Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients.
The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.
Department of Neurochemistry, Max Planck Institute for Psychiatry, Martinsried, Germany.
J W
JW
Hell
L
L
Edelmann
J
J
Hartinger
R
R
Jahn
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002540:Cerebral Cortex / Q000502:physiology; D050485:GABA Plasma Membrane Transport Proteins; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008564:Membrane Potentials; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D027361:Organic Anion Transporters; D011188:Potassium / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D013572:Synaptic Vesicles / Q000502:physiology*; D014634:Valinomycin / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D005971:Glutamates; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D027361:Organic Anion Transporters; D011510:Proteolipids; C020485:proteoliposomes; D014634:Valinomycin; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D011188:Potassium