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Comparative Study
. 2005 May 4;25(18):4672-80.
doi: 10.1523/JNEUROSCI.0549-05.2005.

Galphao2 Regulates Vesicular Glutamate Transporter Activity by Changing Its Chloride Dependence

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Free PMC article
Comparative Study

Galphao2 Regulates Vesicular Glutamate Transporter Activity by Changing Its Chloride Dependence

Sandra Winter et al. J Neurosci. .
Free PMC article

Abstract

Classical neurotransmitters, including monoamines, acetylcholine, glutamate, GABA, and glycine, are loaded into synaptic vesicles by means of specific transporters. Vesicular monoamine transporters are under negative regulation by alpha subunits of trimeric G-proteins, including Galpha(o2) and Galpha(q). Furthermore, glutamate uptake, mediated by vesicular glutamate transporters (VGLUTs), is decreased by the nonhydrolysable GTP-analog guanylylimidodiphosphate. Using mutant mice lacking various Galpha subunits, including Galpha(o1), Galpha(o2), Galpha(q), and Galpha11, and a Galpha(o2)-specific monoclonal antibody, we now show that VGLUTs are exclusively regulated by Galpha(o2). G-protein activation does not affect the electrochemical proton gradient serving as driving force for neurotransmitter uptake; rather, Galpha(o2) exerts its action by specifically affecting the chloride dependence of VGLUTs. All VGLUTs show maximal activity at approximately 5 mm chloride. Activated Galpha(o2) shifts this maximum to lower chloride concentrations. In contrast, glutamate uptake by vesicles isolated from Galpha(o2-/-) mice have completely lost chloride activation. Thus, Galpha(o2) acts on a putative regulatory chloride binding domain that appears to modulate transport activity of vesicular glutamate transporters.

