.gamma.-Aminobutyric acid transport in reconstituted preparations from rat brain: coupled sodium and chloride fluxes

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Abstract
Transport of .gamma.-aminobutyric acid (GABA) is electrogenic and completely depends on the presence of both sodium and chloride ions. These ions appear to be cotransported with .gamma.-aminobutyric acid through its transporter [reviewed in Kanner, B. I. (1983) Biochim. Biophys. Acta 726, 293-316]. Using proteoliposomes into which a partially purified .gamma.-aminobutyric acid transporter preparation was reconstituted, we have been able-for the first time-to provide direct evidence for sodium- and chloride-coupled .gamma.-aminobutyric acid transport. This has been done by measuring the fluxes of 22Na+, 36Cl-, and [3H]GABA. These fluxes have the following characteristics: There are components of the net fluxes of sodium and chloride that are .gamma.-aminobutyric acid dependent. The sodium flux is chloride depenent; i.e., when Cl- is replaced by inorganic phosphate or by SO42-, .gamma.-aminobutyric acid dependent sodium fluxes are abolished. The chloride flux is sodium dependent; i.e., when Na+ is replaced by Tris+ or by Li+, .gamma.-aminobutyric acid dependent chloride fluxes are abolished. Thus, the .gamma.-aminobutyric acid dependent sodium and chloride fluxes appear to be catalyzed by the transporter. Using these fluxes we have attempted to determine the stoichiometry of the process. We measured the initial rate of sodium-dependent .gamma.-aminobutyric acid fluxes and that of .gamma.-aminobutyric acid dependent sodium fluxes. This yields the stoichiometry between sodium and .gamma.-aminobutyric acid (2.58 .+-. 0.99). Similarly, we measured the stoichiometry between chloride and .gamma.-aminobutyric acid, which is found to be 1.27 .+-. 0.12. These results are consistent with those obtained previously when a thermodynamic approach was used [Radian, R., and Kanner, B. I. (1983) Biochemistry 22, 1236-1241]. The half-maximal effect obtained when the .gamma.-aminobutyric acid concentration dependence of Cl- and Na+ transport is determined is much higher (70-80 .mu.M) than the known Km of this system (Km = 4-10 .mu.M). Reexamination of the kinetics of .gamma.-aminobutyric acid transport reveals that there are two transport systems for it. The first one has a high affinity, Km = 2.5-4 .mu.M, and the other one has a low affinity, Km = 200-600 .mu.M. The low-affinity .gamma.-aminobutyric acid transport was characterized. It was found that this process, just as the high-affinity process, is electrogenic, and it is absolutely dependent on Cl- and Na+. The sodium, chloride, and .gamma.-aminobutyric acid fluxes probalby originate from the low-affinity transporter.

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