Enhancement of rates of H +, Na + and K + transport across phospholipid vesicular membrane by the combined action of carbonyl cyanide m-chlorophenylhydrazone and valinomycin: temperature-jump studies

Abstract

Enhancement of ΔpH relaxation rate by the combined action of valinomycin (VAL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) has been studied under a variety of concentration conditions in soyabean phospholipid (SBPL) vesicles after creating a pH gradient across the vesicular membrane ΔpH by temperature jump. After taking note of the changes by VAL and CCCP induced membrane disorder (using nigericin and monensin mediated ΔpH decay as probes) the following could be inferred about the mechanism of enhancement of ΔpH decay rate: (i) in solutions containing KCl, therate limiting species have been identified to be (a) Val-K +-CCCP −, at low [Val] 0 and [CCCP] 0 (with translocation rate constant k 2 ∼ 3.2·10 3 s −1); (b) CCCPH, at high [Val] 0 (with translocation rate constant k 1 ∼ 2·10 5 s −1); (c) the neutral valinomycin species Val, at high [CCCP] 0. (ii) In solutions containing NaCl, in our concentration range, the rate limiting species are Val-Na +-CCCP −. (iii) The apparent dissociation constant K M* of Val-M + decreases with pH in SBPL vesicles but is independent of pH in vesicles prepared from PC + 6% PA. (iv) The differences in the ionic strength dependencies of kinetic data shows that the environments of Na + and K + binding sites on VAL are different. (v) In vesicle solutions containing 100 mM MCl, the cation selectivity of VAL (towards K + in preference to Na +) is reduced when CCCP − is already bound to it in the membrane. The CCCP − dissociation constant of Val-M +-CCCP − is smaller with M += Na + (∼ 0.22 mM at 100 mM NaCl) when compared to that with M + = K + (∼ 2 mM at 100 mM KCl). Attributing these differences to the differences in electrostatic interaction between CCCP − and M + in Val-M +-CCCP −, we can say that CCCP − binds closer to the Na + binding site than to the K + binding site on VAL.