The Putative Ca2+-Acting Site in ANO1

Abstract

Ca2+-activated Cl- channels (CaCC) mediate numerous physiological functions including vectorial Cl- movements across epithelia. Anoctamin1/TMEM16A (Ano1) confers Ca2+-activated Cl- currents. ANO1 having TMs with a putative pore region between TM5 and TM6 shows similar biophysical as well as pharmacological properties as those known for endogenous CaCCs. More importantly, ANO1 is activated by intracellular Ca2+ with EC50 of 2.6 microM at −60 mV. Furthermore, ANO1 activation is also voltage dependent as known for endogenous CaCCs. Because functional importance lies on its activation by intracellular Ca2+, the Ca2+ action site in ANO1 was determined with mutagesis. However, unlike other Ca2+-activated channels such as BK channels, there is no consensus sites for Ca2+ binding except one region that shows weak sequence homology with the Ca2+ action site in BK channel. This region contains many negatively charged amino acids. When we deleted 14 amino acids including the highly negatively charged region, the mutant ANO1 was rarely activated by intracellular Ca2+ with right shift of G-V curves, indicating that this region is important for Ca2+ action. With various mutants in this region, we can localize a sensitive site for Ca2+ response. However, when negatively charged amino acids were replaced by alanine, this mutant showed a comparable sensitivity to Ca2+. Judging from the experimental results of chimera studies with other ANOs, we can conclude that Ca2+ action on this site is essential for its activation.This work was supproted by the WCU program of the Ministry of Education and Science ans Technology of Korea.