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R L Barchi

Publications and source records attributed to R L Barchi.

At least 91 records · Page 5Linked to original sources
PubMed1981

Glycoprotein characteristics of the sodium channel saxitoxin-binding component from mammalian sarcolemma.

The saxitoxin-binding component of the excitable membrane sodium channel exhibits glycoprotein characteristics as evidenced by its specific interaction with various agarose-immobilized lectins. The detergent-solubilized saxitoxin-binding component interacts quantitatively with immobilized wheat germ agglutinin and concanavalin A and fractionally with immobilized Lens culinaris hemagglutinin and Ricinus communis agglutinin. These lectins preferentially bind N-acetylglucosamine and sialic acid (wheat germ agglutinin), mannose (concanavalin A and Lens cunilaris) and galactose (Ricinus communis). Removal of terminal sialic acid residues by neuraminidase markedly decreases binding to immobilized wheat germ agglutinin but uncovers sites capable of interacting with lectins specific for galactose and N-acetylgalactosamine. beta-N-acetylglucosaminidase, an exoglycosidase, has no effect on the binding of the channel protein to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component appears to be a glycoprotein containing terminal sialic acid residues and internal mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine residues. The toxin binding site is spatially separated from the binding sites for the lectins studied. The effect of these sugar moieties must be considered when evaluating the biophysical parameters of the sodium channel.

Animals↗
PubMed1981

20,25-diazacholesterol myotonia: an electrophysiological study.

The electrophysiological characteristics of the myotonic syndrome produced in mammalian skeletal muscle by administration of 20,25-diazacholesterol (20,25-D) were studied in detail. In vivo electromyographic recordings confirmed widespread repetitive electrical activity, but delayed relaxation was evanescent and required isotonic rather than isometric conditions, with long recovery periods between stimuli, for clear demonstration. Subsequent administration of a potent inhibitor of membrane chloride conductance (GCl) induced profound delays in relaxation different from that after chronic 20,25-D alone. Intracellular passive cable analysis revealed only a small decrease in membrane GCl and none in potassium conductance. Potassium current-voltage relationships did not differ in control and treated animals. Intracellular microelectrode recordings consistently showed multiple driven action potentials during long depolarizations but no spontaneous myotonic discharges after cessation of the stimulus. Variations in temperature, buffer, and external ionic concentrations also failed to produce spontaneous activity. Anode break excitation under mild depolarizing conditions, however, did elicit repetitive membrane electrical activity. The myotonia induced by 20,25-D is not due to low membrane GCl. The relationship between delayed mechanical relaxation and membrane repetitive electrical activity remains to be clearly established in this myotonic syndrome.

Action Potentials↗
PubMed1981

Alteration of synaptosomal plasma membrane cholesterol content: membrane physical properties and cation transport proteins.

The level of nerve membrane cholesterol was altered by in vitro incubation of rat brain synaptosomal plasma membrane with liposomes having varying cholesterol contents. The normal plasma membrane cholesterol/phospholipid ratio of 0.3-0.4 (mol/mol) could be decreased by about one-half or increased more than 100%. Fluorescence polarization measurements were made using the probe 1,6-diphenyl-1,3,5-hexatriene. At temperatures below 35 percent C, lowering membrane cholesterol led to increased apparent microviscosity, while raising cholesterol content produced little change. However, at 45 percent C a continuous direct relationship existed between experimental membrane cholesterol/phospholipid ratio (ranging from 0.18 to 0.73) and apparent microviscosity. Under standard liposome-synaptosomal plasma membrane exchange conditions, 80% of the initial specific [(3)H]saxitoxin binding activity to the voltage-dependent sodium channel and at least 95% of the (Mg2+,K+)-p-nitrophenylphosphatase activity were preserved. Our results indicate that neither the characteristics of toxin binding nor the kinetics of this enzyme activity is dependent upon membrane cholesterol content.

4-Nitrophenylphosphatase↗
PubMed1981

Estimate of the molecular weight of the sarcolemmal sodium channel using H2O-D2O centrifugation.

