Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes

Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes

^*Department of Physiology, University of Tuebingen, Gmelinstrasse 5, 72076 Tuebingen, Germany; and ^‡Consiglio Nazionale delle Ricerche Unit for Biomembranes, Department of Biomedical Sciences, University of Padova, Via Colombo 3, 35121 Padova, Italy

Edited by Joseph F. Hoffman, Yale University School of Medicine, New Haven, CT, and approved March 10, 1998 (received for review September 3, 1997)

Abstract

CD95/Fas/APO-1 mediated apoptosis is an important mechanism in the regulation of the immune response. Here, we show that CD95 receptor triggering activates an outwardly rectifying chloride channel (ORCC) in Jurkat T lymphocytes. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces activation of ORCC in cell-attached patch clamp experiments. Activation is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor herbimycin A or by genetic deficiency of p56lck. In vitro incubation of excised patches with purified p56lck results in activation of ORCC, which is partially reversed upon addition of anti-phosphotyrosine antibody. Inhibition of ORCC by four different drugs correlates with a 30–65% inhibition of apoptosis. Intracellular acidification observed upon CD95 triggering is abolished by inhibition of either ORCC or p56lck. The results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes.

Footnotes

↵ † To whom reprint requests should be addressed. e-mail: biomed{at}bpciv.bio.unipd.it (I.S.); florian.lang{at}uni-tuebingen.de (F.L.).
↵ § E.G. and F.L. contributed equally to this study and, therefore, share last authorship.
This paper was submitted directly (Track II) to the Proceedings Office.
Abbreviations: CFTR, cystic fibrosis transmembrane regulator; DIDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; DPC, diphenylamine carboxylate; IAA, indoleacetic acid; ORCC, outwardly rectifying chloride channel; PKA, protein kinase A; V_m, membrane potential; FITC, fluorescein isothiocyanate.