The transport of all classical neurotransmitters into synaptic vesicles depends on a H+ electrochemical driving force (??H+) generated by the vacuolar-type H+-ATPase. However, different transmitters depend to varying extents on the two components of ??H+, the chemical component (?pH) and the electrical component (??). Most of the vesicular transporters depend on ?pH, and mediate exchange of cytosolic transmitter for lumenal H+. However, the vesicular glutamate transporters (VGLUTs) depend primarily on ??, with an unclear role for H+. They also exhibit allosteric activation by Cl- and recent work has conflicted about their expression of a Cl- conductance. Adapting the use of electrophysiology to record currents associated with the VGLUTs at the plasma membrane, we have found that the VGLUTs exhibit a Cl- conductance allosterically activated from the lumenal side by H+ as well as Cl-. The Cl- condutance exhibits many of the properties previously described for vesicular glutamate transport and the two anions compete, suggesting they use a related permeation pathway. The long-term objective of this program is to determine how these mechanisms influence synaptic transmission, and the strategy is to characterize the mechanisms in heterologous expression systems, then test the role of these mechanisms in excitatory neurotransmission. We propose the following aims: 1) Identify residues responsible for allosteric activation of the VGLUTs by Cl- and H+. We will determine the effect of point mutations on the Cl- conductance associated with VGLUT expression in Xenopus oocytes, then extend the analysis to glutamate transport by functional reconstitution of purified protein. 2) Determine the role of allosteric regulation by Cl- and H+ in excitatory neurotransmission. Using VGLUT1/2 double knockout mice, we will rescue excitatory transmission with mutant VGLUTs defect in allosteric activation by Cl- and H+. We will also determine the effect of these and other properties on synaptic vesicle acidification. 3) Characterize the properties of currents associated with vesicular glutamate transport by whole endosome recording. We have recently detected currents associated with the expression of wild type VGLUTs on endosomal membranes. Importantly, these currents reflect the flux of glutamate as well as Cl-, and their characterization will provide basic new information about glutamate transport as well as the associated Clconductance. 4) Identify residues responsible for the differences in ionic coupling by different VGLUT family members (continuation of prior Aim 3). Bacterial members of the SLC17 family mediate the cotransport of organic anions with H+, raising questions about their relationship to the ??-drlven activity of the VGLUTs. In this aim, we will test the relationship between these closely related protein by introducing mutations that convert their coupling to that of the other.
Synaptic transmission involves the regulated exocytosis of vesicles filled with transmitter, but we still understand little about the basic mechanisms that regulate neurotransmitter transport into synaptic vesicles. This program will characterize the mechanism that transports glutamate into synaptic vesicles, and test the effect of properties identified on glutamatergic neurotransmission. The results will have important implications for our understanding of excitatory transmission, behavior and neuropsychiatric disease
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