle chloride channel (ClC-1) is sensitive to extracellular pH. In this study, whole-cell recording of currents from wild-type (WT) ClC-1 and a mutant, R304E, expressed in the Sf-9 insect cell line was used to investigate further the nature of the pH-sensitive residues. 2. Extracellular Cd2+ produced a concentration-dependent block of WT ClC-1 with an IC50 of 1.0 +/- 0.1 mM and a Hill coefficient of 2.0 +/- 0.3. This block was sensitive to external pH, reducing at low pH, with an apparent pKa of 6.8 +/- 0.1 and a Hill coefficient for proton binding of 3.0 +/- 0.3. Anthracene-9-carboxylate (A-9-C) block of WT ClC-1 was also pH sensitive, increasing at low pH, with an apparent pKa of 6.4 +/- 0.1 and a Hill coefficient for proton binding of 1.0 +/- 0.2. 3. Compared with WT ClC-1, R304E had a lower affinity for Cd2+ (IC50, 3.0 +/- 0.3 mM) but it had a similar Hill coefficient for transition metal ion binding. The Hill coefficient for proton binding to the Cd2+ binding site was reduced to 1.4 +/- 0.3. In contrast, the A-9-C binding site in R304E showed the same pH sensitivity and affinity for the blocker as that seen in WT ClC-1. 4. ClC-1 has at least two binding sites for Cd2+, each of which has at least three residues which can be protonated. Binding of A-9-C is influenced by protonation of a single residue. Arg 304 is not sufficiently close to the A-9-C binding site to affect its characteristics, but it does. alter Cd2+ binding, indicating that transition metal ions and aromatic carboxylates interact with distinct sites. 5. The block of ClC-1 by transition metal ions and the apparent pKa of this block, together with the apparent pKa for A-9-C block and gating are all compatible with the involvement of His residues in the pore and gate of ClC-1.


Subject(s)
Cadmium/pharmacology , Chloride Channels/metabolism , Insecta/physiology , Mutation/physiology , Animals , Cell Line , Chloride Channels/drug effects , Chloride Channels/genetics , Electrophysiology , Hydrogen-Ion Concentration , Ion Channel Gating/drug effects , Membrane Potentials/physiology , Metals/pharmacology , Mutagenesis, Site-Directed , Patch-Clamp Techniques , Point Mutation/physiology , Rats
9.
J Physiol ; 497 ( Pt 2): 423-35, 1996 Dec 01.
Article in English | MEDLINE | ID: mdl-8961185

ABSTRACT

1. The influence of Cl- concentration and pH on gating of the skeletal muscle Cl- channel, ClC-1, has been assessed using the voltage-clamp technique and the Sf-9 insect cell and Xenopus oocyte expression systems. 2. Hyperpolarization induces deactivating inward currents comprising a steady-state component and two exponentially decaying components, of which the faster is weakly voltage dependent and the slower strongly voltage dependent. 3. Open probability (Po) and kinetics depend on external but not internal Cl- concentration. 4. A point mutation, K585E, in human ClC-1, equivalent to a previously described mutation in the Torpedo electroplaque chloride channel, ClC-0, alters the I-V relationship and kinetics, but retains external Cl- dependence. 5. When external pH is reduced, the deactivating inward currents of ClC-1 are diminished without change in time constants while the steady-state component is enhanced. 6. In contrast, reduced internal pH slows deactivating current kinetics as its most immediately obvious action and the Po curve is shifted in the hyperpolarizing direction. Addition of internal benzoate at low internal pH counteracts both these effects. 7. A current activated by hyperpolarization can be revealed at an external pH of 5.5 in ClC-1, which in some ways resembles currents due to the slow gates of ClC-0. 8. Gating appears to be controlled by a Cl(-)-binding site accessible only from the exterior and, possibly, by modification of this site by external protonation. Intracellular hydroxyl ions strongly affect gating either allosterically or by direct binding and blocking of the pore, an action mimicked by intracellular benzoate.


