MODELING OF THE INTERACTION OF ETA-(4-[I-125]IODOPHENYL)TROPANE-2-BETA-CARBOXYLIC ACID ISOPROPYL ESTER TO THE DOPAMINE TRANSPORTER( AND K+ WITH THE BINDING OF THE COCAINE ANALOG 3)

The present study examines the interaction of Na+ and K+ with the bind ing of the cocaine analogue 3 beta-(4-[I-125]iodophenyl)tropane-2 beta -carboxylic acid isopropyl ester to dopamine transporters (DATs) in ra t striatal synaptosomal membranes at 37 degrees C. The binding increas es with [Na+] from 10 to 100 mM and decreases with higher [Na+]. The p resence of K+ reduces the maximal stimulatory effect of Na+ and causes a nonlinear EC50 shift for Na+, K+ strongly inhibits the binding at l ow [Na+]. Increasing [Na+] produces a linear IC50 shift for K+. Satura tion analysis indicates a single binding site changing its affinity fo r the radioligand depending on [K+]/[Na+] ratio in the assay buffer. A reduced B-max was observed in the presence of 10 mM Na+ and 30 mM K+. Both high [Na+] and high [K+] accelerate the dissociation of the bind ing, and K+-induced acceleration was abolished by increasing [Na+]. Le ast squares model fitting of equilibrium data and kinetic analysis of dissociation rates reveal competitive interactions between Na+ and Kat two sites allosterically linked on the DAT, One site mediates the s timulatory effect of Na+, and the other site involves the radioligand binding and the inhibitory effect of cations on the binding. Various u ptake blockers and substrates, dopamine in particular, display reduced potency in inhibiting the binding at a higher [K+]/[Na+] ratio.