The time course of endocytotic retrieval

of vATPase has not been directly determined, but this retrieval is likely to be complete during the early recycling stage of clathrin-coated vesicles (Prior and Clague, 1997). Figure 8 presents a model that relates measured stimulation-induced pH changes (YFP fluorescence, top panel) to simultaneous changes in cytosolic [Ca2+] (Figure 6) and vesicle exocytosis/endocytosis. The diagram includes the hypothesized underlying changes in transmembrane H+ and Ca2+ fluxes through plasma membrane channels and transporters. The time labeled A represents the resting terminal, in which the steady-state pH is maintained by extruding metabolically generated H+ via an HCO3−/Na+ cotransporter (NBC) and an NHE. Voltage-dependent Alisertib purchase Ca2+ channels (VDCCs) are closed. The time labeled B represents the acidification phase measured when stimulation starts. VDCCs open, and the elevation in cytosolic [Ca2+] initiates exocytosis and increases activation of the PMCA, which admits H+. At this stage H+ entry via the PMCA exceeds H+ extrusion via NBC, NHE, and the vATPase from exocytosed vesicles. As stimulation and Ca2+ entry continue click here (time labeled C), cytosolic [Ca2+] reaches a plateau, but insertion

of vATPase into the plasma membrane continues. Alkalinization mediated by vATPase now exceeds PMCA-mediated acidification. When stimulation stops (time labeled D), there is a rapid fall in cytosolic [Ca2+], which decreases activation of PMCA and the attendant H+ entry, accounting for the early transient increase in alkalinization. Cytosolic [H+] then falls slowly as endocytosis removes vATPase from the plasma membrane. If this model is valid, then stimulation-induced changes in the fluorescence of transgenically expressed YFP offer a technique to detect the activity of presynaptic terminals: early acidification (fluorescence decrease) indicates Ca2+ entry, the later alkalinization (fluorescence increase) indicates vesicular exocytosis, Megestrol Acetate and the decay

of this fluorescence increase measures an aspect of vesicular endocytosis. Work using synaptic preparations subjected to changes in cytosolic pH imposed by manipulating the external medium suggests that the prolonged poststimulation alkalinization measured in motor terminals may have beneficial effects on presynaptic function. One such effect is enhancement of endocytosis. In lizard and mouse motor terminals, endocytotic uptake (but not exocytotic release) of the styryl dye FM1-43 is reversibly inhibited by cytosolic acidification (Lindgren et al., 1997 and Coleman et al., 2008). Lindgren et al. (1997) estimated that in lizard motor terminals endocytosis is pH sensitive within ∼0.4 pH units of resting pH, equivalent to a 48–190 nM deviation from resting [H+] (calculated for resting pH ranging from 7.5 to 6.9, respectively).