Summary
Direct inhibitory effects of Ca2+ and other ions on the epithelial Na+ channels were investigated by measuring the amiloride-blockable22Na+ fluxes in toad bladder vesicles containing defined amounts of mono- and divalent ions. In agreement with a previous report (H.S. Chase, Jr., and Q. Al-Awqati,J. Gen. Physiol. 81:643–666, 1983) we found that the presence of micromolar concentrations of Ca2+ in the internal (cytoplasmic) compartment of the vesicles substantially lowered the channel-mediated fluxes. This inhibition, however, was incomplete and at least 30% of the amiloride-sensitive22Na+ uptake could not be blocked by Ca2+ (up to 1mm). Inhibition of channels could also be induced by millimolar concentrations of Ba2+, Sr2+, or VO2+, but not by Mg2+. The Ca2+ inhibition constant was a strong function of pH, and varied from 0.04 μm at pH 7.8 to >10 μm at pH 7.0 Strong pH effects were also demonstrated by measuring the pH dependence of22Na+ uptake in vesicles that contained 0.5 μm Ca2+. This Ca2+ activity produced a maximal inhibition of22Na+ uptake at pH≥7.4 but had no effect at pH≤7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over this range. The data is compatible with the model that Ca2+ blocks channels by binding to a site composed of several deprotonated groups. The protonation of any one of these groups prevents Ca2+ from binding to this site but does not by itself inhibit transport. The fact that the apical Na+ conductance in vesicles, can effectively be modulated by minor variations of the internal pH near the physiological value, raises the possibility that channels are being regulated by pH changes which alter their apparent affinity to cytoplasmic Ca2+, rather than, or in addition to changes in the cytoplasmic level of free Ca2+.
Similar content being viewed by others
References
Arruda, J.A.L., Sabatini, S., Westenfelder, C. 1982. Serosal Na/Ca exchange and H+ and Na+ transport by the turtle and toad bladders.J. Membrane Biol. 70:135–146
Burch, R.M., Halushka, P.V. 1984. ADH or theophylline induced changes in intracellular free and membrane-bound calcium.Am. J. Physiol. 247:F939-F945
Chase, H.S., Jr. 1984. Does calcium couple the apical and basolateral membrane permeabilities in epithelia?Am. J. Physiol. 247:F869-F876
Chase, H.S., Jr., Al-Awqati, Q. 1981. Regulation of sodium permeability of the luminal border of toad bladder by intracellular sodium and calcium. Role of sodium-calcium exchange in the basolateral membrane.J. Gen. Physiol. 77:693–712
Chase, H.S., Jr., Al-Awqati, Q. 1983. Calcium reduces the sodium permeability of luminal membrane vesicles from toad bladder. Studies using a fast reaction apparatus.J. Gen. Physiol. 81:643–666
Chase, H.S., Wong, S. 1984. Effect of extracellular Na+ and osmolarity on intracellular Ca2+ in toad bladder cells measured with Quin 2.J. Gen. Physiol. 84:16a (Abstr.)
Civan, M.M., Lin, L.E., Peterson-Yantorno, K., Taylor, J., Deutsch, C. 1984. Intracellular pH of perfused single frog skin: Combined19F and31P NMR analysis.Am. J. Physiol. 247:C506-C510
Crutch, B., Taylor, A. 1983. Measurement of cytosolic free Ca2+ concentration in epithelial cells of toad bladder.J. Physiol. (London) 345:109p (Abstr.)
