Anoctamin 6 differs from VRAC and VSOAC but is involved in apoptosis and supports volume regulation in the presence of Ca2+

C A Juul,I H Lambert,K A Poulsen,T Kyed,E K Hoffmann,E H Larsen,N Hashem,S Grubb

doi:10.1007/s00424-013-1428-4

C A Juul, I H Lambert + Show 6 more

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https://doi.org/10.1007/s00424-013-1428-4

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Abstract

Anoctamin 6 (ANO6), also known as TMEM16F, has been shown to be a calcium-activated anion channel with delayed calcium activation. The cellular function of ANO6 is under debate, and different groups have come to different conclusions about ANO6’s physiological role. Although it is now quite well established that ANO6 is distinct from the volume-regulated anion channel, it is still unclear whether ANO6 or other anoctamins can be activated by cell swelling. In this study, we suggest that ANO1, ANO6, and ANO10 do not contribute to the volume-activated current in ANO-overexpressing HEK293 cells. Furthermore, knock-down of ANO6 in Ehrlich ascites tumor cells (EATC) and Ehrlich–Lettre ascites (ELA) did not decrease but instead significantly increased swelling-activated membrane currents. Knock-down of ANO6 in EATC did not reduce regulatory volume decrease (RVD) in the absence of extracellular calcium, whereas it significantly reduced RVD in the presence of calcium. Interestingly, we found that knock-down of ANO6 in ELA cells resulted in a decrease in cisplatin-induced caspase-3 activity, confirming earlier findings that ANO6 is involved in apoptosis. Finally, knock-down of ANO1 and ANO6 did not affect the volume-sensitive release of taurine in ELA cells. Thus, our data provide evidence that ANO6 cannot be activated directly by cell swelling unless Ca2+ is present. We also conclude that ANO6 carries a current during RVD, provided extracellular calcium is present. Thus, swelling activation of ANO6 requires the presence of free calcium.

Highlights

Anoctamin 6 (ANO6), known as TMEM16F, is a member of the anoctamin family of transmembrane proteins, which comprises ten members in vertebrates (ANO1–10)
The swelling-activated currents were recorded in mock, mANO1, mANO6, and mANO10expressing human embryonic kidney 293 (HEK293) cells, which were visually identified by their EmGFP expression
We expected to see an increase in current if ANO6 contributed to the swelling-activated current, no significant changes in the current was found in either mANO1, mANO6, or mANO10-expressing cells when compared with mocktransfected cells (Fig. 1e)

Summary

Introduction

Anoctamin 6 (ANO6), known as TMEM16F, is a member of the anoctamin family of transmembrane proteins, which comprises ten members in vertebrates (ANO1–10). Several anoctamins are known to be mutated or dysregulated in human diseases, such as musculoskeletal disorders, ataxias, and several types of cancer, which emphasizes the need to thoroughly study them [4]. ANO6 is interesting because it has been shown that mutations in this protein are the cause of the congenital bleeding disorder Scott syndrome in humans [34]. ANO6’s involvement in blood coagulation was confirmed in a study of ANO6 knock-out mice, which showed prolonged bleeding time compared with wild-type (WT) mice [37]. Another knock-out mouse study suggested that ANO6 is important for embryonic development of skeletal tissues [6]. ANO6’s properties differed significantly from those of ANO1, including a markedly higher EC50 for Ca2+ and a delay of several minutes from cytosolic (Ca2+) increase to Cl− current activation

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