11 research outputs found

    Activation of ASIC1a modifies the current kinetics of GABA<sub>A</sub> current.

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    <p>A, The representative current traces recorded from HEK293 cells co-transfected with GABA<sub>A</sub> receptor subunits (α<sub>1</sub> and β<sub>2</sub>) and ASIC1a. ASIC1a activated by pH 6 reversibly altered the overall shape of GABA<sub>A</sub> currents. GABA<sub>A</sub> current traces were superimposed to the right. Red arrow indicates the current activated by pH 6 solution. B, pH 6 solution changed the peak current amplitude but not the shape of GABA<sub>A</sub> currents in HEK293 cells transfected with cDNA of GABA<sub>A</sub> receptor subunits only. C (cotransfected with both plasmids) and D (transfected with cDNA of GABA<sub>A</sub> receptor subunits), bar graph showing the summarized data of rise time of activation (10–90%) (i), desensitization time constant (ii) and deactivation time (iii) of GABA<sub>A</sub> currents in the presence of pH 7.4 or pH 6. ***, paired t-test, <i>p</i><0.001, pH6 group vs. control group, n = 12.</p

    Activation of GABA<sub>A</sub> receptors attenuated the peak current amplitude and enhanced the sustained current of ASIC1a.

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    <p>A, Example traces of a fast-inactivating transient current and a sustained current of ASIC1a activated by pH 3.5. GABA (100 μM) attenuated a fast-inactivating transient current and enhanced the sustained current of ASIC1a in HEK293 cells co-transfected with GABA<sub>A</sub> receptor subunits (α<sub>1</sub> and β<sub>2</sub>) and ASIC1a, which can totally abolished by co-application of picrotoxin (100 μM) with GABA. ASIC1a current traces were superimposed to the right (inset) (B). C, GABA had no effect on ASIC1a currents in HEK293 cells transfected with ASIC1a cDNA only. ASIC1a current traces were superimposed to the right (inset).</p

    Co-immunoprecipitation of ASIC1a and GABA<sub>A</sub> proteins in transfected HEK293 cells and DRG neurons.

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    <p>A. Co-immunoprecipitation of ASIC1a and GABA<sub>A</sub> proteins in transfected HEK293 cells. GABA<sub>A</sub> specifically co-precipitates with ASIC1a in cells co-transfected with ASIC1a and GABA<sub>A</sub> (both). n = 3. B1. DRG neurons were double-labeled with anti-ASIC1a and -GABA<sub>A</sub>R antibodies. ASIC1a (red in left panel) as well as GABA<sub>A</sub>R (green in middle panel) localized to the apical membrane of DRG neurons. The merge (right) of ASIC1a and GABA<sub>A</sub>R images indicates that ASIC1a co-segregates with GABA<sub>A</sub>R in the apical membrane in neurons (yellow). Nuclei were identified by DAPI staining (blue). Scale bars equal 50 µm. B2. GABA<sub>A</sub> precipitates with ASIC 1a in primary cultured DRG neurons. DRG neurons lysates were immunoprecipitated (IP) with anti-GABA<sub>A</sub> β<sub>2/3</sub> subunits polyclonal antibody in 8% SDS-PAGE gel, and the blot was then probed with anti- ASIC 1a polyclonal antibody (IB). In turn, ASIC 1a co-immunoprecipitates with GABA<sub>A</sub>. Cell lysates were immunoprecipitated with ASIC 1a antibody in 6% SDS-PAGE gel, and the blot was probed with GABA<sub>A</sub> β<sub>2/3</sub> antibody. These experiments were repeated three times with identical results.</p

    Activation of GABA<sub>A</sub> receptors reversibly inhibits ASIC1a currents.