Subject(s)
Chloride Channels/genetics , Muscle, Skeletal/chemistry , Animals , Benzoates/pharmacology , Benzoic Acid , Chloride Channels/metabolism , Chlorides/metabolism , Chlorides/pharmacology , Dose-Response Relationship, Drug , Female , Food Preservatives/pharmacology , Hydrogen-Ion Concentration , Ion Channel Gating/physiology , Kinetics , Membrane Potentials/drug effects , Membrane Potentials/physiology , Muscle, Skeletal/metabolism , Mutation/physiology , Oocytes/physiology , Patch-Clamp Techniques , Rats , Xenopus
10.
Biochim Biophys Acta ; 1280(2): 178-86, 1996 Apr 26.
Article in English | MEDLINE | ID: mdl-8639692

ABSTRACT

Using the baculovirus system, the skeletal muscle chloride channel, CIC-1 (rat), and a point mutant replacing arginine 304 with glutamic acid were expressed at high levels in cultured Sf-9 insect cells. Whole-cell patch-clamping revealed large inwardly rectifying currents with maxima up to 15 nA inward and 2.5 nA outward. Saturation was evident at voltage steps positive to +40 mV whilst steps negative to -60 mV produced inactivating currents made up of a steady state component and two exponentially decaying components with tau 1 = 6.14+/- 0.92 ms, tau 2 = 36.5+/- 3.29 ms (S.D) n = 7 for steps to -120 mV. Currents recorded in the outside-out patch configuration were often unexpectedly large and up to 5% of whole-cell currents obtained in the same cell, suggesting an uneven channel distribution in the plasmalemma of Sf-9 cells. The pharmacology of a number of chloride channel blockers, including anthracene-9-carboxylate (A9C), niflumate, and perrhenate, was investigated and showed for the first time that perrhenate is an effective blocker of C1C-1 and that it has a complex mechanism of action. Further, the potency of A9C was found to be dependent on external chloride concentration. As in studies on muscle cells themselves, blockade was rapidly effective and easily reversible, except when applying the indanyloxyacetate derivative, IAA94/95, which took up to 10 min to act, and, consistent with an intracellular site of action, was difficult to reverse by washing. Mutation of the highly conserved arginine at position 304 to a glutamic acid did not significantly alter the behaviour of the channel.


Subject(s)
Chloride Channels/metabolism , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Point Mutation , Animals , Baculoviridae/genetics , Base Sequence , Cell Line , Chloride Channels/genetics , Chloride Channels/physiology , Cloning, Molecular , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Membrane Potentials , Molecular Sequence Data , Muscle Proteins/genetics , Muscle Proteins/physiology , Rats , Spodoptera
11.
Neurosci Lett ; 181(1-2): 83-6, 1994 Nov 07.
Article in English | MEDLINE | ID: mdl-7898777

ABSTRACT

Patch-clamp studies of ion channels in the sarcoball membrane, a relatively pure preparation of sarcoplasmic reticulum, had earlier revealed a high-conductance anion channel with some properties similar to the mitochondrial voltage-dependent anion channel (VDAC). Using post-embedding immunolabelling, the presence of VDAC was investigated in sarcoball preparations from the semitendinosus muscle of the cane toad Bufo marinus. As expected, the outer membrane of mitochondria found within the interior of skinned fibres was decorated with gold label. Surprisingly, sarcoplasmic reticulum membrane was also labelled. The sarcoball membranes, which could arise from either the sarcoplasmic reticulum or from mitochondria, were also labelled. These results indicate the presence of a VDAC-like protein in the sarcoplasmic reticulum.


