Proton-mediated modulations of Anoctamins

Abstract

Ca2+-activated Cl- channels are involved in fluid and electrolyte secretion in epithelial cells. It is closely related to intracellular pH. Anoctamin1 is known to mediate Ca2+-activated Cl- currents. However, little is known whether ANO1 can be modulated by change of intracellular pH. Here, we demonstrated that both ANO1 and ANO2 are inhibited directly by acidic intracellular pH using the inside-out patch clamp technique. The acid-induced modulations of other ANOs were also tested. ANO6 and ANO7 were inhibited by intracellular acid. But intracellular acid failed to block the current of ANO9. Intracellular acid also inhibit the voltage-dependent activation. Intracellular acid induce the decrease of the Ca2+ sensitivity in ANO1. The higher Ca2+-activated ANO1 current required more protons to inhibit. But the voltage-activated and heat-activated ANO1 current was not inhibited by intracellular acid. To identify the location of the acid-induced inhibition, mutagenesis studies were performed. Mutations were made on His residues in cytoplasmic side of ANO1. However, there was no His mutant that showed the reduction of the acid-induced inhibition. Some clusters of Glu and Asp are also dispensable for the acid-induced inhibition. Recently, the Ca2+-binding site of a fungal anoctamin (nhTMEM16) was uncovered by crystallography. The Ca2+-binding residues are present in α6, α7 and α8