Active transport of l-proline by membrane vesicles isolated from rat brain

(1) Active transport of l-proline has been demonstrated in membrane vesicles isolated from rat brain. The process is dependent on external Na⁺ and is observed only under conditions when artificial Na⁺ gradients (C_out >C_in) are imposed across the vesicle's membrane. (2) The transport process, the K_m value of which has been determined to be 140 μM, is strongly inhibited (75–85%) by nigericin. The process is stimulated by valinomycin and, in the absence of a pH gradient, inhibited (about 50%) by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone. These data are consistent with the concept that proline transport is electrogenic. It seems optimal in the presence of a membrane potential (interior negative). In contrast, transport was not affected by ouabain and only slightly inhibited by arsenate. Optimal transport is obtained in the presence of external Cl⁻. This dependency is only partial, but even persists when K⁺-loaded vesicles are used in the presence of valinomycin. In addition, the process is inhibited by alkaloids like veratridine and aconitine and this inhibition is prevented by tetrodotoxin. The results provide direct evidence for Na⁺-coupled active transport by rat brain synaptic plasma membrane vesicles. (3) The proline carrier has been solubilized from the membrane vesicles using cholate. The solubilized transporter was reconstituted in the presence of soybean phospholipids and potassium phosphate using the cholate dialysis technique. The reconstituted proteoliposomes catalyzed Na⁺-dependent proline transport, which was 6–10-fold stimulated by valinomycin.