Novel properties of a mouse gamma-aminobutyric acid transporter (GAT4).
We expressed the mouse gamma-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 (Q10) of 4.3. The GAT4 turnover rate (1.5 s(-l); -50 mV, 21 degrees 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 (ICl(leak)) was observed when Na+ was replaced with choline or tetraethylammonium. The reversal potential of (ICl(leak)) followed the Cl- Nernst potential. (ii) A Li+ leak current (ILi(leak)) was observed when Na+ was replaced with Li+. Both leak currents were inhibited by Na+, and both were temperature-independent (Q10 approximately 1). The two leak modes appeared not to coexist, as Li+ inhibited (ICl(leak)). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na+/Cl-/GABA cotransport, ILi(leak), and ICl(leak), (Ki approximately 30 microM), and the voltage-induced presteady-state charge movements (Ki approximately 440 microM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jicroumps 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.
Biological Sciences Department, California State Polytechnic University, Pomona, CA 91768-4032, USA.
M H
MH
Karakossian
S R
SR
Spencer
A Q
AQ
Gomez
O R
OR
Padilla
A
A
Sacher
D D F
DD
Loo
N
N
Nelson
S
S
Eskandari
D000818:Animals; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050485:GABA Plasma Membrane Transport Proteins; D015870:Gene Expression; D006801:Humans; D017136:Ion Transport / Q000235:genetics / Q000502:physiology; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009865:Oocytes; D012964:Sodium / Q000378:metabolism*; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002712:Chlorides; D004791:Enzyme Inhibitors; D050485:GABA Plasma Membrane Transport Proteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D012964:Sodium; D005680:gamma-Aminobutyric Acid; C079264:Slc6a11 protein, mouse