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Protease activated receptor 1-induced glutamate release in cultured astrocytes is mediated by Bestrophin-1 channel but not by vesicular exocytosis.

Oh SJ, Han KS, Park H, Woo DH, Kim HY, Traynelis SF, Lee CJ - Mol Brain (2012)

Bottom Line: However, whether astrocytes exocytose to release glutamate under physiological condition is still unclear.We demonstrate that upon activation of protease activated receptor 1 (PAR1), an increase in intracellular Ca2+ concentration leads to an opening of Best1 channels and subsequent release of glutamate in cultured astrocytes.These results provide strong molecular evidence for potential astrocyte-neuron interaction via Best1-mediated glutamate release.

View Article: PubMed Central - HTML - PubMed

Affiliation: Korea Institute of Science and Technology, Seoul, South Korea.

ABSTRACT

Background: Glutamate is the major transmitter that mediates the principal form of excitatory synaptic transmission in the brain. It has been well established that glutamate is released via Ca2+-dependent exocytosis of glutamate-containing vesicles in neurons. However, whether astrocytes exocytose to release glutamate under physiological condition is still unclear.

Findings: We report a novel form of glutamate release in astrocytes via the recently characterized Ca2+-activated anion channel, Bestrophin-1 (Best1) by Ca2+ dependent mechanism through the channel pore. We demonstrate that upon activation of protease activated receptor 1 (PAR1), an increase in intracellular Ca2+ concentration leads to an opening of Best1 channels and subsequent release of glutamate in cultured astrocytes.

Conclusions: These results provide strong molecular evidence for potential astrocyte-neuron interaction via Best1-mediated glutamate release.

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Related in: MedlinePlus

Development of Best1 specific genetic tools. A) Left; Topology model of Bestrophin suggested by Tsunenari et al [17]. Blue circle indicates amino acid position of Best1 pore mutant (W93C). Red square indicates target amino acids for Best1 shRNA. Right; nucleotide and amino acid sequences of Best1 shRNA target. Nucleotide sequence differences from wild type Best1 (Best1 WT) and shRNA insensitive Best1 (Best1(sh insens)) were highlightened. B) Schematic diagram for perforated-patch-current recording from cultured astrocytes. C) Representative perforated-patch-current recording from Best1-shRNA and Best1 (sh insens) or Best1-shRNA and shRNA insensitive pore mutant Best1 (Best1-W93C (sh insens)) expressing cultured astrocytes. The current responses were recorded in response to a voltage ramp command (from -100 to +100 mV, 1 s duration, 0.2 Hz; Vh of -70 mV) before and after 30 μM TFLLR treatment. D) The bar graph summarizes the mean current amplitude at Vh = -70 mV ± s.e.m (*p < 0.05, **p < 0.01, unpaired t-test). Among 13 recorded astrocytes which expressed Best1-shRNA and Best1 (sh insens), only 5 cells showed comparable currents. And a big current recorded in an astrocyte expressing Best1-shRNA and Best1-W93C (sh insens) was eliminated in the bar graph. Numbers of determinations are indicated on the bar graph.
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Figure 2: Development of Best1 specific genetic tools. A) Left; Topology model of Bestrophin suggested by Tsunenari et al [17]. Blue circle indicates amino acid position of Best1 pore mutant (W93C). Red square indicates target amino acids for Best1 shRNA. Right; nucleotide and amino acid sequences of Best1 shRNA target. Nucleotide sequence differences from wild type Best1 (Best1 WT) and shRNA insensitive Best1 (Best1(sh insens)) were highlightened. B) Schematic diagram for perforated-patch-current recording from cultured astrocytes. C) Representative perforated-patch-current recording from Best1-shRNA and Best1 (sh insens) or Best1-shRNA and shRNA insensitive pore mutant Best1 (Best1-W93C (sh insens)) expressing cultured astrocytes. The current responses were recorded in response to a voltage ramp command (from -100 to +100 mV, 1 s duration, 0.2 Hz; Vh of -70 mV) before and after 30 μM TFLLR treatment. D) The bar graph summarizes the mean current amplitude at Vh = -70 mV ± s.e.m (*p < 0.05, **p < 0.01, unpaired t-test). Among 13 recorded astrocytes which expressed Best1-shRNA and Best1 (sh insens), only 5 cells showed comparable currents. And a big current recorded in an astrocyte expressing Best1-shRNA and Best1-W93C (sh insens) was eliminated in the bar graph. Numbers of determinations are indicated on the bar graph.

