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 GA BA transporter, GAT1, cloned downstream of a T7 RNA polymerase promote r. Six to sixteen hours after transfection, whole-cell recording with a voltage ramp in the range -90 to 50 mV revealed GABA-induced current s (approximately -100 pA at -60 mV in 100 mu M GABA, 16 h after transf ection at room temperature). No GABA-induced currents were observed in parental HeLa cells or in mock-transfected cells. 2. GABA-induced cur rents were suppressed by extracellular perfusion with GABA-free soluti ons or addition of GAT1 inhibitors SKF89976-A or SKF100330-A. At fixed voltage the GABA dependence of the inward current fitted the Michaeli s-Menten equation with a Hill coefficient, n, near unity and an equili brium constant, K-m, near 3 mu M. The Na+ dependence of the inward cur rents fitted the Michaelis-Menten equation with n approximate to 2 and K-m approximate to 10 mM. The constants n and K-m for GABA and Na+ we re 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 th is result is that GAT1 can mediate electrogenic (electrophoretic) infl ux or efflux of GABA depending on the membrane voltage. The presence o f an outward current in our experiments is consistent with radioactive -labelled flux data from resealed vesicle studies. However, it is inco nsistent with frog oocyte expression experiments using the same clone. In oocytes, GAT1 generates no outward current in a similar voltage ra nge. Smaller intracellular volume or higher turnover rates in the mamm alian 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 c an also facilitate an outward current. This GAT1-mediated outward curr ent occurs only after applying negative potentials to the cell. These data are consistent with the concept that negative potentials drive GA BA and Na+ into the cell, which then leads to electrogenic efflux thro ugh GAT1 at positive voltages. 5. Assuming coupled transport, we estim ate the number of transporters, N, times the turnover rate, r, to be N r approximate to 10(9) s(-1) under nominal conditions (V = -60 mV, 30 mu M GABA, 130 mM Na+ and room temperature). This indicates either ver y high levels of expression (similar to 10(4) mu m(-2)), assuming publ ished turnover rates (similar to 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+ an d blocked by GAT1. antagonists. The channel mode of conduction would e xist in addition to the coupled, fixed-stoichiometry transporter mode of conduction.