| The cocaine and antidepressant-sensitive serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a putative twelve transmembrane domain (TMD) protein critical for the termination of 5-HT signaling in the CNS and the periphery. However, neither the SERT residues comprising a permeation pathway, nor those functional groups on 5-HT contacting these residues is known. I have adopted the methods of SERT species chimeras and site-directed mutagenesis to search for domains and residues controlling antagonist interactions and the permeation of 5HT. Screening of antagonists and tryptamine derivatives against hSERT and dSERT reveals rank order potency differences in the two species variants with differences up to 100-fold for antagonists and 40-fold for substrate derivatives. I have established that an aromatic residue in TMD I (Y95 in hSERT, F90 in dSERT), contributes to the ability of two antagonists, mazindol and citalopram, and tryptamine derivatives with bulky indole nitrogen constituents to discriminate hSERT and dSERT. These and other findings has led us to investigate the possibility of TMD I functioning as a structural motif in a permeation pathway for 5HT and cosubstrates. To test this hypothesis, we mutated individual residues in TMD I of hSERT to cysteine to examine accessibility to methanethiosulfonate (MTS) derivatives. In general, cysteine mutants from the extracellular-facing half of the putative helix exhibit more reactivity to MTS reagents than those mutants on the internal face of the membrane. Furthermore, studies examining the effects of 5HT and cocaine on the accessibility of MTS reagents show that several residues are protected by 5HT and/or cocaine, while others become more exposed to NM in the presence of the ligands. The residues that display sensitivity to replacement by Cys, reactivity to MTS reagents, and are differentially sensitive to MTS reagents in the presence of 5HT and/or cocaine defined a stripe on the TMD I helix, and suggest this is a permeation pathway lining set of amino acids in SERT. |
