Figure 4.
Characterization of ASIC1a antisense oligonucleotides using the F-11 cell line. Native proton-activated inward current was recorded from F-11 cells using the whole-cell configuration of the patch-clamp technique at a holding potential of −50 mV. This current was generated following a rapid decrease of the external pH (from pH 7.4 to the pH value indicated at each current trace). The dotted lines represent the zero current level. A, The pH dependency of this native current is similar to the one of ASIC1a homomeric current, with a half-maximal activation pH0.5 = 6.21 ± 0.05 (n = 13 cells). B, Inhibition of the F-11 cells proton-activated native current by 20 nm of the spider toxin PcTX1 (n = 8 cells; p < 0.01, paired Student's t test). C, Comparison of the proton-activated current recorded from F-11 cells to ASIC1a homomeric current recorded from transfected COS cells. The inactivation time constants of both currents were measured using a monoexponential fit. As shown by the histogram, the homomeric ASIC1a current (black bars) has the same inactivation rates as those of the proton-activated F-11 cells native current (white bars; n = 8–26). D, RT-PCR experiments show that ASIC1a is the only ASIC subunit that is expressed in F-11 cells. E, Typical pH5-evoked native ASIC1a whole-cell currents recorded at −50 mV from F-11 cells treated or not for 24, 48, or 72 h with the AS1 or IS1 oligonucleotides. F, Statistical analysis of the native ASIC1a current amplitudes measured from F-11 cells treated with the AS1 or IS1 oligonucleotides or with the AS2 or IS2 oligonucleotides, or from control F-11 untreated cells (n = 10–36; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, Kruskal–Wallis test followed by Dunns post hoc test). Error bars represent SEM.