An optogenetic actuator for reversible alkalization of acidic presynaptic compartments

Precise control of synaptic vesicle luminal pH offers a way to analyze proton-driven mechanisms involved in neurotransmitter loading, vesicle maturation, and synaptic transmission. Current genetically encoded tools either monitor vesicular pH or optogenetically acidify vesicles; here, we address this gap with a light-driven alkalizing actuator. Here, we developed syp-NsXeR, a genetically encoded optogenetic actuator based on the inward proton pump xenorhodopsin NsXeR, targeted to acidic presynaptic compartments through synaptophysin. Confocal imaging demonstrated enrichment of the construct at presynaptic sites in cultured hippocampal neurons. Illumination with 561-nm light induced rapid and reversible alkalization of acidic presynaptic compartments, monitored by intraluminal pHluorin fluorescence. Optical manipulation occurred without pharmacological inhibition of endogenous V-ATPase activity and produced substantially stronger responses in presynaptic boutons than in neuronal somata. These results establish syp-NsXeR as a genetically encoded optogenetic tool for reversible manipulation of presynaptic vesicular proton gradients with high spatiotemporal precision. The construct provides a platform for future studies of proton-dependent mechanisms regulating presynaptic physiology.