Sodium-dependent GABA-induced currents in GAT1-transfected HeLa cells.
1. HeLa cells were infected with recombinant vaccinia virus containing the T7 RNA polymerase gene and transfected with the cDNA for a rat GABA transporter, GAT1, cloned downstream of a T7 RNA polymerase promoter. Six to sixteen hours after transfection, whole-cell recording with a voltage ramp in the range -90 to 50 mV revealed GABA-induced currents (approximately -100 pA at -60 mV in 100 microM GABA, 16 h after transfection at room temperature). No GABA-induced currents were observed in parental HeLa cells or in mock-transfected cells. 2. GABA-induced currents were suppressed by extracellular perfusion with GABA-free solutions or addition of GAT1 inhibitors SKF89976-A or SKF100330-A. At fixed voltage the GABA dependence of the inward current fitted the Michaelis-Menten equation with a Hill coefficient, n, near unity and an equilibrium constant, K(m), near 3 microM. The Na+ dependence of the inward currents fitted the Michaelis-Menten equation with n approximately equal to 2 and K(m) approximately equal to 10 mM. The constants n and K(m) for GABA and Na+ were independent of voltage in the range -90 to -30 mV. 3. GABA-induced currents reverse direction in the range 5-10 mV. The implication of this result is that GAT1 can mediate electrogenic (electrophoretic) influx or efflux of GABA depending on the membrane voltage. The presence of an outward current in our experiments is consistent with radioactive-labelled flux data from resealed vesicle studies. However, it is inconsistent with frog oocyte expression experiments using the sample clone. In oocytes, GAT1 generates no outward current in a similar voltage range. Smaller intracellular volume or higher turnover rates in the mammalian expression system may explain the outward currents. 4. External GABA induces inward current, and internal GABA induces outward current. However, in cells initially devoid of internal GABA, external GABA can also facilitate an outward current. This GAT1-mediated outward current occurs only after applying negative potentials to the cell. These data are consistent with the concept that negative potentials drive GABA and Na+ into the cell, which then leads to electrogenic efflux through GAT1 at positive voltages. 5. Assuming coupled transport, we estimate the number of transporters, N, times the turnover rate, r, to be Nr approximately 10(9) s-1 under nominal conditions (V = -60 mV, 30 microM GABA, 130 mM Na+ and room temperature). This indicates either very high levels of expression (approximately 10(4) microns-2), assuming published turnover rates (approximately 10 s-1), or turnover rates that are significantly greater than previously reported. As an alternative, a channel may exist in the GAT1 protein that is gated by GABA and Na+ and blocked by GAT1 antagonists. The channel mode of conduction would exist in addition to the coupled, fixed-stoichiometry transporter mode of conduction.
Risso
S
S
Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.
DeFelice
L J
LJ
Blakely
R D
RD
D000818:Animals; D002352:Carrier Proteins / Q000187:drug effects*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D050485:GABA Plasma Membrane Transport Proteins; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects*; D008565:Membrane Proteins / Q000187:drug effects*; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D051381:Rats; D012964:Sodium / Q000494:pharmacology*; D013997:Time Factors; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C493413:SLC6A1 protein, human; C493415:Slc6a1 protein, rat; D005680:gamma-Aminobutyric Acid; D012964:Sodium