Study to determine if human serotonin transporter mutants associated with autism induce conformational changes.
Daryl Jones, Dr. Keith Henry
Autism is a neural development disorder that affects 1 in 88 individuals. Studies have correlated (1) the function of the human serotonin transporter (hSERT) and (2) increased serotonin (5-HT) plasma levels to individuals with the rigidity component (repetitive behavior) of the autism spectrum disorder (ASD). Genetic analyses in human populations have identified five distinct genetic variants (G56A, K605N, I425L, F465L, L550V) coding for non- synonymous single nucleotide polymorphisms (SNPs) of hSERT that occur at a higher frequency in autistic individuals than control subjects. hSERT is involved in maintaining serotonergic tone and its dysregulation has been linked to several mood disorders. Interestingly, these ASD-related SNPs in SERT appear to upregulate SERT activity such that the transporter cannot be down regulated through normal cellular mechanisms. However, the manner by which these SNPs modulate SERT function is unknown. In this study, we will utilize chemical probes and site directed mutagenesis (SDM) to investigate whether the ASD SERT mutations confer conformational changes to the transporter contributing to altered SERT function. To accomplish this we used SDM to generate two SERT mutants, S404C and S277C. To date, all five ASD SNPs have been introduced into the S404C background. These constructs are being analyzed to determine the impact of the SNPs on S404C sensitivity and inactivation by thiol-reactive methanethiosulfonates (MTS) reagents. [3H]5-HT uptake assays were used to evaluate MTS-mediated inactivation in the ASD mutants in comparison to the S404C background alone. Changes in 5-HT transport between the S404C construct and the S404C/SNP mutants will suggest that the SNPs induce a conformational change in SERT and provide a plausible mechanism for how these SNPs alter SERT function.
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