Figure 2. The canonical local anesthetic-binding sites are not conserved in NaChBac. (A) The sequence alignment of S6 of bacterial and eukaryotic S6 segments, highlighting two sites implicated in use-dependent inhibition of eukaryotic channels. (B) Top-down view of the highlighted residues shown on the right. (C) A concentration response curve was generated with the mean of Idrug/Icontrol obtained from the peak currents after exposing cells to 30 μM, 100 μM, 300 μM, 1 mM and 3 mM (for T220A only) lidocaine. The IC₅₀ for lidocaine, according to the concentration response curve, is ~135 μM for WT(), ~788 μM for T220A() and ~214 μM for F227A (). Representative current traces are obtained after depolarization from the holding potential -120 to -20 mV in the presence of various lidocaine concentrations, peak current sizes were reduced upon application of increasing lidocaine concentrations (right).