Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia
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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.
KeywordsTMEM16E Anoctamin5 Anoctamins Phospholipid scramblase Calcium-activated chloride channels Phosphatidylserine
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 , also known as TMEM16E or Ano5, encoding a 913-amino acid integral membrane protein of unknown physiological function. At present, eight GDD-causing TMEM16E mutations have been identified leading to amino acid exchanges at six positions: p.Arg215Gly , p.Cys356Gly, p.Cys356Arg , p.Cys356Tyr [2, 9, 17, 38], p.Cys360Tyr , p.Ser500Phe , p.Thr513Ile  and p.Gly518Glu .
In humans and mice, the TMEM16E gene is highly expressed in skeletal muscle and bone tissues, such as calvaria, femur and mandibule . In particular, it is expressed in human osteoblasts and periodontal ligament cells, consistent with GDD disease phenotypes . Unlike other TMEM16 family members, TMEM16E does not show a clear plasma membrane (PM) localization, at most weakly , but rather in unspecified intracellular vesicles [23, 34]. When heterologously overexpressed, it was found predominantly localized in the ER network [10, 13, 36] or at the PM .
TMEM16E belongs to a family of integral membrane proteins named TMEM16 or anoctamins [16, 26]. According to the recently published crystal structure of the fungal homologue nhTMEM16 , TMEM16 proteins have 10 transmembrane domains. Although they share a similar membrane topology, they can perform diverse cellular functions: TMEM16A and TMEM16B function as Ca2+-activated chloride channels, while TMEM16F acts as a Ca2+-dependent phospholipid scramblase facilitating the movement (scrambling) of phospholipids between the leaflets of the membrane bilayer. There is no such consensus about the functional roles of other family members.
Available data on TMEM16E function are few and not conclusive. Attempts to directly record electrical activity of heterologously expressed TMEM16E failed [10, 35] or revealed small Ca2+-activated currents , and no anion transport activity was detected in halide-sensitive YFP-based fluorescence assays [22, 31]. Based on amino acid conservation between family members, Tran et al.  inserted the GDD-related C356G and C356R exchanges  at the corresponding position in the PM-localized TMEM16A protein. Only the protein carrying the glycine exchange gave rise to currents, which, quite surprisingly, appeared to display cationic selectivity, as opposed to anionic in wild-type TMEM16A . Due to the predominant intracellular localization of TMEM16E, several studies have furthermore relied on chimeric constructs based on the TMEM16A backbone. A chimeric protein carrying the putative TMEM16E channel pore region was detected at the PM, although not conductive , while three different chimeras created by Duran et al.  were retained intracellularly. Recently, following the identification of a specific 35-aa-long scrambling domain necessary and sufficient for TMEM16F activity , Gyobu et al. [13, 14] measured scrambling activity of a chimeric protein carrying the homologous region of TMEM16E in the TMEM16A backbone, indicating that the 35-aa stretch worked as a scrambling domain. Scramblase activity for the TMEM16E wild-type protein still awaits confirmation.
Here, we find partial PM localization of heterologously expressed TMEM16E, which allowed us to perform functional studies on the ion transport and scrambling activity of TMEM16E. Furthermore, we identify a gain-of-function phenotype for a TMEM16E mutation related to gnathodiaphyseal dysplasia.
TMEM16E-EGFP fusion proteins show partial plasma membrane localization
TMEM16E mediates Ca2+-dependent non-selective ionic currents
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 ( V threshold; Fig. 2i), defined as the first potential step at which time-dependent currents were observed, as illustrated in the inset. Average V threshold values were + 88.1 ± 2.8 mV (n = 31) for TMEM16E898-EGFP, + 91.4 ± 7.2 mV (n = 7) for untagged TMEM16E898, both expressed in HEK293 cells, and + 75.0 ± 3.1 mV (n = 8) for TMEM16E898-EGFP expressed in CHO cells. The construct TMEM16E913-EGFP (in HEK293 cells) showed more positive values (+ 118.3 ± 4.7 mV, n = 18), possibly related to its lower PM expression and current amplitudes, which limits the accuracy of threshold determination.
