Structural verification of a human serotonin transporter homology model

Strong evidence implicates the serotonin transporter (SERT) as a target for many antidepressants and drugs of abuse. The structure of this integral membrane protein at the presynaptic membrane is unclear. Our research focused on identifying amino acid residues and specific helices involved in the substrate permeation pathway. Recently, the crystal structure of a bacterial homologue of Na + /Cl − ‐dependent neurotransmitter transporters was determined. Based on this structure, a homology model of SERT was generated. The function of residues predicted to be at the entrance to the permeation pore were explored using the substituted cysteine accessibility method (SCAM). Each cysteine mutant was characterized by using the membrane impermeant [2‐(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) and the novel 4‐(1‐methylpyridinium)phenylmethanethiosulfonate iodide (MTS‐MPP+) reagents. Inactivation by these reagents suggested that these residues reside near the substrate pore. The proximity of the helices shown to be near the permeation pathway was further evaluated by examining the sensitivity of double cysteine mutants to bifunctional MTS cross‐linking reagents, thus confirming the proximity of these residues to each other. Taken together, our studies allow us to refine our homology model of SERT to further explore SERT function. This research supported by grant R01DA018682 from NIH.