Functional roles of glutamic acid E143 and E705 residues in the N-terminus and transmembrane domain 7 of Anoctamin 1 in calcium and noxious heat sensing

Anoctamin 1 (ANO1) is an anion channel that is activated by changes in cytosolic Ca(2+) concentration and noxious heat. Although the critical roles of ANO1 have been elucidated in various cell types, the control of its gating mechanisms by Ca(2+) and heat remain more elusive. To investigate critical amino acid residues for modulation of Ca(2+) and heat sensing, we constructed a randomized mutant library for ANO1. Among 695 random mutants, reduced Ca(2+) sensitivity was observed in two mutants (mutant 84 and 87). Consequently, the E143A mutant showed reduced sensitivity to Ca(2+) but not to high temperatures, whereas the E705V mutant exhibited reduced sensitivity to both Ca(2+) and noxious heat. These results suggest that the glutamic acids (E) at 143 and 705 residues in ANO1 are critical for modulation of Ca(2+) and/or heat responses. Furthermore, these findings help to provide a better understanding of the Ca(2+)-mediated activation and heat-sensing mechanism of ANO1.