TMEM16E exhibits Ca2+-dependent phospholipid scramblase activity
We further performed patch-clamp experiments on TMEM16E898-EGFP transfected HEK293 cells, simultaneously recording ionic currents and imaging PLS activity in the presence of Alexa Fluor 555-conjugated annexin-V in the bath solution (Fig. 5d–g). TMEM16E ion currents became apparent quickly after reaching the whole-cell configuration, activated by 3 μM free Ca2+ in the intracellular solution entering the cytoplasm (Fig. 5e). Currents mediated by TMEM16E remained relatively stable over time, although we noted a time-dependent increase of background currents in these recording conditions (Supplementary Figure 3), possibly partly due to a modification of the membrane properties by PLS activity and/or extracellularly bound annexin-V. Fluorescence signals lining the cell boundaries, indicative of annexin-V binding, became detectable on average after 5.2 ± 0.7 min (n = 10) in the whole-cell configuration and strongly increased over time (Fig. 5f, g). A lag phase of similar length between current activation and PLS detection was also reported for TMEM16F . In non-transfected control cells, no time-dependent currents developed and no annexin-V binding was observed within the 25-min recording period (Fig. 5g and Supplementary Figure 3c).
The GDD-causing T513I substitution affects TMEM16E scrambling and ion transport activities
Numeration of isoforms and the GDD-causing T513I substitution in the TMEM16 proteins used in this study
Total length (aa)
Position of substitution
Surprisingly, these assays showed that annexin-V bound to cell membranes even in the absence of Ca2+ ionophore (Fig. 6c–g), indicating that PLS activity of the TMEM16ET498I mutant protein was no longer dependent on elevated cytosolic Ca2+ concentrations. Patch-clamp recordings fully confirmed this result: differently from the wild-type protein (Fig. 6h, j), TMEM16ET498I mediated large time-dependent currents even at zero Ca2+, which further increased in amplitude at 3 μM free Ca2+ in the pipette solution (Fig. 6i, j). The threshold potential of TMEM16ET498I activation shifted from + 82.3 ± 4.1 mV at zero Ca2+ (n = 11) to + 59.6 ± 3.2 mV at 3 μM Ca2+ (n = 12), while the respective V threshold values for TMEM16EWT were + 125.2 ± 6.1 mV (n = 19) and + 88.1 ± 2.8 mV (n = 31; Fig. 6k), almost identical to the WT data determined in CHO cells (Fig. 4d). V threshold at saturating [Ca2+] was around + 43 mV for the T498I mutant (Fig. 6k), compared to + 62 mV for the WT protein, indicative of a significant shift of the Ca2+ dependence towards more negative membrane potentials. Thus, the data of both current amplitudes and threshold potentials concurrently demonstrate that, as a consequence, TMEM16ET498I activity at zero Ca2+ is comparable to TMEM16EWT activity at 3 μM cytosolic Ca2+, strongly suggesting that the GDD-causing T513I substitution causes a gain-of-function phenotype for the TMEM16E protein.
Minor effects of the T513I substitution introduced into TMEM16B
This study provides direct evidence for phospholipid scrambling and non-selective ion transport activity of TMEM16E, a member of the TMEM16 protein family  which is functionally split into Ca2+-activated Cl− channels (TMEM16A/B) and Ca2+-activated phospholipid scramblases (TMEM16C/D/F/G/J). The identification of TMEM16E function represents a crucial step towards the definition of its physiological role, especially since mutations in the human TMEM16E gene are related to severe genetic diseases.
The prevalently intracellular localization of TMEM16E [10, 23] has significantly delayed the clarification of its function. In accordance with previous reports [10, 13, 36], TMEM16E-EGFP fusion proteins expressed in HEK293 and CHO cells showed strong co-localization with a ER marker; however, in cells with high expression levels, we additionally observed partial PM targeting. PM localization of heterologously overexpressed TMEM16E has also been reported by Tian et al. . Moreover, in patch-clamp recordings, they found a significant increase in the membrane conductance (measured in the limited voltage range of ± 50 mV) upon application of the Ca2+ ionophore ionomycin. The results presented here provide the first detailed characterization of the kinetics, voltage- and Ca2+ dependence and ion selectivity of TMEM16E-mediated ion currents. Compared to previous studies which failed in this regard [10, 34, 35], a combination of factors may have contributed, among which the TMEM16E protein isoform, the expression vector, the time window of optimal expression and, importantly, the exploration of a wider range of positive membrane potentials.
