4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid inhibits CD3-T cell antigen receptor-stimulated Ca²⁺ influx in human T lymphocytes

Stimulation of the CD3-T cell antigen receptor complex on T lymphocytes results in a rapid rise in intracellular calcium from both intra- and extracellular sources. The former is thought to be released from the endoplasmic reticulum in response to inositol trisphosphate, while the latter enters the cells through a membrane potential-sensitive transporter (Oettgen, H. C., Terhorst, C., Cantley, L. C., and Rosoff, P. M. (1985) Cell 40, 583-590). In this report we show that the stilbene disulfonate, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), inhibited the ability of monoclonal anti-CD3 complex antibodies to stimulate an influx of calcium in the human T lymphocyte cell line, Jurkat. DIDS had no effect on either antibody binding to the receptor or receptor-stimulated phosphatidylinositol turnover. The K(i) was ~ 25 μM in the presence of extracellular Cl^- and 10 μM when labeling was performed in the absence of Cl^-, suggesting that DIDS was competing with Cl^- for binding to the cell membrane. The reduced form of DIDS, dihydroDIDS, was only 50% as effective as DIDS itself, and the monoisothiocyanate stilbene, 4-acetamido-4'-isothiocyantostilbene-2,2'-disulfonic acid, was totally ineffective, even to concentrations of 0.750 mM. Removal of extracellular Cl^- also inhibited the antibody-stimulated influx of calcium. These data suggest that the function of the CD3-T cell receptor-activated calcium channel/transporter may be dependent on or regulated by extracellular Cl^-.