Durham, A.C.H. 1983. A survey of readily available chelators for buffering calcium ion concentrations in physiological solutions.Cell Calcium 4:33–46
Fabiato, A. 1981. Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle.J. Gen. Physiol. 78:457–497
Frizzell, R.A., Schultz, S.G. 1978. Effect of aldosterone on ion transport by rabbit colonin vitro.J. Membrane Biol. 39:1–26
Garty, H. 1984. Amiloride blockable sodium fluxes in toad bladder membrane vesicles.J. Membrane Biol. 82:269–279
Garty, H. 1986. Mechanisms of aldosterone action in tight epithelia.J. Membrane Biol. 90:193–205
Garty, H., Asher, C. 1985. Ca dependent, temperature sensitive, regulation of Na channels in tight epithelia. A study using membrane vesicles.J. Biol. Chem. 260:8330–8335
Garty, H., Asher, C. 1986. Ca2+ induced down regulation of Na+ channels in toad bladder epithelium.J. Biol. Chem. 261:7400–7406
Garty, H., Civan, E.D., Civan, M.M. 1985. Effects of internal and external pH on amiloride-blockable Na+ transport across toad urinary bladder vesicles.J. Membrane Biol. 87:67–75
Garty, H., Edelman, I.S. 1983. Amiloride-sensitive trypsinization of apical sodium channels. Analysis of hormonal regulation of sodium transport in toad bladder.J. Gen. Physiol. 81:785–803
Garty, H., Edelman, I.S., 1984. Hormonal regulation of Na+ channels in tight epithelia.In: Regulation and Development of Membrane Transport Processes. J.S. Graves, editor. pp. 106–120. John Wiley and Sons, New York
Garty, H., Edelman, I.S., Lindeman, B. 1983a. Metabolic regulation of apical sodium permeability in toad urinary bladder in the presence and absence of aldosterone.J. Membrane Biol. 74:15–24
Garty, H., Karlish, S.J.D. 1987. Measurements of ion channel mediated fluxes in membrane vesicles. Selective amplification of isotope uptake by electrical diffusion potentials.Methods Enzymol. (In press)
Garty, H., Lindemann, B. 1984. Feedback inhibition of sodium uptake in K+-depolarized toad urinary bladder.Biochim. Biophys. Acta 771:89–98
Garty, H., Rudy, B., Karlish, S.J.D. 1983b. A simple and sensitive procedure for measuring isotope fluxes through ion-specific channels in heterogeneous populations of membrane vesicles.J. Biol. Chem. 258:13094–13099
Gasko, O.D., Knowles, A.F., Shertzor, H.G., Soulina, E.M., Racher, E. 1976. The use of ion-exchange resins for studying ion transport in biological systems.Anal. Biochem. 72:57–65
Grinstein, S., Erlij, D. 1978. Intracellular calcium and the regulation of sodium transport in the frog skin.Proc. R. Soc. London B. 202:353–360
Helman, S.I., Cox, T.C., Van Driessche, W. 1983. Hormonal control of apical membrane Na+ transport in epithelia.J. Gen. Physiol. 82:201–220
Leaf, A., Keller, A., Dempsey, E.F. 1964. Stimulation of sodium transport in toad bladder by acidification of mucosal medium.Am. J. Physiol. 207:547–552
Lewis, S.A., Eaton, D.C., Diamond, J.M. 1976. The mechanism of Na+ transport by rabbit urinary bladder.J. Membrane Biol. 28:41–70
Li, J.H.Y., Palmer, L.G., Edelman, I.S., Lindemann, B. 1982. The role of the sodium-channel density in the natriferic response of the toad urinary bladder to an antidiuretic hormone.J. Membrane Biol. 64:77–89
Lindemann, B. 1984. Fluctuation analysis of sodium channels in epithelia.Annu. Rev. Physiol. 46:497–515
Palmer, L.G., Edelman, I.S., Lindemann, B. 1980. Current-voltage analysis of apical sodium transport in toad urinary bladder: Effects of inhibitors of transport and metabolism.J. Membrane Biol. 57:59–71
Palmer, L.G., Frindt, G. 1986. Regulation of apical Na channels in rat cortical collecting tubule by cytoplasmic pH.Fed. Proc. 45:1010 (Abstr.).
Palmer, L.G., Li, J.H.Y., Lindemann, B., Edelman, I.S. 1982. Aldosterone control of the density of sodium channels in the toad urinary bladder.J. Membrane Biol. 64:91–102
Pershadsingh, H.A., McDonald, J.M. 1980. A high affinity calcium-stimulated magnesium-dependent adenosine triphosphate in rat adipocyte plasma membranes.J. Biol. Chem. 255:4087–4093
Roos, A., Boron, W.F. 1981. Intracellular pH.Physiol. Rev. 61:296–434
Rubinson, K.A. 1981. Concerning the form of biochemically active vanadium.Proc. R. Soc. London B. 212:65–84
Schuldiner, S., Rosengurt, E. 1982. Na+/H+ antiport in Swiss 3T3 cells: Mitogenic stimulation leads to cytoplasmic alkalinization.Proc. Natl. Acad. Sci. USA 79:7778–7782
Schultz, S.G. 1981. Homocellular regulatory mechanisms in sodium transporting epithelia: Avoidance of extinction by “flush-through.”Am. J. Physiol. 241:F579-F590
Schultz, S.G. 1984. A cellular model for active sodium absorption by mammalian colon.Annu. Rev. Physiol. 46:435–451
Taylor, A., Palmer, L.G. 1982. Hormonal regulation of sodium, chloride and water transport in epithelia.In: Biological Regulation and Development. R.F. Goldberger and K.R. Yamamoto, editors. Vol. 3A, pp. 253–298. Plenum, New York-London
Taylor, A., Windhager, E.E. 1979. Possible role of cytosolic calcium and Na−Ca exchange in regulation of transepithelial sodium transport.Am. J. Physiol. 236:F505-F512
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Garty, H., Asher, C. & Yeger, O. Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium, and by other divalent ions. J. Membrain Biol. 95, 151–162 (1987). https://doi.org/10.1007/BF01869160
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01869160