High affinity choline uptake and calcium-dependent acetylcholine release in proteoliposomes derived from rat cortical...

Proteoliposomes were prepared from rat cortical synaptic plasma membranes that retained high and low affinity choline transport. High affinity transport was inhibited by hemicholinium-3 in a competitive manner and was apparently dependent on membrane potential or ion gradients. Proteoliposomes supplemented with an acetylcholine-generating system were able to synthesize [3H]ACh de novo from [3H]choline taken up by high or low affinity transport; however, we were able to specifically load proteoliposomes derived from cholinergic membranes by using a low choline concentration. [3H]ACh, but not [3H]choline, was subsequently released in a calcium-dependent manner by K+ depolarization. This preparation offers a simplified membrane system for studying functional high affinity choline transport and nonvesicular ACh release.