The sodium channel saxitoxin binding component from rat sarcolemma was solubilized with NP-40 and centrifuged on sucrose gradients constructed in either D2O or H2O. When compared with a series of standard proteins the sedimentation behavior of the solubilized channel complex changed from an apparent S20,w of 9.1 in H2O to 6.1 in D2O. From these observations, a true partial specific volume of 0.83 ml/g was calculated for the complex. A Stokes radius of 8.6 nm was estimated from Sepharose 6-B chromatography in NP-40. The calculated protein molecular weight of the lipid-protein-detergent complex based on these data is 560,000. The complex contains about 56% protein, and the calculated molecular weight of this component is 314,000 if a v for the protein of 0.74 ml/g is assumed.

Amphibian Proteins↗
PubMed1980

Size characteristics of the solubilized sodium channel saxitoxin binding site from mammalian sarcolemma.

The sodium channel saxitoxin binding component from rat sarcolemma was solubilized with medium chain length non-ionic detergents including NP-40, Brij-96 and Lubrol-PX. Phospholipid was required for stability of the binding component. Specific saxitoxin binding was significantly temperature sensitive even with optimal levels of phospholipid present. The solubilized saxitoxin binding component chromatographed on Sepharose 6B at a position corresponding to that of a globular protein of 95--10 A Stokes radius, but had an apparent s20,w typical of a smaller molecule (s20,w = 9.2--10). Column behavior and s20,w were independent of the specific detergent used for solubilization. Anomalous column behavior may reflect molecular asymmetry, contribution from bound detergent or similar considerations.

Animals↗
PubMed1980

Purification from rat sarcolemma of the saxitoxin-binding component of the excitable membrane sodium channel.

The saxitoxin-binding component (SBC) of the excitable membrane sodium channel has been solubilized and purified from rat skeletal muscle sarcolemma. Phospholipid was required in mixed micelles with detergent for stability of the mammalian SBC. Even at optimal detergent-to-phospholipid ratio, the solubilized SBC showed significant temperature-dependent loss of specific toxin binding with time, necessitating maintenance of low temperatures during purification. Characteristics of saxitoxin binding to the solubilized material closely resembled those seen in intact membranes. A weak anion-exchange column was synthesized; it provided rapid 10- to 20-fold purification of the solubilized SBC. Additional necessary purification was obtained by chromatography on immobilized wheat germ agglutinin. Specific saxitoxin-binding activity of the purified material averaged approximately 1500 pmol of saxitoxin bound per mg of protein. Three bands were present in this material on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified material sedimented on a sucrose gradient with an apparent s20,w of 9.9 S.

Animals↗
PubMed1980

Ultraviolet irradiation produces loss of saxitoxin binding to sodium channels in rat synaptosomes.

Ultraviolet irradiation (UV) has been shown to cause an electrophysiologically measured inactivation of the rapid, transient sodium conductance system in nerve. Tritiated saxitoxin ([3H]STX) was used as a structural probe to assess the possibility of a corresponding perturbation in the conformation of the STX binding site. UV irradiation caused an irreversible decrease in the total number of high-affinity [3H]STX binding sites in rat synaptosomes, while the dissociation constant of the remaining sites did not change. The receptor loss followed first-order kinetics, and the rate of loss was independent of temperature. The action spectrum for binding loss indicated a peak in spectral sensitivity near 280 nm. A22Na flux assay in irradiated synaptosomes directly demonstrated that [3H]STX binding sites and veratridine-stimulated, STX-blocked 22Na efflux had similar sensitivities to UV radiation. We conclude that the UV inactivation of functional channels includes a modification of the STX binding-site structure.

Animals↗
PubMed1980

Fluorescent probe analysis of erythrocyte membranes in myotonic dystrophy.

Previous electron spin resonance studies in myotonic muscular dystrophy (MyMD) have suggested that an increased cell surface membrane "fluidity" might underlie the widespread abnormalities in this disorder. We attempted to confirm and expand those findings by using another physical probe technique: fluorescence polarization spectroscopy. Spectral characteristics and rotational relaxation rates of four hydrophobic fluorescent probes were monitored in control and MyMD erythrocyte membrane ghosts. Structural unrelated probes were used to minimize the possibility that an alteration in physical properties in a restricted membrane microdomain would pass undetected. All probes were studied between 5 degrees C and 40 degrees C in each preparation to quantitate temperature-dependent changes. We failed to confirm the abnormality in apparent membrane fluidity previously reported in MyMD.

