Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia

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.

Keywords: Anoctamin5; Anoctamins; Calcium-activated chloride channels; Phosphatidylserine; Phospholipid scramblase; TMEM16E.

Fig. 1

Localization of TMEM16E-EGFP fusion proteins. a, b Confocal images of HEK293 cells transfected with the TMEM16E₈₉₈-EGFP (a) or TMEM16E₉₁₃-EGFP (b) fusion constructs and co-stained with the ER marker CellLight ER-RFP. Left, green channel (EGFP); middle, red channel (ER-RFP); right, merge. c–f Co-staining of HEK293 cells expressing TMEM16E₈₉₈-EGFP (c, d) or TMEM16E₉₁₃-EGFP (e, f) with the PM marker FM4-64. From left to right: in transmission light, in the green channel (EGFP), in the red channel (FM4-64), merge of green and red channels. d, f Close-up views of the squared regions indicated in c, e

Fig. 2

Ion transport activity of TMEM16E proteins. a–f Whole-cell patch-clamp recordings with standard intracellular solution containing 3 µM calculated free Ca²⁺, in HEK293 cells transfected with TMEM16E₈₉₈-EGFP (a), TMEM16E₈₉₈ (b), TMEM16E₉₁₃-EGFP (c) and in a non-transfected HEK293 cell (d); in a CHO cell transfected with TMEM16E₈₉₈-EGFP (e) and a non-transfected CHO cell (f). The stimulation protocol (inset in d) consisted of 300-ms voltage steps ranging from − 100 to + 180 mV with 20-mV increments, followed by a 175-ms tail pulse to − 80 mV. Holding potential at 0 mV. g Average I–V relationships in HEK293 and CHO cells, non-transfected and transfected with TMEM16E-EGFP constructs. Symbols as indicated in a–f, n given in brackets. h Average relaxation time constants (τ) of membrane currents are plotted versus the applied membrane potential. Colors as indicated in a–c, e, n given in brackets. i Average threshold potentials (V _threshold) of TMEM16E current activation, for TMEM16E₈₉₈-EGFP in CHO (n = 8; gray bar) and TMEM16E₈₉₈-EGFP (n = 31; black bar), TMEM16E₈₉₈ (n = 7; green bar), TMEM16E₉₁₃-EGFP (n = 18; blue bar) in HEK293. Inset: current traces illustrating the first membrane potential at which time-dependent currents were observed, defined as V _threshold. Error bars indicate sem in all panels

Fig. 3

Ionic selectivity of TMEM16E-mediated currents. a Whole-cell patch-clamp recordings in a HEK293 cell transfected with TMEM16E₈₉₈-EGFP, successively exposed to standard bath solution (140 NaCl), NMDG-Cl and Na-gluconate solution, as indicated. Stimulation protocol as in the inset of Fig. 2d. b Tail current recordings in a HEK293 cell transfected with TMEM16E₈₉₈-EGFP, successively exposed to standard bath solution (140 mM NaCl; left traces) and bath solution containing 10 mM NaCl (right traces). The stimulation protocol (inset) consisted of a prepulse to + 140 mV followed by voltage steps between − 50 and + 50 mV with 10-mV increments. For clarity, only current traces in 20-mV steps are shown. c I–V relationships of the tail current recordings shown in b. d Current amplitudes at the +140 mV prepulse in 140 mM NaCl and 10 mM NaCl bath solution, for individual cells (n = 11) and average (red symbols; paired t test, P = 7.9 × 10⁻⁴). e Tail current recordings as in b, but in a HEK293 cell transfected with TMEM16B-EGFP and using a stimulation protocol (inset) with voltage steps between − 40 and + 80 mV with 10-mV increments. f I–V relationships of the tail current recordings shown in e. g Average reversal potentials (V _rev) of tail current recordings in 140 mM NaCl (dark gray bars) and 10 mM NaCl bath solution (light gray bars), for TMEM16B-EGFP (n = 6; paired t test, **P = 2 × 10⁻⁷) and TMEM16E₈₉₈-EGFP (n = 8; paired t test, P = 0.14, ns not significant). Error bars indicate sem in all panels

Fig. 4

Calcium dependence of TMEM16E-mediated ionic currents. a Whole-cell patch-clamp recordings in CHO cells transfected with TMEM16E₈₉₈-EGFP, in the presence of 1 µM (left), 3 µM (middle) and 240 µM (right) calculated free Ca²⁺ in the intracellular solution. Note the different current amplitude scales. Stimulation protocol as in the inset of Fig. 2d. b Average I–V relationships derived from current recordings at different intracellular free Ca²⁺ concentrations, as partly shown in a. c Ca²⁺ dependence of current amplitudes recorded at + 140 and + 180 mV (data from b). d Threshold potentials (V _threshold) of TMEM16E current activation are plotted as a function of the intracellular free Ca²⁺ concentration (n = 7 for zero Ca²⁺, 9, 100, 240 µM; n = 5 for 1 µM; n = 10 for 3 µM). Data points were fitted with a Hill function (continuous line) yielding a half-maximal concentration of 2.9 µM Ca²⁺ and a Hill coefficient of 1.5. e Average current amplitudes recorded at different intracellular free Ca²⁺ concentrations in non-transfected CHO cells. Error bars indicate sem in all panels

