Na+ stimulates binding of dopamine to the dopamine transporter in cells but not in cell-free preparations
Article first published online: 4 JUL 2003
Journal of Neurochemistry
Volume 86, Issue 3, pages 678–686, August 2003
How to Cite
Chen, N., Rickey, J. and Reith, M. E. A. (2003), Na+ stimulates binding of dopamine to the dopamine transporter in cells but not in cell-free preparations. Journal of Neurochemistry, 86: 678–686. doi: 10.1046/j.1471-4159.2003.01889.x
- Issue published online: 4 JUL 2003
- Article first published online: 4 JUL 2003
- Received March 28, 2003; revised manuscript received April 25, 2003; accepted April 28, 2003.
- cocaine analog;
- dopamine transporter;
- sodium ion
Although Na+ is crucial for the function of the dopamine (DA) transporter (DAT), its role in the substrate binding step has been questioned. To address this issue, we investigated the effect of Na+ on DA binding by measuring the potency of DA in inhibiting the binding of the cocaine analogue [3H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane (CFT) in intact cells expressing DAT in their plasma membranes and in membranes isolated from these cells. In cells, Na+ substantially enhanced the potency of DA in inhibiting CFT binding. This effect of Na+ was independent of buffer compositions and substitutes (sucrose vs. NMDG), more pronounced at 4°C than 25°C, and correlated with its stimulatory effect on DA uptake Km. Removing extracellular Na+ had little effect on intracellular concentrations of Na+ and K+, or on membrane potential. These data suggest that extracellular Na+ most likely acts at the transporter level to enhance the binding of external DA during the transport cycle. In contrast, in cell-free membrane preparations the Na+ stimulation was abolished without impairment of the potency of DA in inhibiting CFT binding, regardless of whether sucrose was used to maintain the buffer osmolarity. The difference in Na+ dependence for DA to inhibit CFT binding between plasma membranes of intact cells and isolated membranes raises the possibility that intracellular ion environment, alone or in combination with other cellular factors, plays a critical role in determining DA–DAT interaction and the integration of Na+ modulation in this interaction.