Erythrocyte Membrane↗
PubMed1979

Muscle surface membranes: preparative methods affect apparent chemical properties and neurotoxin binding.

Considerable disagreement exists between results reported by various authors for lipid composition and enzyme activity in purified muscle membrane fractions presumed to be sarcolemma, although an explanation for these discrepancies has not been presented. We have prepared muscle light surface membrane fractions of comparable density (1.050--1.120) by a low-salt sucrose method and by an LiBr-KCl extraction procedure and compared them for density profile, total lipid and cholesterol content, protein composition and ATPase activity. In addition, sodium channels characteristic of excitable membranes have been quantitated in each preparation using [3H]saxitoxin binding assays, and the density of acetylcholine receptors determined in fractions from control and denervated muscle using alpha-[125I]bungarotoxin. Although both fractions contain predominantly surface membrane, the LiBr fraction consistently shows the higher specific activity of p-nitrophenylphosphatase, higher free cholesterol content, and higher density of sodium channels and acetylcholine receptors. The density distribution of sodium channels appears uniform throughout both fractions. Quantitative differences were seen between sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of membrane proteins from the two preparations although most bands are represented in both. A majority of the low-salt sucrose light membrane proteins were accessible in varying degrees to labelling with diazotized diiodosulfanylic acid in intact muscle. These results suggest that light surface membrane fractions may be mixtures of sarcolemma and T-tubular membranes. Using our preparative methods, the LiBr fraction may contain predominantly sarcolemma while low-salt sucrose light membranes may be enriched in T-tubular elements.

4-Nitrophenylphosphatase↗
PubMed1979

Characteristics of saxitoxin binding to the sodium channel of sarcolemma isolated from rat skeletal muscle.

1. The characteristics of saxitoxin (STX) binding to the mammalian Na channel have been studied in purified sarcolemma isolated from rat skeletal muscle. 2. STX binds specifically to isolated sarcolemma with a Kd of 1.43 x 10(-9) M and Bmax of 7-8 p-mole STX bound/mg membrane protein at 0 degrees C in the presence of 140 mM-NaCl. In rat muscle homogenate under the same conditions the corresponding values are Kd = 1.53 x 10(-9) M and Bmax = 0.15-0.20 p-mole/mg protein (18-20 p-mole/g wet wt.). Membrane purification produced a fortyfold increase in STX binding site concentration per milligram protein. Calculated binding site density in isolated sarcolemma was about 30 sites/micron 2 of membrane surface. 3. Denervation (10-14 days) results in a 43% reduction in the density of high-affinity STX binding sites in purified sarcolemma, but the Kd for this class of sites is not changed. 4. In sarcolemma, the apparent Kd for STX binding is dependent on temperature pH and ionic strength. The Q10 for Kd between 0 and 40 degrees C is 1.3. Protonation of a group having a pK of 6.0 markedly raises Kd without affecting Bmax. Apparent Kd increases eightfold when ionic strength is raised from 20 to 600 mM. 5. Dissociation and association rate constants for STX binding are temperature dependent with Q10 of 2.6 and 1.9 respectively between 0 and 20 degrees C. 6. STX binding is competitively inhibited by monovalent and divalent cations under conditions of constant total ionic strength. An affinity sequence of Tl+ greater than Li+ greater than Na+ greater than K+ greater than Rb+ greater than Cs+ is seen for the monovalent cation-binding site. 7. The STX binding site is relatively stable to heat and to enzymic degradation. A specific modifier of carboxyl residues inactivates subsequent STX binding. This process can be prevented by the presence of STX during the reaction. 8. Characteristics of the STX binding site in isolated sarcolemma are compared to those reported for other isolated excitable membranes and for studies of whole muscle and muscle homogenate. Sarcolemma provides a potential source of enriched Na channels for further purification efforts in a mammalian system.

