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 transport inhibitor hemicholinium‐3, present outside or inside the nerve terminals, to prevent uptake and release of [ 3 H]choline. Because hemicholinium‐3 is membrane‐impermeable, it was previously entrapped into synaptosomes during homogenization of brain tissue. External and internalized hemicholinium‐3 produced similar maximal inhibition (80–90%) of [ 3 H]choline uptake. Also comparable (∼30 nM) are the potency of externally applied hemicholinium‐3 and the estimated potency of the entrapped inhibitor. Exposure to ouabain elicited release of both [ 3 H]acetylcholine and [ 3 H]choline from synaptosomes prelabeled with [ 3 H]choline. The ouabain (300 μM)‐evoked release of [ 3 H]choline only was blocked by externally added (IC 50 ≃ 10 nM) or internalized (estimated IC 50 ≃ 5 nM) hemicholinium‐3. Release of previously taken up [ 3 H]choline elicited by 100 μM external choline (homoexchange) was prevented by external (IC 50 ≃ 30 μM) or entrapped (estimated IC 50 ≃ 20 μM) hemicholinium‐3. The results suggest that the choline carriers fit into the alternating‐access model proposed for classical transmitter transport. Entrapping nonpermeant ligands into synaptosomes could allow investigation of the inward‐facing conformation of native transporters and how cytoplasmic ligands affect the bidirectional transport of neurotransmitters. J. Neurosci. Res. 61:533–540, 2000. © 2000 Wiley‐Liss, Inc.