Figures

Figure 1.
Figure 1.
Inhibition of glutamate uptake into synaptic vesicles by GMP-P(NH)P. A, Glutamate uptake was performed in KGC/ATP (2 mm ATP) for 10 min at 37°C using synaptic vesicles prepared from rat or mouse cortex. Specificity of uptake is shown by omitting ATP (-ATP), by incubating vesicles with KGC/ATP either on ice or in the presence of a mixture of 5 μm nigericine and 20 μm valinomycine (N./V.), or in the presence of 10 μm trypan blue or 10 mm glutamate. Values represent the mean ± SD of triplicates (p < 0.00001 according to Student's t test). B, Glutamate uptake into rat brain vesicles was performed in KGC/ATP for 10 min at 37°C in the absence or presence of 50 μm GMP-P(NH)P. Non specific glutamate binding was subtracted. Values represent the mean ± SD of triplicates (p < 0.001 according to Student's t test). C, Rat brain synaptic vesicles were incubated with increasing glutamate concentrations in either the absence (filled rectangles) or presence (open triangles) of 50 μm GMP-P(NH)P. Note that Vmax (5.358) and Km (4.387) are decreased by GMP-P(NH)P (Vmax of 3.504 and Km of 3.103). Values represent the mean of three determinations, and nonspecific binding was subtracted. The experiment was repeated with Vmax of 5.287 and Km of 4.035 in controls and Vmax of 4.035 and Km of 3.095 in the presence of GMP-P(NH)P.
Figure 2.
Figure 2.
VGLUT activity and its inhibition by GMP-P(NH)P in deletion mutants of Gαo1, Gαo2, Gαq, and Gα11. Synaptic vesicles were prepared from brains of Gαo2-/- (A, B), Gαo1-/- (C), Gαq-/- (D), and Gα11-/- (E) mice and the corresponding wild types (wt). Glutamate uptake was performed in either the absence or presence of 50 μm GMP-P(NH)P. Values represent the mean of three determinations corrected for nonspecific binding. For each deletion mutant, experiments were repeated three times, and the respective GMP-P(NH)P-mediated inhibition of VGLUT activity is given in the smaller graphs inA-E. Note that inhibition in all wild-type experiments was between 25 and 30%, as it was in the Gαo1-/-, Gαq-/-, and Gα11-/- mutants. In Gαo2-/- mice, GMP-P(NH)P did not downregulate VGLUT activity, which was also reduced in untreated controls compared with the wild-type vesicles (A). In Gαo2-/- mice, nonspecific [3H]glutamate accumulation was comparable with the one seen in wild-type mice and rats (compare Fig.1 A), whereas aspartate (10 mm) was almost not transported. *p < 0.04 denotes significance according to Student's t test.
Figure 3.
Figure 3.
Abolition of GMP-P(NH)P-mediated inhibition of VGLUT activity by Gαo2-specific antibodies. A, Monoclonal antibodies clone 101.1 and clone 101.4 generated against recombinant Gαo2 were tested for specificity by Western blotting using synaptic vesicle preparations from wild-type (wt), Gαo1-/-, or Gαo2-/- mice. Whereas clone 101.1 recognizes both splice variants, clone 101.4 is Gαo2 specific. B, C, Synaptic vesicles from rat (B) or mouse brain (C) were incubated with or without (no ab) the indicated dialyzed antibodies for 30 min on ice before they were subjected to glutamate uptake in the absence or presence of 50 μm GMP-P(NH)P. Values are presented as GMP-P(NH)P-mediated inhibition. Each experiment was performed in triplicate, and nonspecific glutamate binding was subtracted. The graphs in B represent the mean of three (clone 101.1) or two (clone 101.4; SNAP-25) independent experiments, and the graphs in C represent the mean of three (clone 101.1) or two (clone 101.4) independent experiments (p < 0.005 according to Student's t test). The final IgG concentrations used in the experiments were 862 μg/ml for SNAP-25 for rat and mouse vesicles and 646 μg/ml for clone 101.1 and 431 μg/ml for clone 101.4 in the experiments with either rat or mouse vesicles, respectively. Note that both Gαo-antibodies overcome the GMP-P(NH)P-mediated downregulation of VGLUT activity with the Gαo2-specific clone 101.4 being more efficient, whereas the antibody against SNAP-25 has no effect.
Figure 4.
Figure 4.
GMP-P(NH)P-mediated inhibition of VGLUT2. Synaptic vesicle-enriched membranes (LP2) were prepared from mouse cerebellum or whole brain either at P9 or from adult brain. Glutamate uptake was performed in either the absence or presence of 50 μm GMP-P(NH)P. Values represent the mean of three determinations corrected for nonspecific binding obtained in the presence of trypan blue and are presented as GMP-P(NH)P-mediated inhibition. Synaptosomal membranes (P2) from the same preparations were subjected to SDS-PAGE and Western blotting using the antibodies indicated. Synaptobrevin (Syb) and Gαo2 were detected with the monoclonal antibody 69.1 or 101.4, respectively. Note that vesicle-enriched membranes from P9 contain only VGLUT2 and no VGLUT1, which is, however, clearly present in adult vesicular membranes. As expected, the amount of synaptobrevin analyzed for comparison was increased in adult synaptosomal membranes, whereas Gαo2 appeared to be downregulated in adulthood.
Figure 5.
Figure 5.
