Functional role of critical stripe residues in transmembrane span 7 of theserotonin transporter - Effects of Na+, Li+, and methanethiosulfonate reagents

Mutations at critical residue positions in transmembrane span 7 (TM7) of th e serotonin transporter affect the Na+ dependence of transport. It was poss ible that these residues, which form a stripe along one side of the predict ed alpha -helix. formed part of a water-filled pore for Na+. We tested whet her cysteine substitutions in TM7 mere accessible to hydrophilic, membrane- impermeant methanethiosulfonate (MTS) reagents. Although all five cysteine- containing mutants tested were sensitive to these reagents, noncysteine con trol 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 wh en tested in the C109A background. It is thus unlikely that the critical st ripe forms part of a water-filled pore. Instead, studies of the ion depende nce of the reaction between Cys-109 and MTS reagents lead to the conclusion that TM7 is involved in propagating conformational changes caused by ion b inding, 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.