Ionic currents mediated by TMEM16E were strikingly similar to those observed for TMEM16F [32, 41], the closest paralogue within the mammalian TMEM16 protein family. They were strongly outward rectifying, activating at highly depolarized membrane potentials, showed slow kinetics of activation and deactivation and were poorly ion selective. For both proteins, ionic currents displayed half-maximal activation at cytosolic [Ca2+] in the low micromolar range, although the Ca2+ dependence appears slightly less steep for TMEM16E . Furthermore, contrarily to TMEM16F , TMEM16E showed low basal ion transport activity at highly depolarized potentials in the absence of cytosolic Ca2+. It is also noteworthy that TMEM16E-mediated currents were observed immediately after the establishment of the whole-cell configuration (even at low [Ca2+]), differently to TMEM16F-dependent currents, which consistently presented an activation delay of several minutes [12, 18, 32, 33, 41], indicative of negative regulation by a putative cytosolic factor in heterologous expression systems.
Importantly, we show here that, alike TMEM16F [32, 41], also TMEM16E has phospholipid scrambling activity in transiently transfected HEK293 cells. The crystal structure of the fungal nhTMEM16 lipid scramblase shows a lateral hydrophilic furrow facing the lipid bilayer, which appears to accommodate the hydrophilic head groups of phospholipids during their translocation across the membrane . This hydrophilic furrow is thought to provide also an unspecific pathway for the ion transport observed in TMEM16F, consistent with its highly non-selective nature . Similarly, the two fungal homologs nhTMEM16 and afTMEM16 show both lipid and non-selective ion transport activity [19, 21]. Based on similarities to TMEM16F currents, we propose the same origin for TMEM16E-mediated currents. Considering furthermore their exclusive activation at highly depolarized membrane potentials, which are unlikely to be experienced by non-excitable cells, one may conclude that ion transport is not among the physiological functions of TMEM16E. It constitutes, nevertheless, a reliable real-time readout of TMEM16E activity, for example in studies of its Ca2+ dependence and structure–function relationships.
Our data collectively support the idea of TMEM16E working as a Ca2+-activated phospholipid scramblase. This finding is an important step towards the identification of its physiological role, but detailed information about its subcellular localization in bone and skeletal muscle cells is now absolutely essential, in order to understand where and under which physiological circumstances its scrambling activity is required. The ability of TMEM16E to carry out PLS has been anticipated by a recent study in which the TMEM16E region corresponding to the 35-aa-long scrambling domain, identified in TMEM16F , was introduced into the TMEM16A backbone [13, 14]. Our data provide the direct demonstration of PLS mediated by the wild-type TMEM16E protein.
The determination of TMEM16E function will be equally instrumental to clarify its involvement in genetic diseases. Mutations in the human TMEM16E gene have been associated both with the autosomal-dominant skeletal dysplasia GDD and with two different forms of recessively inherited muscular dystrophy, proximal LGMD2L (limb-girdle muscular dystrophy-2 l; OMIM: 611307, [15, 20] and Miyoshi myopathy, OMIM: 613319, ), consistent with the highest TMEM16E expression found in bone and skeletal muscle [23, 36, 37]. To date, more than 70 different TMEM16E mutations have been reported in muscular dystrophy patients  and are believed to cause a loss-of-function phenotype, also since some of them are non-sense mutations leading to a truncated protein. Contrarily, based on the type of inheritance and the absence of overlap with muscular dystrophies, GDD-associated mutations may cause a gain of function. The data presented here provide the first direct evidence in favor of this hypothesis: the TMEM16E mutant protein carrying the T513I substitution , while preserving the subcellular localization of the wild-type protein, both mediated PLS that was no longer dependent on elevated cytosolic Ca2+ levels and carried large outward currents even at extremely low intracellular Ca2+ concentrations. These data strongly suggest that constitutive TMEM16E scrambling activity at basal cytosolic Ca2+ levels may lead to the pathological consequences observed in the bone tissue of GDD patients.
Despite poor sequence homology among TMEM16 family members in the extracellular loop connecting transmembrane domain 3 and 4, threonine 513 is conserved in the PM-localized Ca2+-activated Cl− channels TMEM16A and TMEM16B. Substitution of the corresponding position in TMEM16B caused partial intracellular retention, but no major changes in the functional properties and Ca2+ dependence of the mutant protein. This result indicates that, during evolution, the second extracellular loop harboring the T513 residue and two further residues affected in GDD (S500F and G518E; [17, 29] has adopted differential roles in Cl− channel members within the TMEM16 family compared to phospholipid scramblases.