Subject(s)
Membrane Proteins/metabolism , Mitochondria, Muscle/metabolism , Muscle, Skeletal/metabolism , Porins , Sarcoplasmic Reticulum/metabolism , Animals , Bufo marinus , Immunohistochemistry , Voltage-Dependent Anion Channels
12.
Infect Immun ; 62(9): 4054-8, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7520425

ABSTRACT

Two outer membrane proteins of Aeromonas hydrophila A6, isolated in a one-step affinity chromatography process based on carbohydrate reactivity, were found to be pore-forming molecules in artificial planar bilayer membranes. These carbohydrate-reactive outer membrane proteins (CROMPs; M(r)s, 40,000 and 43,000) were subjected to amino acid analysis. The amino acid profiles for these two outer membrane proteins were almost identical. A partial protein sequence of a 14-amino-acid fragment of the 40,000-Da protein revealed homology with outer membrane porins of Escherichia coli and A. hydrophila. CROMPs were compared with carbohydrate-reactive porins also extracted from outer membranes of A. hydrophila A6. These porins were isolated by using standard porin purification techniques (insolubility in 2% sodium dodecyl sulfate, solubility in 0.4 M NaCl, and Sephacryl S-200 gel filtration), and then Synsorb H type 2 affinity chromatography was done. The physical and functional properties of the carbohydrate-reactive porins and CROMPs were found to be identical. On the basis of pore-forming properties in planar lipid bilayers and channel inhibition with maltotriose solutions, a nonspecific, general diffusion porin and a LamB-like maltoporin were identified in both CROMP and carbohydrate-reactive porin preparations. To our knowledge, the use of carbohydrate reactivity to isolate channel-forming proteins from bacterial outer membranes has not been reported previously.


Subject(s)
Aeromonas hydrophila/chemistry , Carbohydrates/pharmacology , Porins/analysis , Aeromonas hydrophila/pathogenicity , Bacterial Outer Membrane Proteins , Carbohydrate Sequence , Ion Channels/drug effects , Molecular Sequence Data , Receptors, Virus/analysis , Virulence , Water Microbiology
13.
J Neurol Neurosurg Psychiatry ; 57(3): 344-7, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8158184

ABSTRACT

Four members of a family in three generations exhibited unusual clinical features of localised transient swelling of muscle induced by percussion (muscle mounding or myoedema) and were able, voluntarily, to induce rhythmic waves of contraction in certain muscles (muscle rippling or rolling). All had raised serum creatine kinase activity. Muscle biopsy in two members showed no specific abnormality. Experimental studies performed on excised intercostal muscle showed that abnormal "after-contractions" and increased sarcolemmal excitability could be demonstrated in vitro.


Subject(s)
Muscle Contraction , Muscles/physiopathology , Muscular Diseases/genetics , Action Potentials , Adult , Child , Creatine Kinase/blood , Humans , In Vitro Techniques , Intercostal Muscles/physiopathology , Male , Middle Aged , Muscular Diseases/diagnosis , Muscular Diseases/physiopathology , Sarcolemma/physiology
14.
J Physiol ; 470: 365-82, 1993 Oct.
Article in English | MEDLINE | ID: mdl-7508505

