High-affinity, sodium-gradient-dependent transport of choline into vesiculated presynaptic plasma membrane fragments from the electric organ of Torpedo marmorata and reconstitution of the solubilized transporter into liposomes

Abstract

Vesiculated fragments of presynaptic plasma membranes have been isolated from the purely cholinergic electromotor nerve terminals of Torpedo marmorata. Synaptosomes, generated from the terminals by homogenization, were separated on a discontinuous Ficoll gradient and then lysed by osmotic shock at 2°C, pH 8.5 in the presence of 0.1 mM MgCl 2. These conditions for lysis were optimal for choline transport. Electron micrographs of lysed synaptosomes showed vesiculated membranes with diameters smaller than those of synaptosomes; occasionally, synaptic vesicles were observed attached to them. Intact mitochondria or synaptosomes and basal laminae were not present. High-affinity ( K T = 1.7 μM) uptake of choline into these vesiculated membrane fragments showed: (1) an absolute dependence on the Na + gradient (outside > inside), (2) a transient Na +-gradient-dependent accumulation of choline over the equilibrium concentration (overshoot), (3) electrogenicity and rheogenicity, since the uptake was further stimulated in the presence of a Na + gradient by valinomycin, (4) dependence on the presence of external Cl −, and partial dependence on a Cl − gradient (outside > inside), (5) high-affinity ( K i = 25 nM) inhibition by hemicholinium-3 and (6) temperature sensitivity. The plasma membranes were further purified by centrifugal density gradient fractionation on a 4–12% Ficoll gradient. Several enzymes and polypeptides copurified with the specific binding sites for choline present in the membranes. The fraction with the most binding sites was one denser than 12% Ficoll. This was also the fraction richest in acetylcholinesterase, 5′-nucleotidase and polypeptides of relative molecular mass, M r (×10 −3) of > 200, 140, 68 (doublet), 57, 54 and 28. Acetylcholinesterase was positively identified as a M r 68 000 component by immune blot. By contrast the ouabain-sensitive ATPase showed a negative correlation with choline binding sites. When the solubilized proteins of the vesiculated membranes were transferred to liposomes, they conferred on the latter the capacity to take up choline in a manner closely resembling its transport in natural membranes but with an initial (one minute) rate of uptake approximately 10-times greater per mg of protein. Several proteins were selectively transferred to the liposomes including ones of M r (×10 −3) 34, 42, 47, 54, 60, 68, 92, 160 and > 200. The polypeptides of M r (×10 −3) 140, 57 and 28 were lost in the transfer. When a correlation was made between transport rates observed in the various liposome preparations tried and the presence of particular polypeptides, only three proteins correlated with the ability of liposomes to transport choline: the M r (×10 −3) > 200, 54 and 34 components.