Reconstituted Cl- pump protein: a novel ion(Cl-)-motive ATPase

Cl- absorption by the Aplysia californica foregut is effected through an active Cl- transport mechanism located in the basolateral membrane of the epithelial absorptive cells. These basolateral membranes contain both Cl(-)-stimulated ATPase and ATP-dependent Cl- transport activities which can be incorporated into liposomes via reconstitution. Utilizing the proteoliposomal preparation, it was demonstrated that ATP, and its subsequent hydrolysis, Mg2+, Cl-, and a pH optimum of 7.8 were required to generate maximal intraliposomal Cl- accumulation, electrical negativity, and ATPase activity. Additionally, an inwardly-directed valinomycin-induced K+ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl- accumulation and electrical potential while an outwardly-directed valinomycin-induced K+ diffusion potential, making the liposome interior electrically negative, decreased both ATP-driven Cl- accumulation and electrical potential compared with proteoliposomes lacking the ionophore. Either orthovanadate or p-chloromercurobenzene sulfonate inhibited both the ATP-dependent intraliposomal Cl- accumulation, intraliposomal negative potential difference, and also Cl(-)-stimulated ATPase activity. Both aspects of Cl- pump transport kinetics and its associated catalytic component kinetics were the first obtained utilizing a reconstituted transporter protein. These results strongly support the hypothesis that Cl(-)-ATPase actively transports Cl- by an electrogenic process.