The Journal of Membrane Biology

, Volume 123, Issue 3, pp 223–233 | Cite as

Response of chloride efflux from skeletal muscle ofRana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment

  • Bruce C. Spalding
  • Patricia Taber
  • John G. Swift
  • Paul Horowicz
Articles

Summary

Efflux of36Cl from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.

For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl efflux. The pH-dependent Cl efflux can be described by the relationu=1/(1+10n(pK a -pH)), whereu is the Cl efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about −15 mV), the apparent pK a is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about −65 mV), the apparent pK a at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK a suggests that the critical pH-sensitive moiety producing the pH-dependent Cl efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl efflux increment on raising external pH.

The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl efflux according to the relation, efflux=exp(−k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k−1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q10 of 2.5. The Cl efflux inhibitor SCN at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl efflux by DEPC. The findings that the apparent pK a is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl efflux, and that this action is retarded by SCN supports the notion that protonation of histidine groups associated with Cl channels is the controlling reaction for the pH-dependent Cl efflux.

Key Words

skeletal muscle Cl efflux Cl channel pH muscle membrane temperature diethylpyrocarbonate 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Blatz, A.L. 1984. Asymmetric proton block of inward rectifier K channels in skeletal muscle.Pfluegers Arch. 401:402–407Google Scholar
  2. Bolton, T.B., Vaughan-Jones, R.D. 1977. Continuous direct measurement of intracellular chloride and pH in frog skeletal muscle.J. Physiol. 270:801–833Google Scholar
  3. Boyle, P.J., Conway, E.J. 1941. Potassium accumulation in muscle and associated changes.J. Physiol. 100:1–63Google Scholar
  4. Brahm, J. 1977. Temperature-dependent changes of chloride transport kinetics in human red cells.J. Gen. Physiol. 70:283–306Google Scholar
  5. Brooks, A.E., Hutter, O.F. 1962. The influence of pH on the chloride conductance of skeletal muscle.J. Physiol. 163:9–10PGoogle Scholar
  6. Dalmark, M., Wieth, J.O. 1972. Temperature dependence of chloride, bromide, iodide, thiocyanate and salicylate transport in human red cells.J. Physiol. 224:583–610Google Scholar
  7. Dugas, H., Penney, C. 1981. Bioorganic Chemistry A Chemical Approach to Enzyme Action. Springer-Verlag, New YorkGoogle Scholar
  8. Fedorcsák, I., Ehrenberg, L. 1966. Effects of diethylpyrocarbonate and methyl methanesulfonate on nucleic acids and nucleases.Acta Chem. Scand. 20:107–112Google Scholar
  9. Greenstein, J.P., Winitz, M. 1961. Chemistry of the Amino Acids. Vol. 1. Wiley, New YorkGoogle Scholar
  10. Gunn, R.B., Wieth, J.O., Tosteson, D.C. 1975. Some effects of low pH on chloride exchange in human red blood cells.J. Gen. Physiol. 65:731–749Google Scholar
  11. Hodgkin, A.L., Horowicz, P. 1959. The influence of potassium and chloride ions on the membrane potential of single muscle fibres.J. Physiol. 148:127–160Google Scholar
  12. Hutter, O.F., Noble, D. 1960. The chloride conductance of frog skeletal muscle.J. Physiol. 151:89–102Google Scholar
  13. Hutter, O.F., Warner, A.E. 1967a. Action of some foreign cations and anions on the chloride permeability of frog muscle.J. Physiol. 189:445–460Google Scholar
  14. Hutter, O.F., Warner, A.E. 1967b. The effect of pH on the36Cl efflux from frog skeletal muscle.J. Physiol. 189:427–443Google Scholar
  15. Hutter, O.F., Warner, A.E. 1967c. The pH sensitivity of the chloride conductance of frog skeletal muscle.J. Physiol. 189:403–425Google Scholar
  16. Hutter, O.F., Warner, A.E. 1972. The voltage dependence of the chloride conductance of frog muscle.J. Physiol. 227:275–290Google Scholar
  17. Kotsias, B.A., Horowicz, P. 1990. Nitrate and chloride ions have different permeation pathways in skeletal muscle fibers ofRana pipiens.J. Membrane Biol. 115:95–108Google Scholar
  18. Loo, D.D.F., McLarnon, J.G., Vaughan, P.C. 1981. Some observations on the behaviour of chloride current-voltage relations inXenopus muscle membrane in acid solutions.Can. J. Physiol. Pharmacol. 59:7–13Google Scholar
  19. Melchior, W.B., Fahrney, D. 1970. Ethoxyformylation of proteins. Reaction of ethoxyformic anhydride with chymotrypsin, pepsin, and pancreatic ribonuclease at pH 4.Biochemistry 9:251–258Google Scholar
  20. Mühlrád, A., Hegyi, G., Horányi, M. 1969. Studies on the properties of chemically modified actin. III. Carbethoxylation.Biochim. Biophys. Acta 181:184–190Google Scholar
  21. Mühlrád, A., Hegyi, G., Tóth, G. 1967. Effect of diethylpyrocarbonate on proteins. I. Reaction of diethylpyrocarbonate with amino acids.Acta Biochim. Biophys. Acad. Sci. Hung. 2:19–29Google Scholar
  22. Ovádi, J., Libor, S., Elödi, P. 1967. Spectrophotometric determination of histidine in proteins with diethylpyrocarbonate.Acta Biochim. Biophys. Acad. Sci. Hung. 2:455–458Google Scholar
  23. Skydsgaard, J.M. 1987. Influence of chloride concentration and pH on the36Cl efflux from depolarized skeletal muscle ofRana temporaria.J. Physiol. 385:49–67Google Scholar
  24. Spalding, B.C., Swift, J.G., Horowicz, P. 1986. Influence of external barium and potassium on potassium efflux in depolarized frog sartorius muscles.J. Membrane Biol. 93:141–156Google Scholar
  25. Vaughan, P., Fong, C.N. 1978. Effects of SITS on chloride permeation inXenopus skeletal muscle.Can. J. Physiol. Pharmacol. 56:1051–1054Google Scholar
  26. Venosa, R.A. 1974. Inward movement of sodium ions in resting and stimulated frog's sartorius muscle.J. Physiol. 241:155–173Google Scholar
  27. Venosa, R.A., Horowicz, P. 1981. Density and apparent location of the sodium pump in frog sartorius muscle.J. Membrane Biol. 59:225–232Google Scholar
  28. Venosa, R.A., Ruarte, A.C., Horowicz, P. 1972. Chloride and potassium movements from frog's sartorius muscle in the presence of aromatic anions.J. Membrane Biol. 9:37–56Google Scholar
  29. Warner, A.E. 1972. Kinetic properties of the chloride conductance of frog muscle.J. Physiol. 227:291–312Google Scholar
  30. Woll, K.H., Liebowitz, M.D., Neumcke, B., Hille, B. 1987. A high-conductance anion channel in adult amphibian skeletal muscle.Pfluegers Arch. 410:632–640Google Scholar
  31. Woll, K.H., Neumcke, B. 1987. Conductance properties and voltage dependence of an anion channel in amphibian skeletal muscle.Pfluegers Arch. 410:641–647Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1991

Authors and Affiliations

  • Bruce C. Spalding
    • 1
  • Patricia Taber
    • 1
  • John G. Swift
    • 1
  • Paul Horowicz
    • 1
  1. 1.Department of Physiology, School of Medicine and DentistryUniversity of RochesterRochester

Personalised recommendations