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Fig. 3. Mutation of the EF-hand like region of ANO2 confers Ca2+ sensitivity. (A) The sequence alignments of EF-hand like regions in the ANO family. ANO1 and ANO2 have consensus sites in the EF-hand like region. Red; negatively-charged, pink; polar uncharged, green; positively charged, and blue; hydrophobic amino acid side chain. (B) Dose-response relationships of ANO1 (red lines) and ANO2 (black lines) activation by various concentrations of Ca2+ applied to the baths of inside-out patches of ANO1 or ANO2-transfected HEK cells (n=5~8) at Ehold −80 mV (straight line) or +80 mV (broken line). ANO1 at +80 mV, EC50=0.8 μM (nh=2.4), ANO1 at −80 mV, EC50=1.4 μM (nh=3.4), ANO2 at +80 mV, EC50=2.6 μM (nh=10.4), ANO2 at −80 mV, EC50=12.5 μM (nh=4.9). (C) Whole-cell currents of ANO2 (upper panel) and of ANO2 D316A/E318A/D320A mutant (lower panel) recorded from HEK cells transfected with ANO2 or its mutant with various [Ca2+]i (from 1 to 300 μM) in the pipette solution. (D) The dose-response characteristics of the whole-cell currents of ANO1 (●), ANO2 (■), and ANO2 D316A/E318A/D320A mutant (▲) (n=5~10). Ehold=−60 mV. ANO1 EC50=1.6 μM, ANO2 EC50=20.0 μM, ANO2 D316A/E318A/D320A mutant EC50=53.4 μM.
Exp Neurobiol 2019;28:658~669
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