An extracellular loop region of the serotonin transporter may be involved in the translocation mechanism.
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.
Stephan
M M
MM
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Chen
M A
MA
Penado
K M
KM
Rudnick
G
G
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins
D002352:Carrier Proteins; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin