Abstract

Anoctamins such as TMEM16A and TMEM16B are Ca2+-dependent Cl− channels activated through purinergic receptor signaling. TMEM16A (ANO1), TMEM16B (ANO2) and TMEM16F (ANO6) are predominantly expressed at the plasma membrane and are therefore well accessible for functional studies. While TMEM16A and TMEM16B form halide-selective ion channels, TMEM16F and probably TMEM16E operate as phospholipid scramblases and nonselective ion channels. Other TMEM16 paralogs are expressed mainly in intracellular compartments and are therefore difficult to study at the functional level. Here, we report that TMEM16E (ANO5), -H (ANO8), -J (ANO9) and K (ANO10) are targeted to the plasma membrane when fused to a C-terminal CAAX (cysteine, two aliphatic amino acids plus methionin, serine, alanin, cystein or glutamin) motif. These paralogs produce Ca2+-dependent ion channels. Surprisingly, expression of the TMEM16 paralogs in the plasma membrane did not produce additional scramblase activity. In contrast, endogenous scrambling induced by stimulation of purinergic P2X7 receptors was attenuated, in parallel with reduced plasma membrane blebbing. This could suggest that intracellular TMEM16 paralogs operate differently when compared to plasma membrane-localized TMEM16F, and may even stabilize intracellular membranes. Alternatively, CAAX tagging, which leads to expression in non-raft compartments of the plasma membrane, may antagonize phosphatidylserine exposure by endogenous raft-located TMEM16F. CAAX-containing constructs may be useful to further investigate the molecular properties of intracellular TMEM16 proteins.

Highlights

  • The TMEM16/Anoctamin family of proteins consists of ten transmembrane paralogs, ranging from TMEM16A/ANO1 to TMEM16K/ANO10

  • We report that TMEM16E (ANO5), -H (ANO8), -J (ANO9) and K (ANO10) are targeted to the plasma membrane when fused to a C-terminal CAAX motif

  • In contrast to TMEM16A, which is well expressed at the plasma membrane, TMEM16E, -H, -J and -K are predominantly expressed in membranes of intracellular compartments, in particular in the endoplasmic reticulum (ER) [4,11,14,18,19]

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Summary

Introduction

The TMEM16/Anoctamin family of proteins consists of ten transmembrane paralogs, ranging from TMEM16A/ANO1 to TMEM16K/ANO10. In addition to their function as Ca2+-activated Cl− channels, some members of this family, like TMEM16F, operate as Ca2+-activated lipid scramblases. In contrast to TMEM16A, which is well expressed at the plasma membrane, TMEM16E, -H, -J and -K are predominantly expressed in membranes of intracellular compartments, in particular in the endoplasmic reticulum (ER) [4,11,14,18,19] This makes it difficult to study the biophysical properties and mechanisms of activation of these TMEM16 paralogs. CAAX-mediated plasma membrane targeting may be helpful to further investigate the molecular properties of intracellular TMEM16 proteins

Results
Materials and Methods
Immunocytochemistry
Iodide Quenching Assay
Whole-Cell Patch Clamp
Materials and Statistic
Conclusions
Full Text
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