Synapto-pHluorins: Chimeras between pH-sensitive mutants of green fluorescent protein and synaptic vesicle membrane proteins as reporters of neurotransmitter release

Abstract

Publisher Summary This chapter discusses chimeras between pH-sensitive mutants of green fluorescent protein (GFP) and synaptic vesicle membrane proteins as reporters of neurotransmitter release. Synapto-pHluorins use pH-sensitive mutants of GFP, termed “pHluorins,” to afford the distinction between resting and exocytosed vesicles. Because total fluorescence is the linear sum of emissions from all synapto-pHluorins in a terminal, increases in the “on” content of this composite spectrum indicate neurotransmitter release. Synapto-pHluorins are chimeric membrane proteins composed of two modules: (1) a pHluorin module that reports local pH and (2) the synaptic vesicle membrane protein VAMP-2 that attaches the pHluorin to the inner vesicle surface. The pHluorin amino terminus is fused to the carboxy terminus of VAMP-2, which is located in the vesicle lumen. This results in type II topology, with a membrane anchor segment that also serves as a noncleavable signal peptide. pHluorins with enhanced brightness, synapto-pHluorins carrying multiple fluorescent modules, and methods to fully substitute endogenous vesicle proteins with pHluorin-tagged derivatives help to maximize the fluorescent signal emitted per released quantum of neurotransmitter.