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 microM L-glutamate induced an inward current (IGLAST) with an amplitude ranging from -5 to -30 nA. IGLAST 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 microM L-glutamate and 41 mM Na+. IGLAST saturated at 100 microM 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, IGLAST 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 IGLAST. 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+.
Klöckner
U
U
Institute of Biochemistry, Medical Faculty of the University of Cologne, Germany.
Storck
T
T
Conradt
M
M
Stoffel
W
W
D027322:Amino Acid Transport System X-AG; D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D009865:Oocytes / Q000378:metabolism* / Q000502:physiology; D011188:Potassium / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000378:metabolism; D014982:Xenopus laevis
D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D005971:Glutamates; D006023:Glycoproteins; D018698:Glutamic Acid; D012964:Sodium; D011188:Potassium