Abstract
During whole-cell patch-clamp recording from normal (nontransformed) human T lymphocytes a chloride current spontaneously activated in >98% of cells (n > 200) in the absence of applied osmotic or pressure gradients. However, some volume sensitivity was observed, as negative pressure pulses reduced the current. With iso-osmotic bath and pipette solutions the peak amplitude built up (time constant ≈23 sec at room temperature), a variable-duration plateau phase followed, then the current ran down spontaneously (time constant ≈280 sec). The anion permeability sequence, calculated from reversal potentials was I−, Br− > NO −3 , Cl− > CH3SO −3 , HCO −3 > CH3COO− > F− > aspartate, gluconate, SO 2−4 and there was no measurable monovalent cation permeability. The Cl− current was independent of time during long voltage steps and there was no evidence of voltage-dependent gating; however, the current showed intrinsic outward rectification in symmetrical Cl− solutions. The conductance of the channels underlying the whole-cell current was calculated from fluctuation analysis, using power-spectral density and variance-vs.-mean analysis. Both methods yielded a single channel conductance of about 0.6 pS at −70 mV (close to the normal resting potential of T lymphocytes). The power spectral density function was best fit by the sum of two Lorentzian functions, with corner frequencies of 30 and 295 Hz, corresponding to mean open times of 0.54 and 5.13 msec. The pharmacological profile included rapid block by external application of flufenamic acid (50 μm), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 100 μm, [6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5-y1) oxy] acetic acid (IAA-94, 250 μm) or 100 μm 1,9-dideoxyforskolin. The stilbene derivatives DIDS (4,4′-diisothiocyano-2,2′ di-sulphonic acid stilbene, 500 μm) and SITS (4-acetamido-4′-isothiocyano-2, 2′-disulphonic acid stilbene, 500 μm) prevented buildup of Cl− current after a 30-min preincubation at 500 μm. When tested in a mitogenic assay, DIDS, flufenamic acid, NPPB and IAA-94 all inhibited T-cell proliferation, suggesting a physiological function in addition to the observed volume sensitivity.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Botchkin, L., Matthews, G. 1993. Chloride current activated by swelling in retinal pigment epithelial cells. Am. J. Physiol. 265:C1037-C1045.
Cahalan, M.D., Lewis, R.S. 1988. Role of potassium and chloride channels in volume regulation by T lymphocytes. In: Cell Physiology of Blood, Society of General Physiologists' Series, R.B. Gunn and J.C. Parker, editors. Vol. 43, pp. 281–301. Rockefeller University Press, New York.
Deutsch, C., Lee, S.C. 1988. Cell volume regulation in lymphocytes. Renal Physiol. Biochem. 3–5:260–276.
Diaz, M., Valverde, M.A., Higgins, C.F., Rucareanu, C., Sepulveda, F.V. 1993. Volume-activated chloride channels in HeLa cells are blocked by verapamil and dideoxyforskolin. Pfleugers Arch. 422:347–353.
Diamond, J.M., Wright, E.M. 1969. Biological membranes: The physical basis of ion and nonelectrolyte selectivity. Rev. Physiol. 31:581–647.
Doroshenko, P., Neher, E. 1992. Volume-sensitive chloride conductance in bovine chromaffin cell membrane. J. Physiol. 449:197–218.
Doroshenko, P., Penner, R., Neher, E. 1991. Novel chloride conductance in the membrane of bovine chromaffin cells activated by intracellular GTPγS. J. Physiol. 436:711–724.
Gray, L.S., Russell, J.H. 1986. Cytolytic T lymphocyte effector function requires plasma membrane chloride flux. J. Immunol. 136:3032–3037.
Kawasaki, M., Uchida, S., Monkawa, T., Miyawaki, A., Mikoshiba, K., Marumo, F., Sasaki, S. 1994. Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells. Neuron 12:597–604.
Koch, M.C., Steinmeyer, K., Lorenz, C., Ricker, K., Wolf, F., Otto, M., Zoll, B., Lehmann-Horn, F., Grzeschik, K-H, Jentsch, T.J. 1992. The skeletal muscle chloride channel in dominant and recessive human myotonia. Science 257:797–800.
