# Functional Interdependence of Anoctamins May Influence Conclusions from Overexpression Studies

## TL;DR

ANO6 interacts with other anoctamins, and its presence affects the results of overexpression studies, especially when calcium levels are high.

## Contribution

The study reveals that ANO6's endogenous expression significantly influences overexpression data of other anoctamins.

## Key findings

- ANO6 affects ion currents, phospholipid scrambling, and calcium signals in overexpression studies.
- Results from overexpressed anoctamins are altered in ANO6-knockout cells compared to wild-type cells.
- ANO6's presence is crucial for interpreting functional data of other anoctamins.

## Abstract

Anoctamin 6 (ANO6, TMEM16F) is a phospholipid (PL) scramblase that moves PLs between both plasma membrane (PM) leaflets and operates as an ion channel. It plays a role in development and is essential for hemostasis, bone mineralization and immune defense. However, ANO6 has also been shown to regulate cellular Ca2+ signaling and PM compartments, thereby controlling the expression of ion channels such as CFTR. Given these pleiotropic effects, we investigated the functional interdependence of the ubiquitous ANO6 with other commonly co-expressed anoctamins. As most expression studies on anoctamins use HEK293 human embryonic kidney cells, we compared ion currents, PL scrambling and Ca2+ signals induced by the overexpression of anoctamins in HEK293 wild-type parental and ANO6-knockout cells. The data suggest that the endogenous expression of ANO6 significantly affects the results obtained from overexpressed anoctamins, particularly after increasing intracellular Ca2+. Thus, a significant interdependence of anoctamins may influence the interpretation of data obtained from the functional analysis of overexpressed anoctamins.