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

Nadine Reichhart,Vladimir M Milenkovic,Olaf Strauß,Eyk Schellenberger,Robert Rauh,Susanne Keckeis,Akvile Haeckel,Ahmad S Alfaar,Norbert Kociok,Sergio Crespo-Garcia,Magdalena Cordes,Simon Schöberl,Enno Klussmann,Gabriele Fels,Kerstin Zühlke,Renate Föckler,Christophe Roubeix,Anja Mataruga

doi:10.1038/s41598-018-37287-y

Abstract

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.

Highlights

Using amino acid sequence analysis combined with measurements of ion channel function, we clarified the so far unknown Ano[4] function as Ca2+-dependent, non-selective monovalent cation channel; heterologous Ano[4] expression in HEK293 cells elicits Ca2+ activated conductance with weak selectivity of K+ > Na+ > Li+
Controversial data suggest a function as a Ca2+-dependent cation channel and/or Cl− channel function for Ano[62,3,13,14,15,16]
The scramblase activity of anoctamins may be responsible for the divergent observations in Ano[6] and in other anoctamins[5]

Summary

Introduction

Using amino acid sequence analysis combined with measurements of ion channel function, we clarified the so far unknown Ano[4] function as Ca2+-dependent, non-selective monovalent cation channel; heterologous Ano[4] expression in HEK293 cells elicits Ca2+ activated conductance with weak selectivity of K+ > Na+ > Li+. Scramblase activity at resting Ca2+ levels was observed for Ano[417]; its ion channel function, has not been defined so far, Schreiber and colleagues reported increased membrane currents after applying ionomycin to Ano4-transfected HEK293 cells[18]. HEK293 cells over-expressing Ano[4] showed increased membrane conductance when intracellular free Ca2+ was increased by the extracellular application of ionomycin (Fig. 1B,C).

Results

Conclusion