Journal: Frontiers in Molecular Neuroscience
Article Title: Development of ASIC1a ligand-gated ion channel drug screening assays across multiple automated patch clamp platforms
Figure Lengend Snippet: Pharmacology of endogenous proton-activated currents in HEK cells. (A) Individual % inhibition values plotted against concentration of PcTx1 applied during QPatch 48 recordings, with mean shown by horizontal black lines. Data was obtained from cumulative IC 50 assays where each cell was exposed to three concentrations of PcTx1, typically in half log unit increments. (B) Potency of Mambalgin-3 to inhibit pH-activated currents plotted as a cumulative concentration-response IC 50 curve. Symbols are mean ± S.D ( n = 2 or 3). (C) Exemplar current traces from a HEK cell in response to pH 6.5 activation before and after pre-incubation in increasing concentrations of Mambalgin-3. Scale bar is 500 pA and 1 s. (D) Inhibition of proton-activated currents in HEK cells by Amiloride. Data was obtained in a similar fashion to C, with each cell stimulated by pH 6.5 solution and then pre-incubated in increasing concentrations of Amiloride to construct a cumulative mini-IC 50 curve. Symbols are mean ± S.D ( n = 3). (E) Endogenous proton-activated currents are insensitive to Ruthenium Red (RR, 1 μM) but completely inhibited by a non-selective concentration of the ASIC/ENaC/Degenerin antagonist Amiloride (100 μM). The HEK cell was stimulated by pH 6.5 solution and then pre-incubated (at pH 7.4) in RR or Amiloride followed by consecutive stimulation with pH 6.5 solution containing RR or Amiloride.
Article Snippet: Amiloride and Benzamil were purchased from Merck (Sigma-Aldrich), Mambalgin-3 from Alomone Labs, and PcTx-1 from Tocris.
Techniques: Inhibition, Concentration Assay, Activation Assay, Incubation, Construct