Reconstitution of carrier-mediated choline transport in proteoliposomes prepared from presynaptic membranes oftorpedo electric organ, and its internal and external ionic requirements

Abstract

Proteoliposomes made by a butanol-sonication technique from electric organ presynaptic membranes showed choline transport activity. In contrast to intact nerve terminals, the uptake of choline was dissociated from its conversion to acetylcholine in this preparation. The kinetics of choline uptake by proteoliposomes was best described by two Michaelis-Menten components. At a low concentration of choline, uptake was inhibited by hemicholinium-3 and required external Na⁺ and, thus, closely resembled high-affinity choline uptake by intact cholinergic nerve terminals. Choline transport could be driven by the Na⁺ gradient and by the transmembrane potential (inside negative) but did not directly require ATP. External Cl⁻, but not a Cl⁻ gradient, was needed for choline transport activity. It is suggested that internal K⁺ plays a role in the retention of choline inside the proteoliposome. Proteoliposomes should prove a useful tool for both biochemical and functional studies of the highaffinity choline carrier.