Activation of ASIC1a modifies the current kinetics of GABA_A current.

<p>A, The representative current traces recorded from HEK293 cells co-transfected with GABA_A receptor subunits (α₁ and β₂) and ASIC1a. ASIC1a activated by pH 6 reversibly altered the overall shape of GABA_A currents. GABA_A 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_A currents in HEK293 cells transfected with cDNA of GABA_A receptor subunits only. C (cotransfected with both plasmids) and D (transfected with cDNA of GABA_A 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_A 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

Co-immunoprecipitation of ASIC1a and GABA_A proteins in transfected HEK293 cells and DRG neurons.

<p>A. Co-immunoprecipitation of ASIC1a and GABA_A proteins in transfected HEK293 cells. GABA_A specifically co-precipitates with ASIC1a in cells co-transfected with ASIC1a and GABA_A (both). n = 3. B1. DRG neurons were double-labeled with anti-ASIC1a and -GABA_AR antibodies. ASIC1a (red in left panel) as well as GABA_AR (green in middle panel) localized to the apical membrane of DRG neurons. The merge (right) of ASIC1a and GABA_AR images indicates that ASIC1a co-segregates with GABA_AR in the apical membrane in neurons (yellow). Nuclei were identified by DAPI staining (blue). Scale bars equal 50 µm. B2. GABA_A precipitates with ASIC 1a in primary cultured DRG neurons. DRG neurons lysates were immunoprecipitated (IP) with anti-GABA_A β_2/3 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_A. Cell lysates were immunoprecipitated with ASIC 1a antibody in 6% SDS-PAGE gel, and the blot was probed with GABA_A β_2/3 antibody. These experiments were repeated three times with identical results.</p

Activation of GABA_A receptors attenuated the peak current amplitude and enhanced the sustained current of ASIC1a.

<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_A receptor subunits (α₁ and β₂) 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

Activation of GABA_A receptors reversibly inhibits ASIC1a currents.

<p>A, ASIC1a were activated by pH 6.0 solution repetitively in HEK293 cells co-transfected with GABA_A receptor subunits (α₁ and β₂) 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_A 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_A receptors in DRG neurons.

<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