Oligomerization of the γ-Aminobutyric Acid Transporter-1 Is Driven by an Interplay of Polar and Hydrophobic Interactions in Transmembrane Helix II

Abstract

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 ␥-aminobutyric acid transporter-1 (GAT1). We tested the ability of the resulting mutants to oligomerize by fluorescence microscopy, Foerster resonance energy transfer, and ␤-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. , 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. ␥-Aminobutyric acid (GABA)1 -mediated inhibitory synaptic transmission is terminated by clearance of GABA from the synaptic cleft by high affinity uptake proteins. The four known GABA transporters (GAT1, BGT1 (betaine/GABA transporter), GAT2, and GAT3) belong to the neurotransmitter:sodium symporter (NSS) (1) family, which also includes carriers for dopamine, serotonin, glycine, etc. (2). All members of the family share sequence similarity, membrane topology, and common functional features (e.g. ion channel-like properties, sodium, and chloride) as transported cosubstrates and bidirectional transport of substrates. The interest in transporters of this family is motivated in part by their clinical relevance; they are targets for drugs of abuse (e.g. cocaine, ecstasy, and metamphetamine) and for therapeutic agents including antidepressants (amitryptiline, desipramine, citalopram, and paroxetine) and anticonvulsants (tiagabine), etc. (reviewed in Ref. 3).With the notable exception of the glycine transporter-1 (4), all members of the neurotransmitter transporter family that have so far been examined form oligomers (reviewed in Ref. 5). This is also true for GAT1, for which oligomers have been visualized in membranes of living cells by fluorescence resonance energy transfer (FRET) microscopy (6). The TM2 of GAT1 contains a leucine heptad repeat that seems to supports oligomer formation (7). The leucine heptad repeat in TM2 is a highly conserved feature throughout the NSS family. Consistent with this finding, TM2-mediated dimer formation was also suggested for dopamine transporter (8).By analogy with leucine zippers in soluble proteins, the stretch of leucine residues in the TM2 of GAT1 lines one side of a hypothetical ␣-helix. If this leucine heptad repeat is to serve as an interface between two transporter molecules, there must be additional forces that stabilize this arrangement. In aqueous solution, the hydrophobic leucine (or isoleucine) side chains are tightly packed to minimize the solve...

Concentrative Export from the Endoplasmic Reticulum of the γ-Aminobutyric Acid Transporter 1 Requires Binding to SEC24D

“…It is also evident that the COOH terminus of DAT is required to stabilize the overall conformation, because several mutations within the COOH terminus disrupt ligand binding (30). In contrast, whereas truncation of the COOH terminus causes retention of GAT1 in the endoplasmic reticulum, it does not impair its ability to translocate substrate or bind inhibitors in vesicular uptake assays (31). Similarly, if GAT1-RL/AS reached the cell surface, the mutated transporter supported substrate influx with an affinity comparable with the wild type, whereas the reduction in V max was accounted for the reduction in cell surface levels.…”

Modulation of monocarboxylate transporter 8 oligomerization by specific pathogenic mutations

“…Results from previous studies have indicated (Biebermann et al 2005) that MCT8 occurs as dimers comprising two interacting protomers, or, presumably, as higher order complexes such as tetramers (Visser et al 2009). In support of this, other members of the MFS have also been reported to form oligomers, including the glycine transporter (Bartholomaus et al 2008), organic cation transporter 1 (Keller et al 2011), g-amino butyric acid transporter 1 (Korkhov et al 2004), multidrugtransporter EmrD (Yin et al 2006), dopamine transporter (Hastrup et al 2001), and serotonin transporter (Kilic & Rudnick 2000). Higher-order complexes can be functionally relevant such as those found for amphetamine transporters, for which influx and efflux occurs via separate but interacting transporter protomers (Seidel et al 2005).…”

“…Moreover, it has been demonstrated that the amino acids from positions 267 to 360 (putative TMH4-6) are not involved in MCT8 protomer contacts (Visser et al 2009). However, several contact regions for membrane-spanning transporter oligomers have already been reported (oligomerization is inhibited by mutation): the bacterial Ca 2C /H C antiporter (Ridilla et al 2012) -contacts at TMH2 and TMH6; the Na C /H C antiporter NhaA (Gerchman et al 2001) -loop transition on the cytoplasmatic side of helix 8; the dopamine transporter (Hastrup et al 2003) -at the extracellular end of TMH6; the g-amino-butyric acid transporter 1 -at TMH2 (Korkhov et al 2004); and the glycine transporter 2 (GlyT2) -extracellular transition of TMH6. Very recently, TMH2 has been identified as a crucial dimerization interface of the bacterial sweet transporter (Xu et al 2014).…”

Interaction between lysine 102 and aspartate 338 in the insect amino acid cotransporter KAAT1

“…Figure 10 also shows that the reducing agent DTT (12 mM), after treatment with 0.2 mM CuPh, significantly restored the activity of the K102C/D338C mutant. Recent studies have indicated that members of the NSS family form constitutive oligomers (12,13,15,17,19,20,31,32), although the physiological meaning of this process is still under investigation (17,25,33,35,37). Since KAAT1 belongs to the NSS family, we also considered the hypothesis that CuPh may actually cause the creation of a disulfide bond between two cysteines in different subunits of a KAAT1 homo-oligomer.…”