Format

Send to

Choose Destination
See comment in PubMed Commons below
Biochemistry. 2003 Jun 10;42(22):6784-93.

A second site rescue mutation partially restores functional expression to the serotonin transporter mutant V382P.

Author information

1
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, USA.

Abstract

Transmembrane span 7 (TM7) of the serotonin transporter (SERT) was previously subjected to random mutagenesis, and the mutation V382P was found to abolish transport activity. Val-382 lies next to a threonine residue in the native sequence, creating a TP motif in this mutant. On the basis of molecular modeling studies, which have shown that the presence of a TP motif produces a very large kink in an alpha-helix, it was hypothesized that this motif could be the source of V382P's deleterious effects. We tested this hypothesis by producing second site mutations in the V382P construct that removed the TP motif: T381A-V382P and T381V-V382P. These mutants were tested for the recovery of serotonin transport and binding activities and for expression at the cell surface. The TM7 alpha-helix was modeled computationally, using Biased Monte Carlo simulations to quantify the conformational preferences of the wild type and mutant helices. The double mutation T381A-V382P, which was predicted by modeling to produce a smaller perturbing bend in TM7, was indeed found to allow partial rescue of transport activity. The double mutation T381V-V382P, on the other hand, did not rescue transport activity. Computational analysis of this mutant predicted a markedly different conformational preference from either the V382P or the T381A-V382P mutants. These studies show that changes in the structure of TM7 exert a strong influence on SERT's ability to achieve a mature, properly folded, cell surface conformation.

PMID:
12779333
DOI:
10.1021/bi0273415
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for American Chemical Society
    Loading ...
    Support Center