Journal: Nature Communications

Article Title: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel

doi: 10.1038/s41467-018-03728-5

Figure Lengend Snippet: Switch of reversal potentials between co-expressed rASIC3 and rP2X3Rs in CHO cells. Whole-cell patch-clamp recordings at a holding potential of −65 mV. a Current traces induced by protons (pH 6.5) and α,β-meATP (10 µM) applied individually onto CHO-rASIC3 (left panel) and CHO-rP2X3R cells (middle panel) or successively onto CHO-rASIC3/P2X3 cells (right panel). Agonist application was at holding potentials which increased stepwise from −60 to + 90 mV in 30 mV increments. Current–voltage relationships were constructed from recordings similar to those shown in ( a ) in order to determine the reversal potentials ( E rev ) ( b ). Means ± S.E.M. of the indicated number of experiments. c Measurement of E rev of protons (left panel) and of α,β-meATP (right panel) by a similar protocol as shown in ( a ) on separate populations of CHO-rASIC3/rP2X3R cells. Under these conditions, the E rev values of these two agonists were interchanged. Current–voltage relationships constructed from recordings similar to those shown in ( c ) in order to determine the E rev ( d ). Means ± S.E.M. of the indicated number of experiments. Blockade of ASIC3 channel activity by the selective antagonist APETx2 (0.1 µM) shifted the E rev of α,β-meATP back near to its original value of around 0 mV. e In CHO-rASIC3/rP2X3R cells, ramps of 200 mV duration (−90 to +90 mV) were delivered at a holding potential of −65 mV to determine the E rev of ASIC3 after a drop of the normal pH from 7.4 to 6.5. Then, a high concentration of α,β-meATP (100 µM) was applied in order to change the distribution of extra- and intracellular ions. Eventually, the E rev was re-determined after washing out α,β-meATP. Original tracing; large capacitive artifacts at the beginning and end of the ramp-induced currents were retouched. f Current–voltage curves were constructed from the indicated number of experiments similar to that shown in ( e ). The scale labels for the vertical bars were 2 nA ( a , left panel), 1 nA ( a , middle and right panels), 1 nA ( c ) and 500 pA ( e ). The scale labels for the horizontal bars were 1 s ( a , c ) and 2 s ( e )

Article Snippet: Cell extracts were immunopurified with a rabbit anti-P2X3 antibody (Santa Cruz Biotechnology, cat. no.: sc-25694) and immunoblotted with rabbit anti-ASIC1, rabbit anti-ASIC2, rabbit anti-ASIC3, or rabbit anti-P2X3 antibodies (Alomone Labs, cat. nos.

Techniques: Patch Clamp, Construct, Activity Assay, Concentration Assay

Journal: Nature Communications

Article Title: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel

doi: 10.1038/s41467-018-03728-5

Figure Lengend Snippet: Immunoreactivity, co-immunoprecipitation and membrane expression of rASIC3 and rP2X3Rs in rat DRG neurons. a ASIC3 and P2X3R immunoreactivities in rat DRG neurons cultured in the presence (upper panel) or absence (lower panel) of nerve growth factor (NGF). Hoechst (Hoe) was used to stain the cell nuclei. b Examples of immunoprecipitation (IP) of DTSSP-treated extracts of primary sensory neurons with P2X3 antibodies revealed in western blots (WB) with anti-ASIC3 antibodies. Incubation of neuronal cultures at pH 6.8 or 7.5 has no effect. ASIC3 signal is not found after immunoprecipitation with unrelated antibodies (IgG). Input ASIC3 and P2X3 contents in total extracts are also shown. β-Actin is used as gel loading control. c Examples of immunoprecipitation of P2X3 receptors (P2X3, upper panel) with ASIC3 channels in control conditions (scramble) and after P2X3 receptor silencing (siP2X3). No P2X3/ASIC3 signal was found after siP2X3 treatment. Western blot with anti-ASIC1 or anti-ASIC2 antibodies gave no signal. Pull down with unrelated antibody (IgG) gave no signal. Quality of input lysates and equal gel loading is shown (lower panel, total lysates). d Surface expression of P2X3Rs. Example of membrane protein biotinylation experiments in CHO cells transfected with plasmids encoding for ASIC3 alone or ASIC3 plus P2X3Rs (upper panel, surface). Quality of total protein extracts and controls for equal gel loading are also shown (bottom panel, total lysates). The scale labels in the right upper and lower panels of ( a ) were 20 µm

Article Snippet: Cell extracts were immunopurified with a rabbit anti-P2X3 antibody (Santa Cruz Biotechnology, cat. no.: sc-25694) and immunoblotted with rabbit anti-ASIC1, rabbit anti-ASIC2, rabbit anti-ASIC3, or rabbit anti-P2X3 antibodies (Alomone Labs, cat. nos.

Techniques: Immunoprecipitation, Membrane, Expressing, Cell Culture, Staining, Western Blot, Incubation, Control, Transfection