ABSTRACT

1. A putative function of the transient outward current (ITO) in cardiac myocytes is to modulate the shape of the action potential and, consequently, cardiac contractility. In addition, it has been suggested that this current may help protect against arrhythmias during periods of cardiac ischaemia. In our investigation of the possible anti-arrhythmic action of ITO, we have examined its response to metabolic inhibition and oxidant stress. 2. Whole-cell recordings were obtained from rat atrial myocytes using standard patch-clamp techniques. Inhibition of metabolism, using 10 mM 2-deoxy-D-glucose (2-DG) to block glycolysis with or without the addition of 2 mM cyanide to block oxidative phosphorylation, led to inhibition of ITO at a holding potential of -70 mV. Shifting the holding potential to -80 mV restored ITO, suggesting that metabolic inhibition had shifted the inactivation curve of ITO in a negative direction. 3. Quasi steady-state inactivation curves revealed a shift in ITO inactivation induced by complete metabolic inhibition with 2-DG and cyanide. Myocytes typically contracted shortly after the shift was observed. In the presence of Ruthenium Red, contraction was delayed and myocytes could undergo several exposures to the metabolic inhibitors, each time displaying a shift in ITO inactivation. The shifts ranged between -7 and -20 mV. 4. Recovery from inactivation was determined using a two-pulse protocol. The time constant of recovery at a holding potential of -80 mV reversibly shifted from 48 +/- 8 to 129 +/- 21 ms during metabolic inhibition (n = 4). 5. The activation of ITO from a holding potential of -100 mV shifted in a negative direction during metabolic inhibition, from a half-activation voltage of 0.3 +/- 3.0 to -14.7 +/- 2.5 mV (n = 5). Such a -15 mV shift increases the amplitude of ITO by approximately 30% at 0 mV. 6. A shift in ITO inactivation similar to that produced by metabolic inhibition could be shown when myocytes were subjected to oxidant stress induced by either 1 mM t-butyl hydroperoxide (TBHP) or the photoactivation of 100 nM Rose Bengal. Furthermore, an increase in pipette concentration of free Ca2+ from 20 to 200 nM also shifted ITO inactivation in a negative direction. 7. These results raise the possibility that the rise in intracellular [Ca2+] occurring during both metabolic inhibition and oxidant stress modifies activation and inactivation of ITO.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Antimetabolites/pharmacology , Ion Channels/metabolism , Myocardium/metabolism , Oxidants/pharmacology , Stress, Physiological/metabolism , Animals , Calcium/metabolism , Calcium/physiology , Cyanides/pharmacology , Deoxyglucose/pharmacology , Electrophysiology , In Vitro Techniques , Membrane Potentials/drug effects , Myocardium/cytology , Rats , Rats, Wistar
17.
Aust Paediatr J ; 24 Suppl 1: 50-4, 1988.
Article in English | MEDLINE | ID: mdl-2462411

ABSTRACT

Membrane channels so far characterized at the single channel level and their possible involvement in muscle dysfunction are reviewed. Experiments using isolated external intercostal muscle from a patient with Torbergsen syndrome and isolated rat muscles with chemically induced myotonia suggest further involvement of membrane channels in muscle disease. Both the presence of unusual channels and the absence of normal channels can confer properties on the sarcolemmal and sarcoplasmic reticular membranes which give rise to peculiar and distinctive defects in muscle behaviour.


Subject(s)
Cell Membrane Permeability , Ion Channels/physiology , Muscles/physiopathology , Myotonia Congenita/physiopathology , Adult , Animals , Female , Humans , Male , Membrane Potentials , Muscle Contraction , Rats , Rats, Inbred Strains , Syndrome
20.
Aust Vet J ; 63(12): 396-401, 1986 Dec.
Article in English | MEDLINE | ID: mdl-3800794

ABSTRACT

The distribution and severity of lesions in the skeletal muscles of 37 Merino sheep with congenital progressive muscular dystrophy (CPMD) are described. An explanation for the clinical signs is offered on the basis of functional defects in regional muscle groups. Lesions in the extensors of the hip, stifle and hock joints and flexors of the digits are primarily responsible for the progressive abnormality of hind limb gait that is characteristic of the clinical syndrome. Lesions in extensors of the elbow and flexors of the shoulder, carpus and digits affected fore limb function in advanced cases. The tendency for some affected sheep to develop ruminal tympany is probably caused by lesions in the diaphragmatic crus. Clinically affected sheep had higher resting and post-exercise concentrations of serum creatine phosphokinase and lactic dehydrogenase than unaffected control sheep. The rise in serum creatine phosphokinase after exercise was greater in affected sheep than in controls. Myotonia was not demonstrated in electromyographic studies in one sheep.


Subject(s)
Muscular Dystrophy, Animal/congenital , Sheep Diseases/congenital , Animals , Aspartate Aminotransferases/blood , Creatine Kinase/blood , Female , L-Iditol 2-Dehydrogenase/blood , L-Lactate Dehydrogenase/blood , Male , Muscular Dystrophy, Animal/enzymology , Muscular Dystrophy, Animal/pathology , Physical Exertion , Sheep , Sheep Diseases/enzymology , Sheep Diseases/pathology
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