Characterization of the chloride carrier in the plasmalemma of the alga Valonia utricularis: the inhibition by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid

Abstract

The effect of the anion transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) on the Cl −-transport system located in the plasmalemma of cells of the giant marine alga Valonia utricularis was studied by using the charge pulse relaxation technique. Analysis of the biphasic relaxation patterns in terms of the kinetic model published previously (Wang, J., Wehner, G., Benz, R. and Zimmermann, U. (1991) Biophys. J. 59, 235–248) demonstrated that extracellular DIDS dramatically reduced the translocation rate, K AS , of the Cl −-carrier complex (maximal inhibition 79%). The translocation rate of the free carrier molecules, K S , as well as the total surface concentration of the carrier, N o , were not affected. A Hill-plot of DIDS inhibition on K AS yielded an half-maximal inhibition concentration (IC 50) of 3.9 · 10 −5 M and a Hill-coefficient of 1.61, suggesting a co-operative binding of the inhibitors to the Cl −-carrier. The maximal inhibition of DIDS was dependent on the extracellular Cl −-concentration. This inhibition was not competitive to chloride, since it increased and did not decrease with increasing chloride concentration. The DIDS effect decreased with increasing pH-value (investigated pH range between 6.5 and 10). Intravacuolar DIDS or SITS (4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid) had no effect on the biphasic voltage relaxation pattern. These results showed that the binding sites of DIDS must be located on the outer surface of the plasmalemma of V. utricularis and, in turn, supported previous conclusions that the Cl −-carrier (which is assumed to be part of the turgor-pressure-sensing mechanism) is only located in the outer membrane.