Study of the bidirectional transport of choline by blocking choline carriers from outside or inside brain nerve terminals

Membrane carriers can operate bidirectionally. We studied, in rat neocortex synaptosomes, the choline carrier by comparing the ability of the transpor t inhibitor hemicholinium-3, present outside or inside the nerve terminals, to prevent uptake and release of [H-3]choline. Because hemicholinium-3 is membrane-impermeable, it was previously entrapped into synaptosomes during homogenization of brain tissue. External and internalized hemicholinium-3 p roduced similar maximal inhibition (80-90%) of [H-3]choline uptake. Also co mparable (similar to 30 nM) are the potency of externally applied hemicholi nium-3 and the estimated potency of the entrapped inhibitor. Exposure to ou abain elicited release of both [H-3]acetylcholine and [H-3]choline from syn aptosomes prelabeled with [H-3]choline. The ouabain (300 mu M)-evoked relea se of [H-3]choline only was blocked by externally added (IC50 similar or eq ual to 10 nM) or internalized (estimated IC50 similar or equal to 5 nM) hem icholinium-3. Release of previously taken up [H-3]choline elicited by 100 m u M external choline (homoexchange) was prevented by external (IC50 similar or equal to 30 mu M) or entrapped (estimated IC50 similar or equal to 20 m u M) hemicholinium-3. The results suggest that the choline carriers fit int o the alternating-access model proposed for classical transmitter transport . Entrapping nonpermeant ligands into synaptosomes could allow investigatio n of the inward-facing conformation of native transporters and how cytoplas mic ligands affect the bidirectional transport of neurotransmitters. (C) 20 00 Wiley-Liss, Inc.