Animals↗
PubMed1978

The pathophysiology of myotonia produced by aromatic carboxylic acids.

A series of nine related aromatic monocarboxylic acids (ACAs) previously shown to inhibit muscle membrane chloride conductance (GCl) selectively in the rat were studied for their ability to produce myotonia. All nine induced characteristic repetitive electrical activity and delayed relaxation in isolated muscle, although the concentrations required for this action varied widely. In each case, myotonia was observed at concentrations that correlated closely with previously determined half-maximal concentrations for inhibition of GCl. Intracellular recordings from muscle made myotonic with ACA revealed prolonged latencies at rheobase, multiple driven spikes, and self-sustaining repetitive activity similar to that previously reported in hereditary goat myotonia. Phase-plane diagrams of membrane action potentials recorded after exposure to the most effective of these compounds suggested little effect on the voltage-dependent sodium system. The changes seen could be duplicated by simple removal of chloride ion. The expression of repetitive electrical activity in the presence of low membrane GCl depends on ambient temperature and on the concentration of calcium ion. Increasing temperature and decreasing Ca++ predispose toward myotonic activity; converse conditions inhibit myotonia. Myotonia induced by ACA is inhibited by concentrations of diphenylhydantoin that are clinically effective in controlling hereditary myotonia in humans.

Action Potentials↗
PubMed1978

Muscle membrane chloride conductance and the myotonic syndromes.

The myotonic syndrome of delayed relaxation in striated muscle associated with repetitive membrane electrical activity is characteristic of a number of hereditary disorders in animals and man. This state of pathologic membrane hyperexcitability is also seen as a consequence of intoxication with several classes of chemicals. In most of these states an increase in muscle membrane resistance due to a specific reduction in sarcolemmal GCl can be demonstrated. Computer simulations have indicated that such a reduction in GCl alone could account for the observed repetitive electrical activity. Controlled reduction of GCl in normal muscle, using aromatic monocarboxylic acids, produces a myotonic syndrome. This myotonia resembles that seen in hereditary disease in its contractile characteristics, its intracellular electrical behavior and its response to diphenylhydantoin and procainamide. The effects of variations in temperature and divalent cation concentration seen in this system parallel those predicted by computer models of myotonia based on the chloride hypothesis. Comparison of the behavior of this and similar model systems with the hereditary myotonias of man and goat allow conclusions to be drawn concerning the underlying pathophysiologic mechanisms at work in the hereditary disorders. The concept of an abnormally reduced GCl as the final common factor in many of these disorders emerges as the most tenable hypothesis, although the mechanism by which this conductance is altered may be unique in each case.

Animals↗
PubMed1977

Determination of muscle cable parameters from a single membrane voltage response.

Computer techniques have been developed to achieve a least-squares fit of the Hodgkin and Rushton one time-constant cable equation to the recorded responses of muscle cells to the intracellular injection of square current pulses. In the rat diaphragm the response of the muscle fiber to square current pulses is well fit by a one time-constant model. This makes possible the estimation of the passive electrical properties of muscle sarcolemma using the response of the fiber to a single stimulus. A comparison of the results of this method and the older method of spatial decay in the rat diaphragm shows no significant differences. Average values obtained using the one-point method for estimating membrane resistance and capacitance are 520 omega cm2 and 4.6 micronF/cm2, respectively. An estimation of the average specific resistivity of the cytoplasm was made using this method, and it was found to be 213 omega cm. At levels of injected currents producing large membrane hyperpolarizations where delayed conductance changes make the spatial decay method useless, the one-point method continues to give consistent results since it utilizes only the early portion of the membrane response. Work with the frog sartorius muscle, which is known to display the characteristics of a two time-constant system, shows that this method is capable of estimating the slower components of this system by using only the later portion of the response curve. The immunity of this method to delayed conductance changes and its experimental facility make it useful in estimating the passive electrical properties of muscle fibers, particularly when working with tissues that are small and delicate or that have poor in vitro viability.

Animals↗