Krapivinsky, G.B., Ackerman, M.J., Gordon, E.A., Krapivinsky, L.D., Clapham, D.E. 1994. Molecular characterization of a swelling-induced chloride conductance regulatory protein, pICln. Biophys. J. 66(2):A167.
Landry, D.W., Akabas, M.H., Redhead, C., Edelman, A., Cragoe, E.J., Al-Awqati, Q. 1989. Purification and reconstitution of chloride channels from kidney and trachea. Science 244:1469–1472.
Lewis, R.S., Ross, P.E., Cahalan, M.D. 1993. Chloride channels activated by osmotic stress in T lymphocytes. J. Gen. Physiol. 101:801–826.
Mathias, R.T., Cohen, I.S., Oliva, C. 1990. Limitations of the whole cell patch clamp technique in the control of intracellular concentrations. Biophys. J. 58:759–770.
Matthews, G., Neher, E., Penner, R. 1989. Chloride conductance activated by external agonists and internal messengers in rat peritoneal mast cells. J. Physiol. 418:131–144.
Nilius, B., Oike, M., Zahradnik, I., Droogmans, G. 1994. Activation of a Cl− current by hypotonic volume increase in human endothelial cells. J. Gen. Physiol. 103:787–805.
Pahapill, P.A., Schlichter, L.C. 1990. Modulation of potassium channels in human T lymphocytes: effects of temperature. J. Physiol. 422:103–126.
Pahapill, P.A., Schlichter, L.C. 1992a. Cl− channels in intact human T lymphocytes. J. Membrane Biol. 125:171–183.
Pahapill, P.A., Schlichter, L.C. 1992b. Modulation of potassium channels in intact human T lymphocytes. J. Physiol. 445:407–430.
Paulmichl, M., Li, Y., Wickman, K., Ackerman, M., Peralta, E., Clapham, D. 1992. New mammalian chloride channel identified by expression cloning. Nature 356:238–241.
Penner, R., Matthews, G., Neher, E. 1988. Regulation of calcium influx by second messengers in rat mast cells. Nature 334:499–504.
Pusch, M., Steinmeyer, K., Jentsch, T.J. 1994. Low single-channel conductance of the major skeletal muscle chloride channel, CIC-1. Biophys. J. 66:149–152.
Rosoff, P.M., Hall, C., Gramates, L.S., Terlecky, S.R. 1988. 4,4′-diisothio-cyanatostilbene-2, 2′-disulphonic acid inhibits CD3-T cell antigen receptor-stimulated Ca2+ influx in human T lymphocytes. J. Biol. Chem. 263:19535–19540.
Sarkadi, B., Parker, J.C. 1991. Activation of ion-transport pathways by changes in cell volume. Biochim. Biophys. Acta. 1071:407–427.
Schlichter, L.C., Grygorczyk, R., Pahapill, P.A., Grygorczyk, C. 1990. A large, multiple-conductance Cl− channel in normal human T lymphocytes. Pfleugers Arch. 416:413–421.
Schlichter, L.C., Schumacher, P.A., Sakellaropoulos, G. 1994. Are small conductance Cl− channels shape and volume sensors? Biophys. J. 66(2):A168.
Stoddard, J.S., Steinbach, J.H., Simchowitz, L. 1993. Whole cell Cl− currents in human neutrophils induced by cell swelling. Am. J. Physiol. 265:C156-C165.
Valverde, M.A., Diaz, M., Sepulveda, F.V., Gill, D.R., Hyde, S.C., Higgins, C.F. 1992. Volume-regulated chloride channels associated with the human multidrug-resistance P-glycoprotein. Nature 322:467–470.
Author information
Authors and Affiliations
Additional information
We are grateful to Dr. P.S. Pennefather for helpful discussions and comments on this manuscript. Supported by grants to L.C.S. from the Medical Research Council and the National Cancer Institute of Canada and an Ontario Graduate Scholarship to P.A.S.
Rights and permissions
About this article
Cite this article
Schumacher, P.A., Sakellaropoulos, G., Phipps, D.J. et al. Small-conductance chloride channels in human peripheral T lymphocytes. J. Membarin Biol. 145, 217–232 (1995). https://doi.org/10.1007/BF00232714
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00232714