Mentions: We have recently demonstrated that Best1 channel is activated by intracellular Ca2+ (EC50 = 150 nM) with considerable permeability to GABA [26]. To test whether Ca2+- induced glutamate release is also mediated by Best1 in native astrocytes, we adopted a gene silencing tool using the short hairpin RNA (shRNA) specifically targeted to mouse Best1 transcript [25,26]. We recorded whole cell currents in cultured astrocytes under gramicidin-D perforated patch configuration (Figure 2B). We found the significant Ca2+-induced conductance carried by the efflux of glutamate at -70 mV in astrocyte with scrambled-shRNA. Silencing the Best1 gene via specific shRNA for Best1 significantly eliminated PAR1 induced whole-cell current in cultured astrocytes. This effect was fully rescued by the cotransfection of Best1-shRNA, along with a shRNA-insensitive form of Best1 (Figure 2C and D). However, the addition of a pore mutation at position 93, from tryptophan to cystein (Best1-W93C sh insens) [29] failed to rescue the current (Figure 2C and D). These results indicate that pore of Best1 channel is responsible for the permeation of anions such as Cl- and glutamate. We further tested the effect of Best1-shRNA on glutamate release using conventional high-performance liquid chromatography (HPLC) from the solution samples collected after 5 min application of TFLLR in cultured astrocytes. In this experiment, we firstly prepared astrocytes with the same cell number of cells that were transfected with Sc-shRNA or Best1-shRNA. Since we found that Best1-shRNA did not affect astrocytic cell growth or survival (data not shown), we assumed that total number of astrocytes in each sample after transfection of Sc-shRNA or Best1-shRNA should be similar. We found a decrease in glutamate release by Best1-shRNA using HPLC method (Figure 3). These results indicate that the Best1 channel is the molecular identity of glutamate permeable CAAC and suggest that the Best1 channel might be the molecular machinery for Ca2+-dependent glutamate release from astrocytes.


Protease activated receptor 1-induced glutamate release in cultured astrocytes is mediated by Bestrophin-1 channel but not by vesicular exocytosis.

Oh SJ, Han KS, Park H, Woo DH, Kim HY, Traynelis SF, Lee CJ - Mol Brain (2012)