Fig. 5

Phospholipid scrambling activity of TMEM16E. a–c Confocal images of live HEK293 cells transfected with TMEM16F-EGFP (a) or TMEM16E₈₉₈-EGFP (b, c) and treated with the Ca²⁺ ionophore A23187 (5 µM) for 4 min, in the presence of 5 mM extracellular Ca²⁺ and Cy3-conjugated annexin-V. From left to right, transmission light, green channel (EGFP), red channel (Cy3). In c, Cy3 fluorescence before and after A23187 treatment. d–g Activation of PLS activity in whole-cell patch-clamp recordings. In HEK293 cells expressing TMEM16E₈₉₈-EGFP (d), membrane currents were elicited immediately after reaching the whole-cell configuration, applying voltage steps from − 100 to + 140 mV in 20-mV increments, from a holding potential of 0 mV (e). Fluorescence images (f) were recorded at the indicated whole-cell time points, with 3 μM free Ca²⁺ in the patch pipette and Alexa Fluor 555-conjugated annexin-V in the bath solution. g Average normalized fluorescence intensity change (∆F _norm) of recordings shown in f, after background fluorescence subtraction, for TMEM16E₈₉₈-EGFP expressing cells (n = 10; filled circles) and non-transfected control cells (n = 4; open circles). Error bars indicate sem

Fig. 6

TMEM16E gain-of-function caused by the GDD-related T513I substitution. a Putative TMEM16E membrane topology, based on nhTMEM16 and mTMEM16A protein structures [6, 25]. The position of the T513I amino acid exchange, corresponding to T498I in TMEM16E₈₉₈, is indicated. b Confocal images of HEK293 cells transfected with TMEM16E₈₉₈^T498I-EGFP showing impaired cell adhesion and round shape (arrows). Left, transmission light; right, green channel (EGFP). c–g Cy3-conjugated annexin-V binding to HEK293 cells transfected with TMEM16E₈₉₈^T498I-EGFP in the absence of Ca²⁺ ionophore. Live cell sample in c, fixed samples in d, f. From left to right: in transmission light, in the green channel (EGFP), in the red channel (Cy3), merge of green and red channels. e, g Close-up views of the squared regions indicated in d, f. h, i Whole-cell patch-clamp recordings in HEK293 cells expressing WT (h) or T498I mutant (i) TMEM16E₈₉₈-EGFP, with intracellular solutions containing zero Ca²⁺ (upper traces) or 3 µM free Ca²⁺ (lower traces). The stimulation protocol consisted in 300-ms voltage steps ranging from − 100 to + 140 mV with 20-mV increments, followed by a 175-ms tail pulse to − 80 mV. Inset in h: current traces showing time-dependent currents at + 140 mV. Scale bars, 100 pA/100 ms. j Average I–V relationships derived from recordings as shown in h, i, for WT (n = 18 at zero Ca²⁺; n = 35 at 3 µM) and T498I mutant protein (n = 11 at zero Ca²⁺; n = 17 at 3 µM). k Threshold potentials (V _threshold) of TMEM16E current activation at zero Ca²⁺ (n = 19 WT, n = 11 T498I), 3 µM (n = 31 WT, n = 12 T498I), 9 µM (n = 9 T498I) and 100 µM free Ca²⁺ (n = 12 T498I). Mann–Whitney U test, **P = 2 × 10⁻⁵ at zero Ca²⁺ and 3 × 10⁻⁷ at 3 µM Ca²⁺. Error bars indicate sem in all panels

Fig. 7

Expression of TMEM16B carrying the GDD-related T489I substitution. a, b Confocal images of HEK293 cells transiently expressing TMEM16B-EGFP (a) and TMEM16B^T489I-EGFP (b). c, d Whole-cell patch-clamp recordings with standard intracellular solution containing zero Ca²⁺ (upper traces) or 3 µM free Ca²⁺ (lower traces), in HEK293 cells expressing TMEM16B-EGFP (c) and TMEM16B^T489I-EGFP (d). Inset in d, stimulation protocol. e Average steady-state I–V relationships of recordings as shown in c, d, for WT (n = 6 in each condition) and T489I mutant protein (n = 7 at zero Ca²⁺, n = 19 at 3 µM Ca²⁺). f Relaxation time constants (τ) of currents recorded at 3 µM free Ca²⁺ are plotted versus the applied membrane potential, for WT (n = 6) and T489I mutant protein (n = 16). Symbols in e, f as indicated in c, d. Error bars indicate sem in all panels