Differences in interactions with the dopamine transporter as revealed by diminishment of Na+ gradient and membrane potential:: Dopamine versus other substrates

In heterologous cells expressing the dopamine transporter (DAT), simultaneous elevation of intracellular Na+ and depolarization of the membrane with gramicidin reduced the potency of various DAT substrates, including dopamine, D-amphetamine, beta-phenethylamine, p-tyramine, and MPP+, in inhibiting binding of the cocaine analog [H-3]CFT, with the greatest reduction observed for D-amphetamine. In rat striatal synaptosomes, gramicidin exerted similar effects; in addition, the potency of D-amphetamine was reduced by the Na+-channel activator veratridine. The latter effect was counteracted by the Na+-channel blocker tetrodotoxin. In broken membranes, where, as the situation with gramicidin, both sides of the non-polarized membrane were exposed to 130 mM Na+, gramicidin was ineffective. Dopamine had a potency for membrane preparations that was not significantly different from that for control cells or synaptosomes, while other substrates had potencies for membrane preparations that were reduced to a level similar to those observed in gramicidin-treated cells or synaptosomes. These results suggest that diminishing Na+ gradient and membrane potential may convert DAT to a conformational state that dopamine could easily bind to when gaining free access to its intracellular portion. In contrast, non-dopamine substrates may not be able to readily interact with this state from either side of the membrane. (c) 2005 Elsevier Ltd. All rights reserved.