Functionally independent subunits in the oligomeric structure of the GABA cotransporter rGAT1.
We have combined structural and functional approaches to investigate the role of oligomerization in the operation of the GABA transporter rGAT1. Xenopus laevis oocytes were induced to express, either separately or simultaneously, the wild-type form of rGAT1 and a mutated (Y140W) form, unable to translocate GABA and to generate transport currents, although its intramembrane charge movement properties are only slightly affected. These characteristics, together with the insensitivity of Y140W to the blocking action of SKF89976A, were used to study the possible functional interaction of the two forms in an heteromeric structure. The electrophysiological data from oocytes coexpressing wild-type and Y140W rGAT1 were consistent with a completely independent activity of the two forms. Oligomerization was also studied by fluorescence resonance energy transfer (FRET) in tsA201 cells expressing the transporters fused with cyan and yellow fluorescent proteins (ECFP and EYFP). All combinations tested (WT-ECFP/WTEYFP, Y140W-ECFP/Y140W-EYFP and WT-ECFP/ Y140W-EYFP) were able to give rise to FRET, confirming the formation of homo- as well as heterooligomers. We conclude that, although rGAT1 undergoes structural oligomerization, each monomer operates independently.
Soragna
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Laboratory of Cellular and Molecular Physiology, Department of Structural and Functional Biology, and Center for Neurosciences, University of Insubria, Via Dunant 3, 21100, Varese, Italy. andrea.soragna@uninsubria.it
Bossi
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E
Giovannardi
S
S
Pisani
R
R
Peres
A
A
D000818:Animals; D002460:Cell Line; D004594:Electrophysiology / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000502:physiology*; D018014:Gene Transfer Techniques; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000737:chemistry / Q000502:physiology; D051381:Rats; D014982:Xenopus laevis
D050485:GABA Plasma Membrane Transport Proteins; D021122:Protein Subunits