UCSF

Synaptic transmission relies on the transport of neurotransmitters into specialized endocytic compartments, synaptic vesicles. Vesicular glutamate transporters (VGLUTs) package glutamate, the major excitatory neurotransmitter, primarily using the membrane potential. Knockout of either of the two major VGLUT isoforms results in perinatal lethality. Two peculiar and controversial properties of VGLUTs are the extraordinary dependence on Cl and a putative Cl conductance. This study investigated this essential transport process and the relationship of Cl, the Cl conductance, and protons using precise electrophysiology techniques.

Two-electrode voltage clamping of Xenopus Oocytes expressing internalization-defective VGLUTs revealed a pH- and Cl-dependent Cl conductance. This Cl conductance was sensitive to both a drug that inhibits vesicular glutamate transport as well as glutamate. Although vesicular glutamate transport was not observed in Xenopus Oocytes, patching on HEK293T endolysosomes expressing VGLUTs revealed both glutamate and Cl conductances. These conductances required luminal Cl and were potentiated by cytoplasmic Cl. Using site-directed mutagenesis of conserved, charged amino acids, we identified key transmembrane residues that regulate anion and glutamate conductances including one responsible for activation by luminal Cl. This finding demonstrates that the mechanism for allosteric activation by Cl is distinct from permeation. In addition, Cl competes with vesicular glutamate transport. This competition is modulated by luminal protons. This study is the first to electrophysiologically reveal the regulatory mechanisms behind the transport of a neurotransmitter, and can be used as a model for the study of other vesicular neurotransmitter transporters.