Glutamate transport into synaptic vesicles. Roles of membrane potential, pH gradient, and intravesicular pH.
Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, is transported into bovine synaptic vesicles in a manner that is ATP dependent and requires a vesicular electrochemical proton gradient. We studied the electrical and chemical elements of this driving force and evaluated the effects of chloride on transport. Increasing concentrations of Cl- were found to increase the steady-state ATP-dependent vesicular pH gradient (delta pH) and were found to concomitantly decrease the vesicular membrane potential (delta psi). Low millimolar chloride concentrations, which cause 3-6-fold stimulation of vesicular glutamate uptake, caused small but measurable increases in delta pH and decreases in delta psi, when compared to control vesicles in the absence of chloride. Nigericin in potassium buffers was used to alter the relative proportions of delta pH and delta psi. Compared to controls, at all chloride concentrations tested, nigericin virtually abolished delta pH and increased the vesicle interior positive delta psi. Concomitantly, nigericin increased ATP-dependent glutamate uptake in 0-1 mM chloride but decreased glutamate uptake in 4 mM (45%), 20 mM (80%), and 140 mM (75%) Cl- (where delta pH in the absence of nigericin was large). These findings suggest that either delta psi, delta pH, or a combination can drive glutamate uptake, but to different degrees. In the presence of 4 mM Cl-, where uptake is optimal, both delta psi and delta pH contribute to the driving force for uptake. When the extravesicular pH was increased from 7.4 to 8.0, more Cl- was required to stimulate vesicular glutamate uptake. In the absence of Cl-, as extravesicular pH was lowered to 6.8, uptake was over 3-fold greater than it was at pH 7.4. As extravesicular pH was reduced from 8.0 toward 6.8, less Cl- was required for maximal stimulation. Decreasing the extravesicular pH from 8.0 to 6.8 in the absence of Cl- significantly increased glutamate uptake activity, even though proton-pumping ATPase activity actually decreased about 45% under identical conditions. In the absence of chloride, nigericin increased glutamate uptake at all the pH values tested except pH 8.0. Glutamate uptake at pH 6.8 in the presence of nigericin was over 6-fold greater than uptake at pH 7.4 in the absence of nigericin. We conclude from these experiments that optimal ATP-dependent glutamate uptake requires a large delta psi and a small delta pH.(ABSTRACT TRUNCATED AT 400 WORDS)
Tabb
J S
JS
Mental Health Research Institute, University of Michigan, Ann Arbor 48109.
Kish
P E
PE
Van Dyke
R
R
Ueda
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism*; D002712:Chlorides / Q000494:pharmacology; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D008744:Methylamines / Q000378:metabolism; D009550:Nigericin / Q000494:pharmacology; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology
D002712:Chlorides; D005971:Glutamates; D008744:Methylamines; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; C027451:methylamine; D009550:Nigericin