Abstract
The plasma membrane serotonin (5-HT) transporter (SERT, SLC6A4) clears 5-HT after release at nerve termini and is targeted by both antidepressant medications and psychostimulants (e.g. MDMA, cocaine). Homology modeling of human SERT (hSERT), based on high resolution structures of the microbial SLC6 family member LeuT(Aa), along with biochemical studies of wild type and mutant transporters, predicts transmembrane (TM) domains 1, 3, 6, and 8 comprise the 5-HT-binding pocket. We utilized the substituted cysteine accessibility method along with surface and site-specific biotinylation to probe TM6 for aqueous accessibility and differential interactions with 5-HT and psychostimulants. Our results are consistent with TM6 being composed of an aqueous-accessible, alpha-helical extracellular domain (TM6a) that is separated by a central, unwound section from a cytoplasmically localized domain (TM6b) with limited aqueous accessibility. The substitution G338C appears to lock hSERT in an outward-facing conformation that, although accessible to aminoethylmethanethiosulfonate-biotin, 5-HT, and citalopram, is incapable of inward 5-HT transport. Transport of 5-HT by G338C can be partially restored by the TM1 mutation Y95F. With regard to methanethiosulfonate (MTS) inactivation of uptake, TM6a Cys mutants demonstrate Na(+)-dependent [2-(trimethylammonium)ethyl]-MTS sensitivity. Studies with the centrally located substitution S336C reveal features of a common binding pocket for 5-HT and 3,4-methylenedioxymethamphetamine (MDMA). Interestingly, the substitution I333C reveals an MDMA-induced conformation not observed with 5-HT. In the context of prior studies on TM1, our findings document shared and unique features of TM6 contributing to hSERT aqueous accessibility, ligand recognition, and conformational dynamics.
Highlights
After vesicular release, thereby constraining the indolamine actions on pre- and postsynaptic 5-HT receptors [1]
Effects of Cys Substitution on human SERT (hSERT) Expression and Function— Before characterizing the accessibility of hSERT TM6 Cys substitutions to MTS reagents, the effect of Cys substitution was examined for impact on 5-HT transport activity and transporter surface expression
Similar to the effects of Cys substitution observed in TM1 [42], Cys substitution produced a greater impact on uptake activity in TM6a than the relatively unperturbed TM6b
Summary
Site-directed Mutagenesis—Site-directed mutagenesis of hSERT C109A was performed using the QuikChange mutagenesis kit and protocol (Stratagene). [3H]Citalopram Competition Binding Assays—hSERT C109A and hSERT C109A mutant-transfected cells were washed once with 4 °C PBS buffer followed by the addition of 4 °C homogenization buffer (50 mM HEPES, 2.5 mM MgCl2, 2 mM EGTA, 100 M ascorbic acid, pH 7.4). Sensitivity of hSERT TM6 Cys Mutants to MTS Reagents— Transiently transfected HEK-293T cells in 24-well tissue culture plates were washed twice with 500 l of room temperature PBS/CM buffer (PBS, 0.1 mM CaCl2 and 1.0 mM MgCl2, pH 7.4) and incubated in PBS/CM buffer with/without MTS reagents for 10 min at room temperature. 48 h after transfection, cells were washed 4 times with ice-cold PBS/CM buffer and incubated with 1.0 mg/ml NHS-SS-biotin in PBS/CM for 30 min at 4 °C. Cys mutants cDNAs were transiently transfected in HEK-293T cells at equal concentrations and assayed for uptake at a single 3H-5-HT concentration (see “Experimental Procedures”). Multiple exposures were captured in all experiments to ensure collection of data in the film linear range
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