CRITICAL AMINO-ACID-RESIDUES IN TRANSMEMBRANE SPAN-7 OF THE SEROTONINTRANSPORTER IDENTIFIED BY RANDOM MUTAGENESIS

Transmembrane span 7 of the rat brain serotonin transporter was subjec ted to random mutagenesis, Of the 27 amino acid residues mutated, six were identified as functionally important by their sensitivity to nonc onservative mutations. These residues were Asn-368 and Tyr-385, where substitutions that retained hydrogen-bonding ability were preferred; G ly-376 and Gly-384, where only glycine was accepted; Phe-380, where a phenyl ring was preferred; and Met-386, where hydrophobic substitution s were preferred. Mutations that did not preserve these structural cha racteristics were highly detrimental to serotonin transport activity. These six residues form a stripe that runs at an angle down the side o f the putative cy-helix, lending support to this structural prediction . Mutations at some of these positions also specifically impaired tran sport activity under low Na+ conditions. Other mutations at nearby pos itions in transmembrane span 7 also impaired activity in low Na+, alth ough 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.