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

Abstract

Cl⁻ absorption by theAplysia 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 valinomycininduced K⁺ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl⁻ accumulation and electrical potential while an outwardly-directed valinomycininduced 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 orp-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.