Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins

In neural systems, information is often carried by ensembles of cells rather than by individual units. Optical indicators(1) provide a powerful means to reveal such distributed activity, particularly when protein-based and encodable in DNA(2-4): encodable probes can be introduced into cells, tissues, or transgenic organisms by genetic manipulation, selectively expressed in anatomically or functionally defined groups of cells, and, ideally, recorded in situ, without a requirement for exogenous cofactors, Here we describe sensors for secretion and neurotransmission that fulfill these criteria We have developed pH-sensitive mutants of green fluorescent protein ('pHluorins') by structure-directed combinatorial mutagenesis, with the aim of exploiting the acidic pH inside secretory vesicles(5,6) to monitor vesicle exocytosis and recycling. When linked to a vesicle membrane protein, pHluorins were sorted to secretory and synaptic vesicles and reported transmission at individual synaptic boutons, as well as secretion and fusion pore 'flicker' of single secretory granules.