Comparison of the amounts of VGLUTs in synaptic vesicle-enriched membranes from wild-type, Gαo1-/-, and Gαo2-/- mice. A, Synaptic vesicle-enriched membranes were prepared from whole brains of respective wild-type (wt) and either Gαo2-/- or Gαo1-/- mice and subjected to SDS-PAGE and Western blotting using the antibodies indicated. Quantification was performed by calculating the ratio between the amount of synaptophysin used as internal standard and the respective transporters or chloride channel proteins. Synaptophysin-based ratios obtained from wild-type animals were set as 100%. The graphs represent the mean ratios of vesicle preparations obtained from three animals ±SD. There was a slight reduction of VGLUT2 in the Gαo2-/- mice and no changes in the amounts of VGLUT1, VGLUT3, or the chloride channel proteins ClC3 and ClC7. B, Synaptosomal membranes (P2) were prepared from whole brains of respective wild-type and either Gαo2-/- or Gαo1-/- mice and extracted using Triton X-114 partitioning. One, 3, or 9 μg of protein were subjected to SDS-PAGE and Western blotting using the antibodies indicated. Quantification was performed by calculating the ratio between the amount of synaptophysin used as internal standard and the respective transporters. The relative optical densities (OD) were given for wild-type and either Gαo1-/-- or Gαo2-/--derived membranes. Values represent the mean ratios of synaptosomal preparations obtained from three animals ± SD. There was no difference between the amounts of VGLUT1 or VGLUT2 in the deletion mutants compared with wild type.
Figure 6.
Figure 6.
Acidification of synaptic vesicles from wild-type (wt) and Gαo2-/- mice. Synaptic vesicles form brains of either genotype were subjected to ATP-induced acidification using either glutamate or chloride. A, Addition of 10 mm glutamate followed by an addition of 30 mm chloride resulted in an identical acidification (indicated by a downward reflection) in both wild-type (black line) and Gαo2-/- (gray line) vesicle preparations. B, Similar data were obtained when 150 or 30 mm chloride was added directly. Addition of 10 mm (NH4)2SO4 reverted the signal caused by neutralization of the vesicular pH.
Figure 7.
Figure 7.
Chloride dependence of VGLUT activity in deletion mutants of Gαo1, Gαo2, Gαq, and Gα11. Synaptic vesicles were prepared from brains of Gαo2-/- (A), Gαo1-/- (C), Gαq-/- (D), and Gα11-/- (E) mice and the corresponding wild types (wt) or from wild-type, heterozygous, or knock-out Gαo2 littermates (B). Glutamate uptake was performed in KGC/ATP buffer using the indicated chloride concentrations. Values represent the mean of three determinations, and nonspecific binding was subtracted. Experiments were performed at least two or three times. Note that 5 mm chloride increases glutamate uptake approximately twofold in wild-type as well as in Gαo1-/- (C), Gαq-/- (D), and Gα11-/- (E) mice. To exclude the influence of a different genetic background and to analyze the effects of gene doses, the chloride dependency was repeated using littermates generated from heterozygous Gαo2+/- mice (B). There is no chloride dependence in Gαo2-/- mice and a reduced chloride dependence in Gαo2+/- heterozygous ones (p < 0.01).
Figure 8.
Figure 8.
Abolition of the chloride dependence of VGLUT activity by GMP-P(NH)P. A, Synaptic vesicles from rat brain were subjected to glutamate uptake using KGC/ATP buffer containing no chloride, 5 mm chloride, or 30 mm chloride as given by the abscissa. Uptake for each chloride concentration was performed in either the absence (filled rectangles) or presence (open squares) of 50 μm GMP-P(NH)P. Values represent the mean of three determinations, and nonspecific uptake in the absence of ATP was subtracted. Note that chloride dependence is abolished in the presence of GMP-P(NH)P (p < 0.006). B, Synaptic vesicles from rat brain were subjected to glutamate uptake using KGC/ATP buffer containing 1, 2, or 5 mm chloride as given by the abscissa in the absence (filled rectangles) or presence (filled squares) of 50 μm GMP-P(NH)P. Experiment was performed in triplicate, and nonspecific glutamate binding was subtracted. G-protein activation stimulates glutamate uptake at 1 mm, but it inhibits it at 5 mm chloride. C, Synaptic vesicles from rat brain were incubated with increasing chloride concentrations (abscissa) in the absence or presence of 0.5 μm nigericin. Values represent the mean of three determinations corrected for nonspecific uptake obtained in the presence of trypan blue. Note that clamping ΔpH by nigericine increases chloride sensitivity of VGLUT. D, Synaptic vesicles from either wild-type (filled rectangles) or Gαo2-/- brains were subjected to glutamate uptake in the presence of the chloride concentrations given at the abscissa. Experiment was performed in triplicate, and nonspecific glutamate binding was subtracted. There is no chloride activation in Gαo2-/- mice but a decrease in the VGLUT activity with increasing chloride concentrations. E, Synaptic vesicles from rat brain were incubated without or with dialyzed Gαo2 antibody 101.4 for 30 min on ice before they were subjected to glutamate uptake using KGC/ATP buffer containing no chloride or 5 mm chloride either in the absence or presence of 50 μm GMP-P(NH)P. Values are expressed as percentage of glutamate uptake obtained in the nominal absence of chloride (set as 100%) and represent the mean of triplicates. Nonspecific glutamate binding was subtracted. Note that the Gαo2-specific antibody prevented GMP-P(NH)P-mediated inhibition at 5 mm chloride (p < 0.05) without changing the activation of VGLUT activity by chloride. Generally one of two or three experiments is shown.

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