ELECTROGENIC L-GLUTAMATE UPTAKE IN XENOPUS-LAEVIS OOCYTES EXPRESSING A CLONED RAT-BRAIN L-GLUTAMATE L-ASPARTATE TRANSPORTER (GLAST-1)

The transport of L-glutamate into Xenopus laevis oocytes expressing the cloned L-glutamate/L-aspartate transporter (GLAST-1) from rat brain was studied using the voltage clamp technique. At a holding potential of -90 mV, a bath application of 100 muM L-glutamate induced an inward current (I(GLAST)) with an amplitude ranging from -5 to -30 nA. I(GLAST) did not require extracellular Ca2+, Mg2+, or Cl-, was larger at negative potentials, and did not reverse up to +80 mV. The current was dependent on external L-glutamate and Na+ with half-maximal amplitudes at 11 muM L-glutamate and 41 mM Na+. I(GLAST) saturated at 100 muM L-glutamate and 80 mM Na+. The Hill coefficient for Na+ and L-glutamate was 3.3 and 1.3, respectively, suggesting that 3 Na+ accompany the transport of 1 L-glutamate molecule. At low [Na+]o, I(GLAST) was enhanced by reducing [K+]o, an indication for the countertransport of K+. Reducing external pH from 7.4 to 6.0 did not change the amplitude of I(GLAST). This argues against a glutamate/proton cotransport. The results provide evidence for GLAST-1 carrying out a high affinity, sodium-dependent L-glutamate transport with a proposed stoichiometry of 3 Na+, 1 L-glutamate-/1 K+.