The role of external loop regions in serotonin transport. Loop scanning mutagenesis of the serotonin transporter external domain.

Chimeric transporters were constructed in which the predicted external loops of the serotonin transporter (SERT) were replaced one at a time with a corresponding sequence from the norepinephrine transporter (NET). All of the chimeric transporters were expressed at levels equal to or greater than those of wild type SERT, but the transport and binding activity of the mutants varied greatly. In particular, mutants in which the NET sequence replaced external loops 4 or 6 of SERT had transport activity 5% or less than that of wild type, and the loop 5 replacement was essentially inactive. In some of these mutants, binding of a high affinity cocaine analog was less affected than transport, suggesting that the mutation had less effect on the initial binding steps in transport than on subsequent conformational changes. The more severely affected mutants also displayed an altered response to Na(+). In contrast to the dramatic reduction in transport and binding, the specificity of ligand binding was essentially unchanged. Chimeric transporters did not gain affinity for dopamine, a NET substrate, or desipramine, an inhibitor, at the expense of affinity for serotonin or paroxetine, a selective SERT inhibitor. The results suggest that external loops are not the primary determinants of substrate and inhibitor binding sites. However, they are not merely passive structures connecting transmembrane segments but rather active elements responsible for maintaining the stability and conformational flexibility of the transporter.