TMEM16E/ANO5 mutations related to bone dysplasia or muscular dystrophy cause opposite effects on lipid scrambling

A new study conducted by a group of researchers of the Institute of Biophysics of Genoa, Anna Boccaccio, Eleonora Di Zanni, Antonella Gradogna, Cristiana Picco, Joachim Scholz-Starke, has obtained promising results on the function of TMEM16E / ANO5, a membrane protein involved in two rare genetic diseases. Mutations in the TMEM16E gene are associated to two different forms of muscular dystrophy and to a skeletal dysplasia, gnatodiaphyseal dysplasia (GDD), characterized by bone fragility and malformation of the jaw and of the long bones. The study, funded by the Telethon Foundation and the Compagnia di San Paolo Foundation, was published in the Human Mutation and highlighted on the journal’s website as an “Editor’s Choice” article (https://doi.org/10.1002/humu.24006) .

The research concerns the study of seven mutations associated with bone dysplasia, and two mutations associated with muscular dystrophy. Functional tests revealed that mutations associated with muscular dystrophy cause a complete loss of protein function, while all those mutations associated with bone disease cause protein hyperactivity.

In conclusion: different mutations cause opposite effects on the protein function according to the type of disease to which they are associated. The pathophysiological role of this protein, both in bone and in muscle, is still unknown and will be the subject of further studies.

Muscular dystrophy‐associated mutations cause loss of TMEM16E activity. (a) Putative membrane topology of TMEM16E (898‐aa isoform) indicating the position of the MD‐causing amino acid exchanges p.Arg532Gln and p.Ser540Ile. (b) Protein sequence alignment of the scrambling domain in two human TMEM16E isoforms (16E898 and 16E913), and mouse TMEM16F. The position of the p.Arg547Gln and p.Ser555Ile amino acid exchanges, corresponding to p.Arg532Gln and p.Ser540Ile in TMEM16E898, is highlighted in red. Putative trans‐membrane domain residues (based on different TMEM16 structures, fungal nhTMEM16 (Brunner et al., 2014) and mTMEM16A protein structures (Dang et al., 2017; Paulino, Kalienkova et al., 2017; Paulino, Neldner, et al., 2017) are marked in gray. (c) Confocal images of HEK293 cells transfected with TMEM16E898‐EGFP wild‐type (WT), p.Arg532Gln or p.Ser540Ile constructs and treated with the Ca2+ ionophore A23187 (5 µM) in the presence of 5 mM extracellular Ca2+ and Alexa555‐conjugated Annexin‐V. The duration of ionophore treatment was 5 min for WT and up to 10 min for p.Arg532Gln and p.Ser540Ile mutants. Red staining of nontransfected cells (middle row) is due to excessive Ca2+ ionophore stimulation leading to cell death. From left to right, transmission light, green channel (EGFP), red channel (Alexa555). (d) Whole‐cell patch‐clamp recordings with standard intracellular solution containing 3 µM calculated free Ca2+, in HEK293 cells transfected with TMEM16E898‐EGFP wild‐type, p.Arg532Gln or p.Ser540Ile constructs, as indicated. The stimulation protocol (inset) consisted in 300‐ms voltage steps ranging from −60 to +160 mV with 20‐mV increments, followed by a 175‐ms tail pulse to −80 mV. Holding potential at 0 mV. Bottom right: steady‐state current amplitudes at +160 mV for transfected and non‐transfected control cells (nt). Data represent mean ± standard error of mean, for n  = 10 WT, n  = 8 p.Arg532Gln, n  = 7 p.Ser540Ile, n  = 5 nt; Kruskal‐Wallis test with post‐hoc Dunn‐Holland‐Wolfe test, **p  < .01