UC Berkeley

Chemical synaptic transmission is the primary mode of communication between neurons, and involves release of neurotransmitter from synaptic vesicles in the presynaptic cell, which then activates receptors on the postsynaptic cell. The amount of neurotransmitter stored in a synaptic vesicle can determine the size of the postsynaptic response, but the factors regulating vesicle filling remain poorly understood. A proton electrochemical gradient (Δμ_H+) generated by the vacuolar H⁺-ATPase drives accumulation of classical transmitters into synaptic vesicles. The chemical component of Δμ_H+ (ΔpH) has received particular attention for its role in the vesicular transport of cationic transmitters, as well as protein sorting and degradation. Thus, considerable work has addressed the factors that promote ΔpH. Although the electrical component of Δμ_H+ (ΔΨ) drives uptake of the principal excitatory transmitter glutamate into synaptic vesicles, the mechanisms that promote ΔΨ remain poorly understood.

We thus employed biochemical methods on isolated synaptic vesicles to better understand the fluxes that occur across vesicle membranes. We used fluorescent dyes to measure ΔpH and ΔΨ dynamics across synaptic vesicles and their dependence on cations, and radioisotopes to measure uptake of glutamate and sodium into synaptic vesicles. To assess the physiological relevance of our in vitro data, we then tested the findings using electrophysiological recordings at a central synapse. We also performed immunofluorescence studies on transfected primary hippocampal cultures to test the synaptic localization of some candidate proteins. We find that synaptic vesicles exhibit a cation/H⁺ exchange activity that converts ΔpH into ΔΨ, thus promoting synaptic vesicle filling with glutamate. Manipulating presynaptic K⁺ concentration at a glutamatergic synapse influences quantal size, demonstrating that K⁺ regulates glutamate release and synaptic transmission. Some proteins tested display synaptic localization and are thus potential molecular candidates for the phenomenon we report.