Figure 3

E775 is a crucial amino -acid for Ano4 ion selectivity. (A) Upper part: Sequence alignment of amino acids (between aa 768 and 797 in murine Ano4) of mouse, rat and human Ano1, 2 and 4. Conserved amino acids are colored in black. Negatively charged amino acids in Ano4 are highlighted in green, and positively charged amino acids in Ano1 and 2 are highlighted in red. Lower part: Corresponding sequence of the two mutations E775G and E775K. hs: Homo sapiens; mm: Mus musculus; rn: Rattus norvegicus. Detailed sequence information is shown in Supp. Fig. 3 and Supp. Table 1. (B) Confocal image of HEK293 cells expressing E775G-c-Myc. Anti-Ano4 antibody staining (red). Nuclei stained with DAPI. Scale bar represents 10 µm. (C) Confocal image of HEK293 cells expressing E775K-c-Myc. Staining with an antibody against Ano4 (red). Nuclei stained with DAPI. Scale bar represents 10 µm. (D) Left panel: Raw currents through HEK293 cells heterologously expressing E775G in Ringer solution before and after the application of ionomycin (1 µM) as indicated by the bar. Middle panel: Current density-voltage plot before (filled circles) and after the application of ionomycin (open circles). The values are given as the mean ± SEM. Right panel: Maximum current density at −140 mV and at +60 mV before (black bars) and after the application of ionomycin (grey bars). ^**p < 0.01. (E) Left panel: Raw currents through HEK293 cells heterologously expressing E775K (extracellular Ringer-NMDG and intracellular asymmetric Cl⁻) before and after the application of ionomycin (1 µM) as indicated by the bar. Middle panel: Current density-voltage plot before (filled circles) and after the application of ionomycin (open circles). The values are given as the mean ± SEM. Right panel: Maximum current density at −140 mV and at + 60 mV before (black bars) and after the application of ionomycin (grey bars). ^*p < 0.05. (F) Left panel: Raw currents through HEK293 cells heterologously expressing E775K (extracellular Ringer-NMDG and intracellular symmetric Cl⁻) before and after the application of ionomycin (1 µm) as indicated by the bar. Middle panel: Current density-voltage plot before (filled circles) and after the application of ionomycin (open circles). The values are given as the mean ± SEM. Right panel: Bar chart illustrating the maximum current density at −140 mV and at + 60 mV before (black bars) and after the application of ionomycin (grey bars). ^***p < 0.001. (G) Reversal potentials of the data shown in E (left), F (middle) and G (right) before (black bars) and after (grey bars) the application of ionomycin. ^**p < 0.01.