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J. Vigh, C. Guatimosim, M.A. Prado, H. von Gersdorff; Synaptic vesicle trafficking and glutamate retention is independent of the vesicular pH gradient in bipolar cell terminals . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1329.
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Purpose: Synaptic vesicles are acidified by a group of ATP–dependent H+ pumps (V–ATPases) that establish transmembrane pH and electrical gradients. These gradients provide the driving force for vesicular transporters to concentrate various transmitters inside vesicles. Here we have investigated whether the vesicular pH gradient influences exocytosis and/or glutamate retention in synaptic vesicles at the bipolar cell terminal. Methods: Voltage–clamped current and capacitance measurements were performed directly from bipolar cell terminals in a goldfish retinal slice preparation. Vesicular pH was imaged with a pH sensitive, indicator dye (Lysotracker) in acutely dissociated bipolar cells. Results: The terminal of the dissociated bipolar cells was strongly and selectively labeled with Lysotracker indicating the presence of acidic compartments. The membrane–permeable weak base methylamine has been shown to accumulate in acidic compartments and discharge the pH gradient without affecting the electrical gradient (Cousin and Nicholls, 1997). When methylamine (5–10 mM) was applied through the recording pipette, the inhibitory effect of exocytosed protons on the calcium current (Palmer et al, 2003) was eliminated within 1–2 min. Interestingly, methylamine had no effect on the number of vesicles released for up to 10 min, as evidenced by capacitance measurements, or on the glutamate evoked reciprocal GABA feedback from amacrine cells. Conclusions: Elimination of the transmembrane pH gradient in the synaptic vesicles has no effect on the vesicle trafficking, or on the glutamate retention of vesicles in the bipolar cell terminal.
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