Mutations within an Intramembrane Leucine Heptad Repeat Disrupt Oligomer Formation of the Rat GABA Transporter 1

Abstract

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 ␥-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 Fö rster 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. Naϩ /Cl Ϫ -dependent neurotransmitter transporters (e.g. the transporters for dopamine, serotonin, or GABA) 1 retrieve neurotransmitters from the synaptic cleft into the presynaptic specialization (1). The medical relevance of these proteins is obvious; for instance, it has long been known that antidepressant drugs block the transporter for norepinephrine and serotonin (2). Likewise, tiagabine, an inhibitor of GABA transport, is used as an anticonvulsant in the treatment of epileptic seizures (3). Transporters support bidirectional flux of substrate, i.e. not only do they mediate influx of substrate but they also allow for non-exocytotic release of substrate (4). Compounds that induce reverse transport enjoy widespread popularity among illicit drug users; this is particularly true for amphetamine and its congeners, including ecstasy.Increasing evidence suggests that neurotransmitter transporters are oligomers (5, 6). Constitutive oligomerization has been visualized in intact cells by FRET microscopy (7); the oligomeric nature of neurotransmitter transporters has also been demonstrated by other, more disruptive, approaches, e.g. co-immunoprecipitation from detergent extracts and crosslinking (8 -10) and by freeze-fracture electron microscopy (11). However, the functional role of oligomer formation remains enigmatic. The hypothesis has been formulated that neurotransmitter transporters function in a manner analogous to ligand-gated ion channels, as binding of substrate induces an ion current (12-14). There is still an ongoing debate whether the GAT operates in a channel-like mode (15, 16) or according to the conservative "alternating ac...

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

“…We also show that a mutant transporter, which is unable to interact with Sec24D, exerts a dominant negative effect on the surface expression of the wild type transporter. These observations are consistent with the hypothesis that ER export requires oligomeric assembly of transporters (12), provide for a mechanistic explanation by linking oligomer formation to efficient recruitment of the COPII components, and may be relevant for other membrane proteins such as G protein-coupled receptors (18).…”

“…The plasmid encoding hamster Sar1a was a gift from W. E. Balch (Department of Cell Biology, The Scripps Research Institute, La Jolla, CA). In DAT-L2A, Leu 113 and Leu 120 were replaced by alanine using the QuikChange II XL Site-directed mutagenesis kit (Stratagene) to create a mutant analogous to GAT1-L2A (12); this mutant is defective in oligomerization (not shown) similar to the related mutants produced by Torres et al (13). Sar1a-T39N was also generated using the QuikChange II XL kit.…”

“…The interaction between cargo and Sec24D takes place in the ER. Accordingly, the transporter of interest must be trapped in the ER: if the leucine repeat in transmembrane segment 2 of GAT1 is disrupted by substitution with alanines, the resulting GAT1-L2A is defective in oligomerization and retained in the ER (12). This mutant still interacts with Sec24D (17).…”

“…We and others previously showed that ER export of neurotransmitter transporters is contingent on their oligomeric assembly (12)(13)(14)(15)(16). We also located a sequence element composed of 10 amino acids (Leu 553 -Gln 572 ) in the GAT1 carboxyl terminus, which mediated an interaction with Sec24D (17).…”

Dopamine transporter endocytic determinants: Carboxy terminal residues critical for basal and PKC-stimulated internalization

“…All members of the family share a similar predicted topology of twelve transmembrane domains, cytoplasmic amino-and carboxy-terminals, a large second extracellular loop with multiple N-linked glycosylation sites, and cytoplasmic phosphorylation sites. Recent crystallographic studies focusing on LeuT Aa , a bacterial SLC6 transporter homolog, support the predicted DAT topology and reveal a dimeric transporter assembly (Yamashita et al, 2005), consistent with FRET and co-immunoprecipitation studies that suggest SLC6 transporter homooligomerization (Schmid et al, 2001;Scholze et al, 2002;Sorkina et al, 2003;Torres et al, 2003b).…”