Mutations within an intramembrane leucine heptad repeat disrupt oligomer formation of the rat GABA transporter 1.
Na(+)/Cl(-)-dependent neurotransmitter transporters form constitutive oligomers, the significance of which is not known. In soluble proteins, leucine heptad repeats drive dimerization; the rat gamma-aminobutyric acid transporter GAT-1 (rGAT) contains a motif reminiscent of a leucine heptad repeat in the second transmembrane helix (TM2). We substituted leucine residues in TM2 of rGAT by alanine and tested the ability of the resulting mutants to form oligomers by three methods of Förster resonance energy transfer (FRET) microscopy. Replacement of one leucine (L97A) resulted in considerable loss of energy transfer, replacing two or more ablated it completely. Furthermore, intracellular trapping increased with the number of leucine substitutions. Only rGAT-L97A reached the cell surface to a sufficient amount such that, in intact cells, it was indistinguishable from wild type rGAT with respect to substrate transport, binding of inhibitors, and regulation by protein kinase C. However, in membrane vesicles prepared from transfected cells, all mutants were still functional. In addition, FRET was readily detected during maturation of wild type rGAT, when the bulk of the protein resided in the endoplasmic reticulum. Hence, our findings strongly argue for a role of oligomer formation during biosynthesis and subsequent delivery of the multimer from the endoplasmic reticulum to the plasma membrane.
Scholze
Petra
P
Institute of Pharmacology, University of Vienna, Währinger Strasse 13a, Austria.
Freissmuth
Michael
M
Sitte
Harald H
HH
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation; D050485:GABA Plasma Membrane Transport Proteins; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D027361:Organic Anion Transporters; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D002352:Carrier Proteins; D018076:DNA, Complementary; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C493413:SLC6A1 protein, human; C493415:Slc6a1 protein, rat; D005680:gamma-Aminobutyric Acid; D011493:Protein Kinase C; D007930:Leucine