Vesicular ATPase Inserted into the Plasma Membrane of Motor Terminals by Exocytosis Alkalinizes Cytosolic pH and Facilitates Endocytosis

Abstract

Key components of vesicular neurotransmitter release, such as Ca2+ influx and membrane recycling, are affected by cytosolic pH. We measured the pH-sensitive fluorescence of Yellow Fluorescent Protein transgenically expressed in mouse motor nerve terminals, and report that Ca2+ influx elicited by action potential trains (12.5-100 Hz) evokes a biphasic pH change: a brief acidification (∼13 nM average peak increase in [H+]), followed by a prolonged alkalinization (∼30 nM peak decrease in [H+]) that outlasts the stimulation train. The alkalinization is selectively eliminated by blocking vesicular exocytosis with botulinum neurotoxins, and is prolonged by the endocytosis-inhibitor dynasore. Blocking H+ pumping by vesicular H+-ATPase (with folimycin or bafilomycin) suppresses stimulation-induced alkalinization and reduces endocytotic uptake of FM1-43. These results suggest that H+-ATPase, known to transfer cytosolic H+ into prefused vesicles, continues to extrude cytosolic H+ after being exocytotically incorporated into the plasma membrane. The resulting cytosolic alkalinization may facilitate vesicular endocytosis. © 2010 Elsevier Inc.