Identification of the Differentiation-Associated Na⁺/P_ITransporter as a Novel Vesicular Glutamate Transporter Expressed in a Distinct Set of Glutamatergic Synapses

Abstract

Glutamate transport into synaptic vesicles is a prerequisite for its regulated neurosecretion. Here we functionally identify a second isoform of the vesicular glutamate transporter (VGLUT2) that was previously identified as a plasma membrane Na+-dependent inorganic phosphate transporter (differentiation-associated Na+/P(I) transporter). Studies using intracellular vesicles from transiently transfected PC12 cells indicate that uptake by VGLUT2 is highly selective for glutamate, is H+ dependent, and requires Cl- ion. Both the vesicular membrane potential (Deltapsi) and the proton gradient (DeltapH) are important driving forces for vesicular glutamate accumulation under physiological Cl- concentrations. Using an antibody specific for VGLUT2, we also find that this protein is enriched on synaptic vesicles and selective for a distinct class of glutamatergic nerve terminals. The pathway-specific, complementary expression of two different vesicular glutamate transporters suggests functional diversity in the regulation of vesicular release at excitatory synapses. Together, the two isoforms may account for the uptake of glutamate by synaptic vesicles from all central glutamatergic neurons.

Lentiviral Delivery of a Vesicular Glutamate Transporter 1 (VGLUT1)-Targeting Short Hairpin RNA Vector Into the Mouse Hippocampus Impairs Cognition

“…The observed pattern of VGLUT1 immunoreactivity in the dorsal hippocampus of control mice was consistent with previous reports (Bellocchio et al, 1998;Fremeau et al, 2001;Varoqui et al, 2002) demonstrating widespread expression, with the exception of granule and pyramidal cell layers, and particular enrichment in stratum oriens and radiatum of CA1 as well as the molecular and polymorphic layers of the dentate gyrus (Figure 4a, left). All VGLUT1 staining was abolished by preadsorption of the primary antibody with a VGLUT1 control peptide (data not shown).…”

“…These mice exhibit anxiety and a 'depressive-like' phenotype accompanied by deficits in working, social, and visual recognition memory and impaired PPI, reversal learning, fear extinction, and hippocampal long-term potentiation (Tordera et al, 2007;Garcia-Garcia et al, 2009;Balschun et al, 2010;Elizalde et al, 2010;Inta et al, 2012;Callaerts-Vegh et al, 2013). The latter is consistent with the fact that VGLUT1 localizes to synapses with low release probability (that exhibit long-term potentiation), whereas VGLUT2 localizes to synapses with high release probability (that exhibit longterm depression; Varoqui et al, 2002). In the current study, localized targeting of VGLUT1 expression within the dorsal hippocampus induced deficits in visual recognition and spatial memory that were actually more marked than those observed in VGLUT1 heterozygotes.…”

“…VGLUT1 is expressed in a distinct laminar pattern throughout the dorsal hippocampus, with particular enrichment in the polymorphic and molecular cell layers. Localization to strata oriens and radiatum of CA1 suggests expression in terminals of the Schaffer collateral system, presence in stratum lucidum of CA3 represents expression in mossy fiber terminals from dentate granule cells, and stronger labeling in outer regions of the molecular layer of the dentate gyrus indicates preferential localization to perforant path inputs from the entorhinal cortex (Bellocchio et al, 1998;Fremeau et al, 2001;Varoqui et al, 2002). Despite low VGLUT2 expression in the pyramidal layer of CA2 and the granular layer of the dentate gyrus (Fremeau et al, 2001), there is no evidence for coexpression of both transporters within individual nerve terminals (Herzog et al, 2001), and a recent detailed study found no evidence for VGLUT1 expression in hippocampal astrocytes (Li et al, 2013).…”

An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size

“…In addition to their close resemblance at the sequence level, VGLUT1, VGLUT2, and VGLUT3 are highly similar with respect to substrate specificity, kinetics, and pharmacology (1)(2)(3)(4)(5)(6)(7)(8)(9). Glutamate transport by all three isoforms shows a characteristic biphasic dependence on chloride concentration (1,5,8,9), that was first identified in the glutamate uptake activity of native synaptic vesicles (13)(14)(15).…”

“…Glutamatergic neurons express at least one of three known vesicular glutamate transporters, VGLUT1, VGLUT2, or VGLUT3. These transporters mediate glutamate uptake into synaptic vesicles and are driven by a proton electrochemical gradient generated by the vacuolar H ϩ -ATPase (1)(2)(3)(4)(5)(6)(7)(8)(9). VGLUTs were initially identified as members of the type I Na ϩ ͞inorganic phosphate transporter family (10,11).…”