Molecular insights into the translocation mechanism for substrates by the serotonin transporter
Abstract
The serotonin transporter (SERT) is responsible for regulating the serotonin (5-HT) concentration in the synapse. I used human and Drosophila serotonin transporters (hSERT and dSERT, respectively) to explore differences in substrate recognition. hSERT and dSERT showed similar K m values for 5-HT transport, suggesting similar recognition of 5-HT by the two species variants. Interestingly, another substrate, N-methyl-4-phenylpyridinium (MPP+), was transported only by hSERT. However, MPP + inhibited 5-HT uptake in both species variants with similar potencies. Guided by studies using hSERT/dSERT chimeras, the region encompassing transmembrane domains (TMDs) V to IX was implicated in substrate recognition and transport. Residues in the TMDs V to VIII region divergent between hSERT and dSERT as well the two phenylalanines at position 551 and 556 of hSERT were mutated. Mutants V366S, M370L, S375A, T381 S, and F556S showed a decrease in transport capacity for 5-HT and MPP+ uptake in comparison with wild-type. Residues V366, M370, and T381 are predicted to face the same region of TMD VII, whereas S375 is localized on the other side of this α-helix. F556 is predicted to be localized in TMD XI, close to the extracellular side. To further explore the role of these residues in the transport process, I generated cysteine mutants at positions S365, V366, M370, S375, F380, T381, F551 and F556. Pretreatment with the cysteine-modifying reagent MTSET ([2-(trimetylammonium)ethyl]methanethiosulfonate) caused a decrease in transport of [3H]5-HT in V366C, M370C, and F556C mutants. MTSET also disrupted [3H]MPP+ uptake in F556C. Replacement of sodium with lithium did not prevent MTSET from interacting with V366C, M370C, or F556C mutants. However, 5-HT partially protects these sites from MTS interaction in the presence of lithium. My data suggest that V366, M370, and F556 are localized in or near the permeation pathway used for substrate translocation. These studies as well as mutagenesis and SCAM data from monoamine transporters were used to generate models for substrate permeation and TMD arrangement for SERT. Supported by NIH grant MH60221 and APA Minority Fellowship in Neuroscience.
Degree
Ph.D.
Advisors
Barker, Purdue University.
Subject Area
Neurology
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