Functional reconstitution of the. gamma. -aminobutyric acid transporter from synaptic vesicles using artificial ion gradients
- Max-Planck-Inst. for Psychiatry, Martinsried (West Germany)
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{prime}-diisopropylthiodicarbocyanine iodide, and changes of the H{sup +} gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K{sup +} gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of ({sup 3}H)GABA which was saturable. Similarly, ({sup 3}H)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{sup +}-loaded proteoliposomes in a buffer free of K{sup +} or Na{sup +} ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H{sup +} ATPase by incubation of K{sup +}-loaded proteoliposomes in equimolar K{sup +} 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, these 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.
- OSTI ID:
- 5687111
- Journal Information:
- Biochemistry; (United States), Vol. 30:51; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
AMINOBUTYRIC ACID
MEMBRANE TRANSPORT
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
TRITIUM COMPOUNDS
UPTAKE
ALANINES
ENZYME ACTIVITY
GLUTAMIC ACID
GLYCINE
LIPOSOMES
RATS
AMINO ACIDS
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
CARBOXYLIC ACIDS
DRUGS
HYDROGEN COMPOUNDS
MAMMALS
NEUROREGULATORS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PROTEINS
RODENTS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques