Oligomerization of the {gamma}-aminobutyric acid transporter-1 is driven by an interplay of polar and hydrophobic interactions in transmembrane helix II.
The available evidence indicates that members of the neurotransmitter:sodium symporter family form constitutive oligomers. Their second transmembrane helix (TM2) contains a leucine heptad repeat proposed to be involved in oligomerization. In artificial transmembrane segments, interhelical interactions are stabilized by polar residues. We searched for these hydrogen bond donors in TM2 by mutating the five polar residues in TM2 of the gamma-aminobutyric acid transporter-1 (GAT1). We tested the ability of the resulting mutants to oligomerize by fluorescence microscopy, Foerster resonance energy transfer, and beta-lactamase fragment complementation. Of all generated mutants, only Y86A- (but not Y86F-), E101A-, E101Q-, and E101D-GAT1 were judged by these criteria to be deficient in oligomerization and were retained intracellularly. The observations are consistent with a model where the leucine heptad repeat in TM2 drives a homophilic association that is stabilized by Tyr(86) and Glu(101); Tyr(86) participates in hydrophobic stacking. Glu(101) is in the a-position of the leucine heptad repeat (where positions 1-7 are denoted a-g, and each leucine is in the central d-position). Thus, Glu(101) is in the position predicted for the hydrogen bond donor (i.e. sandwiched between Leu(97) and Leu(104), which are one helical turn above and below Glu(101)). These key residues, namely Tyr(86) and Glu(101), are conserved in related transporters from archaeae to humans; they are therefore likely to support oligomeric assembly in transporter orthologs and possibly other proteins with multiple transmembrane segments.
Korkhov
Vladimir M
VM
Institute of Pharmacology, Medical University of Vienna, Waehringer Strasse 13a, Vienna A-1090, Austria.
Farhan
Hesso
H
Freissmuth
Michael
M
Sitte
Harald H
HH
D000595:Amino Acid Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D050485:GABA Plasma Membrane Transport Proteins; D005816:Genetic Complementation Test; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008027:Light; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000737:chemistry; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D001618:beta-Lactamases / Q000378:metabolism
D018076:DNA, Complementary; D050485:GABA Plasma Membrane Transport Proteins; D026901:Membrane Transport Proteins; C493413:SLC6A1 protein, human; D018698:Glutamic Acid; D014443:Tyrosine; D012964:Sodium; D001618:beta-Lactamases; D007930:Leucine