Development of Best1 specific genetic tools. A) Left; Topology model of Bestrophin suggested by Tsunenari et al [17]. Blue circle indicates amino acid position of Best1 pore mutant (W93C). Red square indicates target amino acids for Best1 shRNA. Right; nucleotide and amino acid sequences of Best1 shRNA target. Nucleotide sequence differences from wild type Best1 (Best1 WT) and shRNA insensitive Best1 (Best1(sh insens)) were highlightened. B) Schematic diagram for perforated-patch-current recording from cultured astrocytes. C) Representative perforated-patch-current recording from Best1-shRNA and Best1 (sh insens) or Best1-shRNA and shRNA insensitive pore mutant Best1 (Best1-W93C (sh insens)) expressing cultured astrocytes. The current responses were recorded in response to a voltage ramp command (from -100 to +100 mV, 1 s duration, 0.2 Hz; Vh of -70 mV) before and after 30 μM TFLLR treatment. D) The bar graph summarizes the mean current amplitude at Vh = -70 mV ± s.e.m (*p < 0.05, **p < 0.01, unpaired t-test). Among 13 recorded astrocytes which expressed Best1-shRNA and Best1 (sh insens), only 5 cells showed comparable currents. And a big current recorded in an astrocyte expressing Best1-shRNA and Best1-W93C (sh insens) was eliminated in the bar graph. Numbers of determinations are indicated on the bar graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 2: Development of Best1 specific genetic tools. A) Left; Topology model of Bestrophin suggested by Tsunenari et al [17]. Blue circle indicates amino acid position of Best1 pore mutant (W93C). Red square indicates target amino acids for Best1 shRNA. Right; nucleotide and amino acid sequences of Best1 shRNA target. Nucleotide sequence differences from wild type Best1 (Best1 WT) and shRNA insensitive Best1 (Best1(sh insens)) were highlightened. B) Schematic diagram for perforated-patch-current recording from cultured astrocytes. C) Representative perforated-patch-current recording from Best1-shRNA and Best1 (sh insens) or Best1-shRNA and shRNA insensitive pore mutant Best1 (Best1-W93C (sh insens)) expressing cultured astrocytes. The current responses were recorded in response to a voltage ramp command (from -100 to +100 mV, 1 s duration, 0.2 Hz; Vh of -70 mV) before and after 30 μM TFLLR treatment. D) The bar graph summarizes the mean current amplitude at Vh = -70 mV ± s.e.m (*p < 0.05, **p < 0.01, unpaired t-test). Among 13 recorded astrocytes which expressed Best1-shRNA and Best1 (sh insens), only 5 cells showed comparable currents. And a big current recorded in an astrocyte expressing Best1-shRNA and Best1-W93C (sh insens) was eliminated in the bar graph. Numbers of determinations are indicated on the bar graph.
Mentions: We have recently demonstrated that Best1 channel is activated by intracellular Ca2+ (EC50 = 150 nM) with considerable permeability to GABA [26]. To test whether Ca2+- induced glutamate release is also mediated by Best1 in native astrocytes, we adopted a gene silencing tool using the short hairpin RNA (shRNA) specifically targeted to mouse Best1 transcript [25,26]. We recorded whole cell currents in cultured astrocytes under gramicidin-D perforated patch configuration (Figure 2B). We found the significant Ca2+-induced conductance carried by the efflux of glutamate at -70 mV in astrocyte with scrambled-shRNA. Silencing the Best1 gene via specific shRNA for Best1 significantly eliminated PAR1 induced whole-cell current in cultured astrocytes. This effect was fully rescued by the cotransfection of Best1-shRNA, along with a shRNA-insensitive form of Best1 (Figure 2C and D). However, the addition of a pore mutation at position 93, from tryptophan to cystein (Best1-W93C sh insens) [29] failed to rescue the current (Figure 2C and D). These results indicate that pore of Best1 channel is responsible for the permeation of anions such as Cl- and glutamate. We further tested the effect of Best1-shRNA on glutamate release using conventional high-performance liquid chromatography (HPLC) from the solution samples collected after 5 min application of TFLLR in cultured astrocytes. In this experiment, we firstly prepared astrocytes with the same cell number of cells that were transfected with Sc-shRNA or Best1-shRNA. Since we found that Best1-shRNA did not affect astrocytic cell growth or survival (data not shown), we assumed that total number of astrocytes in each sample after transfection of Sc-shRNA or Best1-shRNA should be similar. We found a decrease in glutamate release by Best1-shRNA using HPLC method (Figure 3). These results indicate that the Best1 channel is the molecular identity of glutamate permeable CAAC and suggest that the Best1 channel might be the molecular machinery for Ca2+-dependent glutamate release from astrocytes.

Bottom Line: However, whether astrocytes exocytose to release glutamate under physiological condition is still unclear.We demonstrate that upon activation of protease activated receptor 1 (PAR1), an increase in intracellular Ca2+ concentration leads to an opening of Best1 channels and subsequent release of glutamate in cultured astrocytes.These results provide strong molecular evidence for potential astrocyte-neuron interaction via Best1-mediated glutamate release.

View Article: PubMed Central - HTML - PubMed

Affiliation: Korea Institute of Science and Technology, Seoul, South Korea.

ABSTRACT

Background: Glutamate is the major transmitter that mediates the principal form of excitatory synaptic transmission in the brain. It has been well established that glutamate is released via Ca2+-dependent exocytosis of glutamate-containing vesicles in neurons. However, whether astrocytes exocytose to release glutamate under physiological condition is still unclear.

Findings: We report a novel form of glutamate release in astrocytes via the recently characterized Ca2+-activated anion channel, Bestrophin-1 (Best1) by Ca2+ dependent mechanism through the channel pore. We demonstrate that upon activation of protease activated receptor 1 (PAR1), an increase in intracellular Ca2+ concentration leads to an opening of Best1 channels and subsequent release of glutamate in cultured astrocytes.

Conclusions: These results provide strong molecular evidence for potential astrocyte-neuron interaction via Best1-mediated glutamate release.

Show MeSH
Related in: MedlinePlus