LIGAND-BINDING TO THE SEROTONIN TRANSPORTER - EQUILIBRIA, KINETICS, AND ION DEPENDENCE

The effects of Na+ and Cl- on the binding of [H-3]imipramine and the c ocaine analog [I-125]- beta-carbomethoxy-3 beta-(4-iodophenyl)tropane( [I-125]-beta-CIT) to the human platelet serotonin transporter have bee n measured. The ion dependence of beta-CIT binding is consistent with binding of beta-CIT together with one Na+ ion, but not in an ordered s equence. Imipramine affinity, like beta-CIT affinity, is increased by Na+, but imipramine binding involves at least two Na+ ions. This concl usion is based on the observation that both imipramine association rat e constants and equilibrium affinity constants show a sigmoidal Na+ de pendence. As with beta-CIT, the imipramine and Na+ binding sequence is not strictly ordered. Cl- increases imipramine affinity, apparently b y slowing dissociation. beta-CIT binding occurs even in the absence of Na+ and Cl-. This provided a means to measure substrate and inhibitor affinity in both the presence and absence of cotransported ions. Nont ransported inhibitors, such as imipramine and citalopram, as well as t he transport substrates serotonin and 3,4-(methylenedioxy)methamphetam ine all displaced beta-CIT binding in the absence of NaCl. In the abse nce of Cl-, Na+ increased the affinity of nontransported inhibitors bu t not of substrates. The results suggest that Na+ and Cl- induce indep endent changes in the transporter binding site and that binding of sub strates and inhibitors is affected differently by these changes.