Ca(2+) regulation of the carrier-mediated gamma-aminobutyric acid release from isolated synaptic plasma membrane vesicles.
The regulation of the carrier-mediated gamma-aminobutyric acid (GABA) efflux was studied in isolated synaptic plasma membrane (SPM) vesicles, which are particularly useful to study neurotransmitter release without interference of the exocytotic machinery. We investigated the effect of micromolar intravesicular Ca(2+) on the GABA release from SPM vesicles under conditions of basal release (superfusion with 150 mM NaCl), homoexchange (superfusion with 500 microM GABA) and K(+) depolarization-induced release (superfusion with 150 mM KCl). We observed that, in the presence of intravesicular Ca(2+) (10 microM), the maximal velocity (J(max)) of K(+) depolarization-induced GABA release is decreased by about 64%, and this effect was abolished in the presence of the channel blocker, La(3+). In contrast, the other mechanisms were not significantly altered by these cations. In agreement with our earlier results, inhibition of GABA uptake by intravesicular Ca(2+) was also observed by determining the kinetic parameters (K(0.5) and J(max)) of influx into the SPM vesicles. Under these conditions, the J(max) of GABA uptake was 17.4 pmol/s per mg protein, whereas in control experiments (absence of Ca(2+)), this value achieved 25.5 pmol/s per mg protein. The inhibitory effect of Ca(2+) on translocation of GABA across SPM appears to be mediated by calcium/calmodulin activation of protein phosphatase 2B (calcineurin), since it was completely relieved by W7 (calmodulin antagonist) and by cyclosporin A (calcineurin inhibitor). These results show that the GABA transport system, operating either in forward or backward directions, requires phosphorylation of internally localized calcineurin-sensitive sites to achieve maximal net translocation velocity.
Cordeiro
J M
JM
Centro de Neurociências, Universidade de Coimbra, 3004-504 Coimbra, Portugal.
Meireles
S M
SM
Vale
M G
MG
Oliveira
C R
CR
Gonçalves
P P
PP
D000818:Animals; D019703:Calcineurin / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D050485:GABA Plasma Membrane Transport Proteins; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000494:pharmacology; D012756:Sheep; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology*; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D005680:gamma-Aminobutyric Acid; D019703:Calcineurin; D002122:Calcium Chloride; D011188:Potassium; D002118:Calcium