Abstract
Mutations in the human TMEM16E (ANO5) gene are associated both with the bone disease gnathodiaphyseal dysplasia (GDD; OMIM: 166260) and muscle dystrophies (OMIM: 611307, 613319). However, the physiological function of TMEM16E has remained unclear. We show here that human TMEM16E, when overexpressed in mammalian cell lines, displayed partial plasma membrane localization and gave rise to phospholipid scrambling (PLS) as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials. While the activity of wild-type TMEM16E depended on elevated cytosolic Ca2+ levels, a mutant form carrying the GDD-causing T513I substitution showed PLS and large time-dependent ion currents even at low cytosolic Ca2+ concentrations. Contrarily, mutation of the homologous position in the Ca2+-activated Cl− channel TMEM16B paralog hardly affected its function. In summary, these data provide the first direct demonstration of Ca2+-dependent PLS activity for TMEM16E and suggest a gain-of-function phenotype related to a GDD mutation.
Highlights
Gnathodiaphyseal dysplasia (GDD; OMIM: 166260) is a rare autosomal-dominant generalized skeletal syndrome characterized by fibro-osseous lesions of the jawbones and associated with long and tubular bone dysplasia and fragility
A similar pattern was found in cells transiently expressing the full-length (913-aa-long) TMEM16E isoform, the plasma membrane (PM) localization was less evident in this case (Fig. 1b, e, f)
Since TMEM16E ion currents activated at relatively high positive membrane potentials and did not reach saturation within the applied voltage range, we evaluated current activation from the threshold potential (Vthreshold; Fig. 2 Ion transport activity of TMEM16E proteins. a–f Whole-cell patch-clamp recordings with standard intracellular solution containing 3 μM calculated free C a2+, in HEK293 cells transfected with TMEM16E898-EGFP (a), TMEM16E898 (b), TMEM16E913-EGFP (c) and in a non-transfected HEK293 cell (d); in a chinese hamster ovary (CHO) cell transfected with TMEM16E898-EGFP (e) and a non-transfected CHO cell (f)
Summary
Gnathodiaphyseal dysplasia (GDD; OMIM: 166260) is a rare autosomal-dominant generalized skeletal syndrome characterized by fibro-osseous lesions of the jawbones and associated with long and tubular bone dysplasia and fragility. GDD patients show facial deformity, begin to experience frequent bone fractures around puberty and are susceptible to purulent osteomyelitis in jawbones during adult life [1, 28]. This syndrome has been associated to mutations in the GDD1 gene [36], known as TMEM16E or Ano, encoding a 913-amino acid integral membrane protein of unknown physiological function. The TMEM16E gene is highly expressed in skeletal muscle and bone tissues, such as calvaria, femur and mandibule [36] It is expressed in human osteoblasts and periodontal ligament cells, consistent with GDD disease phenotypes [36]. Unlike other TMEM16 family members, TMEM16E does not show a clear plasma membrane (PM) localization, at most weakly [23], but rather in unspecified intracellular vesicles [23, 34]
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