The prolonged desensitization of the ASIC2a mutants is not due to an internalization of the ion channels in response to extracellular protons. (A) Currents elicited by three consecutive applications of pH 4.0 for 20 s in HEK293T cells expressing either Mut2 (upper) or Mut3 (lower) in the absence (blue) or presence (red) of 50 µM primaquine, begun 1 h before the recordings. (B) Peak current densities in cells expressing either Mut2 (upper) or Mut3 (lower) were normalized to those evoked by the initial acidic stimulus in the absence (blue) or presence (red) of 50 µM primaquine. n = 5 for each group. Acidic stimuli were delivered by switching the bath solution from pH 7.4 to pH 4.0 at 2-min intervals. (C and D) Western blotting showing the amounts of GFP-tagged Mut2 and Mut3 in the plasma membrane (PM) and total cell lysate (Total) following application of pH 7.4 versus pH 4.0. E-cadherin and GAPDH are markers used to normalize the amounts of Mut2 and Mut3 in the plasma membrane and total cell lysate, respectively. n = 3 for each group. n.s. not significant; Mann–Whitney U test.