Folimycin was then added to isolate the acidifying component (second record). Subsequent removal of HCO3−/CO2 buffering (substituting HEPES buffer in TSA HDAC order 100% O2) increased the magnitude of the acidification (third record). Further addition of the NHE inhibitor amiloride increased the stimulation-induced acidification still further (fourth record). The average peak magnitudes of the acidifying component in multiple terminals in each condition are indicated by circles near

each record (vertical lines indicate ±SEM). The calculated stimulation-induced increase in [H+] from rest (Δ[H+]) increased from 22 to 79 nM following removal of HCO3−/CO2 buffering, and further increased to 138 nM following subsequent inhibition of NHE. Figure 7B shows another test of the importance of the HCO3−/CO2 buffer system. The effectiveness of this buffer system relies on rapid equilibration between CO2 and H+/HCO3−, which

is catalyzed by intracellular carbonic anhydrase. The records in Figure 7B show that a membrane-permeable carbonic anhydrase inhibitor, acetazolamide, increased the stimulation-induced Caspase cleavage acidification. These data indicate that HCO3−/CO2 buffering and H+ extrusion via the NHE contribute importantly to limiting the magnitude of the stimulation-induced acidification in motor terminals. Work presented here measured stimulation-induced changes in cytosolic pH in motor nerve terminals using the pH sensitivity of the fluorescence of transgenically expressed

YFP. We demonstrate that trains of action potentials evoke an early acidification followed by a pronounced and prolonged alkalinization. Acidification following stimulation has also been reported in neuronal somata and dendrites (see Introduction), but these structures do not show the later alkalinization that is so prominent in motor terminals. We present evidence that this alkalinization is caused by H+ extrusion via vATPase inserted into the plasma membrane during exocytosis. This hypothesis further suggests that restoration of cytosolic pH reflects endocytotic retrieval ablukast of vATPase from the plasma membrane. If the above scenario is true, then the vATPase must be capable of pumping protons not only in synaptic vesicles, but also when the vesicular membrane becomes (temporarily) incorporated into the plasma membrane following exocytosis. Evidence for this idea in motor terminals is our finding that the stimulation-induced alkalinization is blocked by inhibitors of exocytosis (BoNTs) and by inhibitors of vATPase (folimycin, bafilomycin). Exocytotic insertion of functional H+-ATPases into plasma membranes has been demonstrated in certain nonneuronal cells.