Protein kinase C modulates synaptic vesicle acidification in a ribbon type nerve terminal in the retina.
The driving force for neurotransmitter accumulation into synaptic vesicles is provided by the generation of a transmembrane electrochemical gradient (DeltamicroH+) that has two components: a chemical gradient (DeltapH, inside acidic) and an electrical potential across the vesicular membrane (DeltaPsi, inside positive). This gradient is generated in situ by the electrogenic vacuolar H(+)-ATPase, which is responsible for the acidification and positive membrane potential of the vesicle lumen. Here, we investigate the modulation of vesicle acidification by using the acidic-organelle probe LysoTracker and the pH-sensitive probe LysoSensor at goldfish Mb-type bipolar cell terminals. Since phosphorylation can modulate secretory granule acidification in neuroendocrine cells, we investigated if drugs that affect protein kinases modulate LysoTracker staining of bipolar cell terminals. We find that protein kinase C (PKC) activation induces an increase in LysoTracker-fluorescence. By contrast, protein kinase A (PKA) or calcium/calmodulin kinase II (CaMKII) activation or inhibition did not change LysoTracker-fluorescence. Using a pH-dependent fluorescent dye (LysoSensor) we show that the PKC activation with PMA induces an increase in LysoSensor-fluorescence, whereas the inactive analog 4alpha-PMA was unable to cause the same effect. This increase induced by PMA was blocked by PKC inhibitors, calphostin C and staurosporine. These results suggest that phosphorylation by PKC may increase synaptic vesicle acidification in retinal bipolar cells and therefore has the potential to modulate glutamate concentrations inside synaptic vesicles.
Abreu
Bento J
BJ
Department of Morphology, Biological Sciences Institute, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
Guimarães
Maila
M
Uliana
Livia C
LC
Vigh
Jozsef
J
von Gersdorff
Henrique
H
Prado
Marco A
MA
Guatimosim
Cristina
C
D000588:Amines; D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D006054:Goldfish; D066298:In Vitro Techniques; D009411:Nerve Endings / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D012160:Retina / Q000378:metabolism*; D051245:Retinal Bipolar Cells / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism*
D000588:Amines; D005456:Fluorescent Dyes; C493330:Red DND-99; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2