Glutamate Transport into Synaptic Vesicles

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The quantal nature of synaptic transmission depends on the transport of neurotransmitter into synaptic vesicles (SVs), an activity driven by a H+ electrochemical gradient (∆µH+). In contrast to relatively stable ionic gradients across the plasma membrane, ∆µH+ and other ions including Cl- fluctuate with the exo- and endocytosis of SVs. Vesicle filling requires coordination with these changing conditions and hence regulation of transport. In contrast to the SV uptake of most transmitters that relies primarily on the chemical component of ∆µH+ (∆pH), uptake of the principal excitatory transmitter glutamate depends predominantly on membrane potential. The vesicular glutamate transporters (VGLUTs) also exhibit unusual properties, including allosteric regulation by lumenal H+, cytosolic and lumenal Cl- and an associated Cl- conductance. We hypothesize that these mechanisms coordinate glutamate flux with different steps in the exo- and endocytic recycling of synaptic vesicles. The long-term objective of this proposal is to understand how these properties of the VGLUTs contribute to excitatory neurotransmission. The strategy is to determine how these mechanisms regulate VGLUT activity, and use this information to characterize their physiological role. This program takes advantage of our previous work identifying these regulatory mechanisms, assays we developed to study them, recent structural information and VGLUT knockout neurons that we can use to test rescue by mutants. Aim 1: Elucidate the mechanism and physiological role of pH in vesicular glutamate transport. The requirement for allosteric activation of the VGLUTs by lumenal H+ suggests a mechanism to prevent tonic efflux of glutamate across the plasma membrane that would degrade the quantal signal. We recently identified a single residue that confers the pH requirement of vesicular glutamate transport. We will now use this information to determine how pH regulates glutamate transport and how this regulation influences excitatory transmission. Aim 2: Determine how Cl- allosterically regulates vesicular glutamate transport. We recently found that an extensive cytoplasmic interaction network influences the allosteric regulation by lumenal pH on the other side of the SV membrane, suggesting that the alternating access involved in glutamate transport depends on the balance in strength between cytoplasmic and lumenal gates. We hypothesize that Cl- also affects the two gates, either directly or indirectly. We will thus determine how the cytoplasmic interaction network and lumenal residues contribute to allosteric regulation of glutamate flux by cytoplasmic and lumenal Cl-. Aim 3: Aim 3: Determine how lumenal Cl- affects glutamate storage and release. Removal of extracellular Cl- prevents recovery from the synaptic depression that normally follows strong stimulation. To determine whether this reflects a requirement for the efflux of lumenal Cl- mediated by a VGLUT-associated conductance, we will rescue VGLUT1/2 double knockouts with mutants lacking the conductance, and monitor the effects on glutamate release and SV pH.