Molecular and Functional Analysis of a Novel Neuronal Vesicular Glutamate Transporter

Abstract

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Packaging and storage of glutamate into glutamatergic neuronal vesicles requires ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. VGLUT1, the first identified vesicular glutamate transporter, is only expressed in a subset of glutamatergic neurons. We report here the molecular cloning and functional characterization of a novel glutamate transporter, VGLUT2, from mouse brain. VGLUT2 has all major functional characteristics of a synaptic vesicle glutamate transporter, including ATP dependence, chloride stimulation, substrate specificity, and substrate affinity. It has 75 and 79% amino acid identity with human and rat VGLUT1, respectively. However, expression patterns of VGLUT2 in brain are different from that of VGLUT1. In addition, VGLUT2 activity is dependent on both membrane potential and pH gradient of the electrochemical proton gradient, whereas VGLUT1 is primarily dependent on only membrane potential. The presence of VGLUT2 in brain regions lacking VGLUT1 suggests that the two isoforms together play an important role in vesicular glutamate transport in glutamatergic neurons.Neurotransmission depends on the regulated exocytotic release of vesicular transmitter molecules to the synaptic cleft, where they interact with postsynaptic receptors that subsequently transduce the information. Two types of neurotransmitter transporters have been identified based on membrane localization on plasma membrane or vesicular membrane. Removal of the transmitter from the synaptic cleft results in termination of the signal, and this requires destruction of transmitter or reuptake of transmitter back to the presynaptic terminal or glial cells via a sodium-dependent uptake system on the plasma membrane (1). Packaging and storage of neurotransmitters into specialized secretory vesicles in neurons ensures their regulated release. This storage is also crucial for protecting the neurotransmmitter molecules from leakage or intraneuronal metabolism and for protecting the neuron from possible toxic effects. This process is mediated by specific transporters on the vesicular membranes. At least four different types of vesicular transporters have been functionally identified that are specific for transport of classic neurotransmitters: monoamines, acetylcholine, ␥-aminobutyric acid (GABA), and glutamate (2, 3). Unlike the plasma membrane transporters, which rely on a sodium gradient across the plasma membrane, all of these vesicular transport processes depend on the proton electrochemical gradient (⌬ Hϩ ) 1 generated by a Mg 2ϩ -activated vacuolar H ϩ -ATPase (V-ATPase) on the vesicular membrane (4). When protons are pumped into the vesicular lumen, a proton gradient (⌬pH) and a membrane potential (⌬) occur across the membrane to form ⌬ Hϩ, which favors the exchange of luminal protons for cytoplasmic transmitter. The transport of monoamines and acetylcholine rely predominantly on ...

Amino Acid Metabolism, β-Cell Function, and Diabetes

“…It has been suggested that glutamate could be transported into secretory granules, thereby promoting Ca 2ϩ -dependent exocytosis (47,53). Such a model has been substantiated by demonstration that clonal ␤-cells express vesicular glutamate transporters and that glutamate transport characteristics are similar to neuronal transporters (54). Other evidence in support of the L-glutamate hypothesis comes from work with ␤-cells overexpressing L-glutamate decarboxylase (GAD): overexpression of GAD reduced L-glutamate content in INS-1E and islet ␤-cells and reduced secretory responses to high glucose (48).…”

Vesicular Glutamate Transporter-Dependent Glutamate Release from Astrocytes

“…Hence, astrocytic Ca 2ϩ -dependent glutamate release can be blocked with bafilomycin A 1 (Araque et al, 2000;Bezzi et al, 2001;Pasti et al, 2001), which specifically interferes with V-ATPase, leading to alkalinization of vesicular lumen and collapsing the proton gradient necessary for VGLUTs to transport glutamate into vesicles. Brain tissue expresses VGLUT 1 and 2 isoforms (Ni et al, 1994(Ni et al, , 1995Hisano et al, 2000;Bai et al, 2001) in glutamatergic neurons (Bellocchio et al, 1998;Fremeau et al, 2001;Fujiyama et al, 2001;Herzog et al, 2001;Sakata-Haga et al, 2001;Kaneko et al, 2002;Varoqui et al, 2002). Recently, there have been reports indicating the presence of a VGLUT 3 isoform in subpopulations of GABAergic (Fremeau et al, 2002), cholinergic, and monoaminergic neurons (Fremeau et al, 2002;Gras et al, 2002;Schafer et al, 2002) and some astrocytes (Fremeau et al, 2002).…”

Pharmacological Topics of Bone Metabolism: Glutamate as a Signal Mediator in Bone

“…Moreover, vesicular Glu transporters (VGLUTs) are essential for the signal output through the condensation of Glu into vesicular constituents for subsequent exocytotic release upon stimulation. Within the CNS, both VGLUT1 (13) and VGLUT2 (14) isoforms are supposed to suffice for the definition of an excitatory neuronal phenotype, while VGLUT3 is expressed in a number of cells shown to release Glu through exocytosis including dopaminergic, GABAergic, and serotonergic neurons as well as astrocytes (15).…”