Summary
Whole-cell patch-clamp recordings were made from freshly isolated human platelets. The pipette contained a high concentration of divalent cations, which permitted easy disruption of cell-attached membrane patches by suction. Single-channel currents were measured when the pipette contained isotonic BaCl2 or MgCl2 saline; over 30 sec −5 min an increasing number of channels appeared until conductance steps through individual channels could no longer be distinguished. The current-voltage relationship was curvilinear; chord conductance at −35 mV was 25 pS increasing to 45 to 52 pS at +45 mV. Ion substitution experiments showed the current to be primarily carried by Cl−.E rev was shifted 30 mV/10-fold change in external Cl− (replaced by gluconate), was similar with BaCl2 or MgCl2 in the pipette and was not significantly shifted by replacing external Na+ with K+. Addition of 1mm BAPTA to the MgCl2 pipette saline prevented activation of Cl− currents; with isotonic CaCl2 internal saline, current appeared immediately upon patch rupture, suggesting that the Cl− channels are dependent on internal Ca2+, 5-nitro-2-(3-phenylpropylamino)-benzoate, reported to block a Cl− conductance in studies of rat epithelial cells, caused a potent flickery block and may be a useful tool with which to investigate the physiological role of Cl− currents in human platelets.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bosma, M.M. 1989. Anion channels with multiple condcutance levels in a mouse B lymphocyte cell line.J. Physiol. (London) 410:67–90
Cahalan, M.D., Lewis, R.S. 1988. Role of potassium and chloride channels in volume regulation by T lymphocytes.In Cell Physiology of Blood. R. Gunn and J. Parker, editors. Rockefeller University Press, New York.
Chen, J.H., Shulman, H., Gardner, P. 1989. A cAMP-regulated chloride channel in lymphocytes that is affected in cystic fibrosis.Science 243:657–660
Grinstein, S., Clarke, C.A., Dupre, A., Rothstein, A. 1982. Volume-induced increase of anion permeability in human lymphocytes.J. General Physiol. 80:801–823
Hallam, T.J., Rink, T.J. 1985. Responses to adenosine diphosphate in human platelets loaded with the fluorescent calcium indicator quin 2.J. Physiol. (London) 368:131–146
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.Pfluegers Arch. 391:85–100
Livne, A., Grinstein, S., Rothstein, A. 1987. Volume-regulating behaviour of human platelets.J. Cell. Physiol. 131:354–363
MacIntrye, D.E., Rink, T.J. 1982. The role of platelet membrane potential in the initiation of platelet aggregation.Thromb. Haemost. 47:22–26
Mahaut-Smith, M.P. 1990. Whole-cell patch-clamp recordings of chloride currents from human platelets.J. Physiol. (London) 422:42P
Mahaut-Smith, M.P., Rink, T.J., Sage, S.O. 1989. Single channels in human platelets activated by ADP.J. Physiol. (London) 415:24P
Manning, S.D., Williams, A.J. 1989. Conduction and blocking properties of a predominantly anion-selective channel from human platelet surface membrane reconstituted into planar phospholipid bilayers.J. Membrane Biol. 109:113–122
Marty, A. 1989. The physiological role of calcium-dependent channels.Trends Neurosci. 12:420–424
Maruyama, Y. 1987. A patch-clamp study of mammalian platelets and their voltage-gated potassium current.J. Physiol. (London) 391:467–485
Meech, R.W. 1980. Ca++-activated K+ conductance.In: Molluscan Nerve Cells: From Biophysics to Behaviour. Cold Spring Harbor Report in Neurosciences.Cold Spring Harbor Laboratory 1:93–103
Oberhauser, A., Alvarez, O., Latorre, R. 1988. Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane.J. Gen. Physiol. 92:67–86
Pipili, E. 1985. Platelet membrane potential: Simultaneous measurement of diSC3(5) fluorescence and optical density.Thromb. Haemost. 54:645–649
Robinson, R.A., Stokes, R.H. 1955. Electrolyte Solutions. (2nd ED.) Butterworth, London
Sage, S.O., Rink, T.J. 1986. Kinetic differences between thrombin-induced and ADP-induced calcium influx and release from internal stores in fura-2 loaded platelets.Biochem. Biophys. Res. Commun. 136:1124–1129
Tsien, R.Y. 1980. New calcium indicators and buffers with high selectivity against magnesium and protons: Design, synthesis and properties of prototype structures.Biochemistry 19:2396–2404
Wangemann, P., Wittner, M., Di Stefano, A., Englert, H.C., Lang, H.J., Schlatter, E., Greger, R. 1986. Cl− channel blockers in the thick ascending limb of the loop of Henle. Structure activity relationship.Pfluegers Arch. 407:S128-S141
Worrell, R.T., Butt, A.G., Cliff, W.H., Frizzell, R.A., 1989. A volume-sensitive chloride conductance in human colonic cell line T84.Am. J. Physiol. 256:C1111-C1119
Zschauer, A., van Breeman, C., Buhler, F.R., Nelson, M.T. 1988. Calcium channels in thrombin-activated human platelet membrane.Nature (London) 334:703–705
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Mahaut-Smith, M.P. Chloride channels in human platelets: Evidence for activation by internal calcium. J. Membrain Biol. 118, 69–75 (1990). https://doi.org/10.1007/BF01872205
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01872205