Anoctamin-4 is a bona fide Ca²⁺-dependent non-selective cation channel

Changes in cell function occur by specific patterns of intracellular Ca²⁺, activating Ca²⁺-sensitive proteins. The anoctamin (TMEM16) protein family has Ca²⁺-dependent ion channel activity, which provides transmembrane ion transport, and/or Ca²⁺-dependent phosphatidyl-scramblase activity. Using amino acid sequence analysis combined with measurements of ion channel function, we clarified the so far unknown Ano4 function as Ca²⁺-dependent, non-selective monovalent cation channel; heterologous Ano4 expression in HEK293 cells elicits Ca²⁺ activated conductance with weak selectivity of K⁺ > Na⁺ > Li⁺. Endogenously expressed Ca²⁺-dependent cation channels in the retinal pigment epithelium were identified as Ano4 by KO mouse-derived primary RPE cells and siRNA against Ano4. Exchanging a negatively charged amino acid in the putative pore region (AA702-855) into a positive one (E775K) turns Ano4-elicited currents into Cl^- currents evidencing its importance for ion selectivity. The molecular identification of Ano4 as a Ca²⁺-activated cation channel advances the understanding of its role in Ca²⁺ signaling.