Modeling of the interaction of Na+ and K+ with the binding of dopamine and[H-3]WIN 35,428 to the human dopamine transporter

Although much is known about the effects of Na+, K+, and Cl- on the functio nal activity of the neuronal dopamine transporter, little information is av ailable on their role in the initial event in dopamine uptake, i.e,, the re cognition step. This was addressed here by studying the inhibition by dopam ine of the binding of [H-3]WIN 35,428 {2 beta-carbomethoxy-3 beta-(4-fluoro phenyl)[H-3]tropane} a phenyltropane analogue of cocaine, to the cloned hum an dopamine transporter expressed in HEK-293 cells. The decrease in the aff inity of dopamine (or WIN 35,428) binding affinity with increasing [K+] cou ld be fitted to a competitive model involving an inhibitory cation site (1) overlapping with the dopamine (or WIN 35,428) domain. The K+ IC50 for inhi biting dopamine or WIN 35,428 binding increased linearly with [Na+], indica ting a K-D,K-Na+ of 30-44 mM and a K-D,K-K+ of 13-16 mM for this cation sit e. A second Na+ site (2), distal from the WIN 35,428 domain but linked by p ositive allosterism, was indicated by model fitting of the WIN 35,428 bindi ng affinities as a function of [Na+]. No strong evidence for this second si te was obtained for dopamine binding in the absence or presence of low (20 mM) Cl- and could not be acquired for high [Cl-] because of the lack of a s uitable substitute ion for Na+. The K-D but not B-max of [H-3]WIN 35,428 bi nding increased as a function of the [K+]/[Na+] ratio regardless of total [ Cl-] or ion tonicity. A similar plot was obtained for the K-i of dopamine b inding, with Cl- greater than or equal to 140 mM decreasing the K-i. At 290 mM Cl- and 300 mM Na+ the potency of K+ in inhibiting dopamine binding was enhanced as compared with the absence of Cl- in contrast to the lack of ef fect of Cl- up to 140 mM (Nat up to 150 mM). The results indicate that Cl- at its extracellular level enhances dopamine binding through a mechanism no t involving site 1, The observed correspondence between the WIN 35,428 and dopamine domains in their inclusion of the inhibitory cation site explains why many of the previously reported interrelated effects of Na+ and K+ on t he binding site of radiolabeled blockers to the dopamine transporter are ap plicable to dopamine uptake in which dopamine recognition is the first step .