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    <p>A, ASIC1a were activated by pH 6.0 solution repetitively in HEK293 cells co-transfected with GABA<sub>A</sub> receptor subunits (α<sub>1</sub> and β<sub>2</sub>) and ASIC1a. GABA (100 μM) reversibly attenuated ASIC1a currents. Red arrow indicates the current activated by GABA. B, co-application of bicuculline (BIC, 30 μM) or of picrotoxin (PIC, 100 μM) with GABA largerly abolished the GABA-induced inhibition of ASICs. C, GABA had no effect on ASIC1a currents in HEK293 cells transfected with cDNA of ASIC1a only. D, statistic graph shows relative ASIC currents that were affected by GABA but reversed by antagonists of GABA<sub>A</sub> receptors. n = 6, ***, <i>p</i><0.001, <i>T</i>-test, before vs. after drug; ###, <i>p</i><0.001, <i>one-way ANOVA</i>, GABA plus GABA antagonists vs. GABA alone.</p

    Interregulation of ASIC1a and GABA<sub>A</sub> receptors in DRG neurons.

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    <p>A. Example traces of ASICs current activated by pH 6.0 solution in DRG neurons. GABA (100 μM) reversibly attenuated acid-evoked currents (n = 12). B. The representative current traces of GABA induced currents in DRG neurons. The pH 6.0 solution induced an inward current (enlarge inset). GABA-induced current was enhanced in the presence of pH 6.0 solution (n = 7).</p

    Effects of Cl<sup>−</sup> and Ca<sup>2+</sup>-free solution on CQ-evoked increase in <i>I<sub>SC</sub></i> in rat ileum.

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    <p>CQ-induced increases in <i>I<sub>SC</sub></i> was greatly reduced in the absence of Cl<i><sup>−</sup></i>(n = 6,A). In the absence of Ca<sup>2+</sup>, CQ-induced increases in <i>I<sub>SC</sub></i> were totally abolished, whereas a decrease in basal <i>I<sub>SC</sub></i> was seen in this Ca<sup>2+</sup>-free condition(n = 5,B). A similar effect was obtained in response to pretreatment with thapsigargin(10<sup>−6</sup>M), a Ca<sup>2+</sup> pump inhibitor(n = 6, C). **<i>P</i><0.01 by unpaired <i>t</i>-test.</p

    A working model of CQ in intestinal epithelial cells.

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    <p>CQ binds to T2Rs activating a G-protein to produce phospholipase C (PLC) in the basement membrane of intestinal epithelial cells followed by Ca<sup>2+</sup> release from the sarcoplasmic reticulum (SR). Local Ca<sup>2+</sup> entry through store-operated Ca<sup>2+</sup> release-activated Ca<sup>2+</sup> channels(CRAC) drives Cl<i><sup>−</sup></i> secretion by stimulating Ca<sup>2+</sup>-activated Cl<i><sup>−</sup></i> channels(CaCC) in the apical membrane of intestinal epithelial cells. The intracellular-free chloride concentration is maintained by a Na<sup>+</sup>-K<sup>+</sup>-2Cl<sup>−</sup> co-transporter that actively accumulates Cl<sup>−</sup>.</p

    CQ evoked an increase in <i>I<sub>SC</sub></i> in rat ileum.

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    <p>CQ dose-dependently increased basal <i>I<sub>SC</sub></i> at low concentrations(≤5×10<sup>−4 </sup>M), however, it markedly decreased basal <i>I<sub>SC</sub></i> at high concentrations (≥10<sup>−3</sup> M) (n = 6, A). The serosal addition of CQ (3×10<sup>−4</sup>M) increased basal <i>I<sub>SC</sub></i>, whereas CQ to mucosal bathing solution had no effect on basal <i>I<sub>SC</sub></i> (n = 6,B)<sub>.</sub> The CQ-induced increase in <i>I<sub>SC</sub></i> was not influenced by TTX(n = 4,C). *<i>P</i><0.05; **<i>P</i><0.01 compared with control or apical addition; <sup>#</sup><i>P</i><0.05 compared with 100 µM group; n.s: no significance by the one-way ANOVA or unpaired <i>t</i>-test.</p
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