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

Abstract

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, β-phenethylamine, p-tyramine, and MPP +, in inhibiting binding of the cocaine analog [ 3H]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.