A second site rescue mutation partially restores functional expression to the serotonin transporter mutant V382P.
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.
Visiers
Irache
I
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, USA.
Weinstein
Harel
H
Rudnick
Gary
G
Stephan
Megan M
MM
D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D003198:Computer Simulation; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D009010:Monte Carlo Method; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D011392:Proline / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D050486:Serotonin Plasma Membrane Transport Proteins; D013816:Thermodynamics; D014633:Valine / Q000235:genetics
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D011392:Proline; D014633:Valine