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Mutations at critical residue positions in transmembrane span 7 (TM7) of the serotonin transporter affect the Na(+) dependence of transport. It was possible that these residues, which form a stripe along one side of the predicted alpha-helix, formed part of a water-filled pore for Na(+). We tested whether cysteine substitutions in TM7 were accessible to hydrophilic, membrane-impermeant methanethiosulfonate (MTS) reagents. Although all five cysteine-containing mutants tested were sensitive to these reagents, noncysteine control mutants at the same positions were in most cases equally sensitive. In all cases, MTS sensitivity could be traced to changes in accessibility of a native cysteine residue in extracellular loop 1, Cys-109. Moreover, none of the TM7 cysteines reacted with the biotinylating reagent MTSEA-biotin when tested in the C109A background. It is thus unlikely that the critical stripe forms part of a water-filled pore. Instead, studies of the ion dependence of the reaction between Cys-109 and MTS reagents lead to the conclusion that TM7 is involved in propagating conformational changes caused by ion binding, perhaps as part of the translocation mechanism. The critical stripe residues on TM7 probably represent a close contact region between TM7 and one or more other TMs in the transporter's three-dimensional structure.

Transmembrane span 7 of the rat brain serotonin transporter was subjected to random mutagenesis. Of the 27 amino acid residues mutated, six were identified as functionally important by their sensitivity to nonconservative mutations. These residues were Asn-368 and Tyr-385, where substitutions that retained hydrogen-bonding ability were preferred; Gly-376 and Gly-384, where only glycine was accepted; Phe-380, where a phenyl ring was preferred; and Met-386, where hydrophobic substitutions were preferred. Mutations that did not preserve these structural characteristics were highly detrimental to serotonin transport activity. These six residues form a stripe that runs at an angle down the side of the putative alpha-helix, lending support to this structural prediction. Mutations at some of these positions also specifically impaired transport activity under low Na+ conditions. Other mutations at nearby positions in transmembrane span 7 also impaired activity in low Na+, although the activity of the mutants in high Na+ was similar to wild type. These results suggest that at least some of the six critical residues play a role in Na+ binding or perhaps in the coupling of Na+ binding to later steps in the transport cycle. These residues may be important in other aspects of the transporter's function as well.

The serotonin transporter (SERT) is a member of a highly homologous family of proteins responsible for the reuptake of biogenic amines from the synaptic cleft. We took advantage of native restriction sites in SERT to construct a chimeric transporter containing a small (34 amino acid) region of the norepinephrine transporter. The substituted region corresponds to about half of the largest extracellular loop. This chimera transports serotonin very slowly compared to wild type SERT. However, it binds serotonin and the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane with a high affinity indistinguishable from wild type. It has the same specificity as wild type SERT for the antidepressants paroxetine and desipramine. The low rate of transport does not appear to be due to poor expression, since the chimeric transporter is expressed at the membrane surface at close to wild type levels as measured by cell surface biotinylation. These observations lead us to conclude that, rather than playing a role in substrate or drug binding, this region of the large extracellular loop may be involved in the conformational changes associated with substrate translocation into the cell.