Novel properties of a mouse γ-aminobutyric acid transporter (GAT4)

M. H. Karakossian, S. R. Spencer, A. Q. Gomez, O. R. Padilla, A. Sacher, D. D.F. Loo, N. Nelson, S. Eskandari*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

We expressed the mouse γ-aminobutyric acid (GABA) transporter GAT4 (homologous to rat/human GAT-3) in Xenopus laevis oocytes and examined its functional and pharmacological properties by using electrophysiological and tracer uptake methods. In the coupled mode of transport (Na+/Cl -/GABA cotransport), there was tight coupling between charge flux and GABA flux across the plasma membrane (2 charges/GABA). Transport was highly temperature-dependent with a temperature coefficient (Q 10) of 4.3. The GAT4 turnover rate (1.5 s-1; -50 mV, 21°C) and temperature dependence suggest physiological turnover rates of 15-20 s-1. No uncoupled current was observed in the presence of Na+. In the absence of external Na+, GAT4 exhibited two distinct uncoupled currents. (i) A Cl- leak current (IleakCl ) was observed when Na+ was replaced with choline or tetraethylammonium. The reversal potential of (IleakCl) followed the Cl - Nernst potential. (ii) A Li+ leak current (I leakLi) was observed when Na+ was replaced with Li+. Both leak currents were inhibited by Na+, and both were temperature-independent (Q 10 ≈ 1). The two leak modes appeared not to coexist, as Li+ inhibited (Ileak Cl). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na +/C1-/GABA cotransport, IleakLi), and IleakCl), (K i ≈ 30 μM), and the voltage-induced presteady-state charge movements (K i ≈ 440 μM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jumps revealed that slow Na+ binding to the transporter is followed by rapid GABA-induced translocation of the ligands across the plasma membrane. Thus, Na+ binding and associated conformational changes constitute the rate-limiting steps in the transport cycle.

Original languageEnglish
Pages (from-to)65-82
Number of pages18
JournalJournal of Membrane Biology
Volume203
Issue number2
DOIs
StatePublished - Jan 2005

Funding

FundersFunder number
National Institute of General Medical SciencesS06GM053933

    Keywords

    • Channel mode
    • GABA
    • Mouse GAT4
    • Neurotransmitter Transporter
    • Pharmacology

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