In summary, we exploited the partial plasma membrane localization of heterologously overexpressed TMEM16E (1) to directly demonstrate phospholipid scrambling and non-selective ion transport activity of this elusive member of the TMEM16 family and (2) to identify a gain-of-function phenotype for the T513I substitution related to the autosomal-dominant skeletal dysplasia GDD. These results pave the way for the identification of its physiological role and the functional characterization of further TMEM16E mutants related to GDD and muscular dystrophies.
Materials and methods
DNA constructs, cell cultures and transfection
The cDNA clone of human TMEM16E, isolated from the Saos-2 osteosarcoma cell line (kindly provided by Dr. Galietta, Istituto Giannina Gaslini, Genoa, Italy; ; alternative splicing isoform of exon 4 encoding a protein of 898 aa; XP_005252878.2) was subcloned into the vector pFROG  for heterologous expression (referred to as TMEM16E898) and an enhanced green fluorescent protein (EGFP) tag was attached to the TMEM16E C-terminus (referred to as TMEM16E898-EGFP). Full-length human TMEM16E (913 aa, UniProt: Q75V66; , subcloned into the pCR8/GW/TOPO vector, was kindly provided by Dr. Tobiumi (Hiroshima University, Japan) and carried an EGFP tag at its C-terminus as well (; referred to as TMEM16E913-EGFP).
The retinal isoform of mouse TMEM16B [4, 7, 8, 27], subcloned into pCMV-Sport6 (ImaGenes GmbH; NP_705817.1), was used and an EGFP tag was attached to its C-terminus (referred to as TMEM16B-EGFP). Mouse TMEM16F carrying an EGFP tag, after subcloning into the expression vector pEGFP-N1, was kindly provided by Dr. L. Jan (Howard Hughes Medical Institute, San Francisco, USA; ; referred to as TMEM16F-EGFP).
PCR-based mutagenesis using the QuikChange XL site-directed mutagenesis kit (Agilent Technologies Italia) was performed to introduce the amino acid substitutions into TMEM16E898-EGFP and TMEM16B-EGFP and confirmed by subsequent DNA sequencing of the coding region.
Transient transfection of human embryonic kidney HEK293T and chinese hamster ovary (CHO) cells was done using Effectene reagent (Qiagen, Milan) and 400 ng of plasmid DNA. For expression of TMEM16E898, cells were co-transfected with 50 ng pcDNA3.1-E2GFP/DsRed plasmid DNA  for fluorescence identification of transfected cells.
Confocal fluorescence microscopy
Transiently transfected HEK293 cells were seeded in glass-bottom petri dishes (custom-made or purchased from IBL Baustoff + Labor GmbH, Austria). Live cell imaging was performed using a Leica TCS-SL confocal laser scanning microscope equipped with 40× or 63× oil immersion objectives (numerical aperture 1.25 and 1.45, respectively). Final images are the average of 4–12 acquisitions. No filtering was applied.
Endoplasmic reticulum was stained using CellLight ER-RFP BacMam 2.0 (Thermo Fisher Scientific), applied to the cell dish 36 h before visualization. The PM marker FM4-64 (Thermo Fisher Scientific) was added at a final concentration of 10 μM in cold solution, and cells were imaged immediately.
Current recordings were performed in the whole-cell patch-clamp configuration between 48 and 96 h after transfection. Patch pipettes were made of borosilicate glass (Hilgenberg, Malsfeld, Germany or Harvard Bioscience) and had resistances of 3–5 MΩ. Currents were recorded with Axopatch 200 or Axopatch 200B amplifiers (Molecular Devices, Sunnyvale, USA) controlled by the custom acquisition program GePulse (by Dr. Michael Pusch, Institute of Biophysics, CNR, Genoa, Italy; freely available at http://users.ge.ibf.cnr.it/pusch/programs-mik.html). Experiments were performed at room temperature (20–24 °C). In experiments requiring solution exchange, the bath was grounded via a 1 M NaCl–agar salt bridge connected to a Ag/AgCl reference electrode. Applied voltages were not corrected for liquid junction potentials.
The extracellular solution contained (in mM): 140 NaCl, 5 K-gluconate, 2 CaSO4, 2 MgSO4, 10 HEPES, pH 7.4. 10–30 mM glucose was added to reduce volume-regulated chloride currents. In selectivity experiments, the NaCl concentration was reduced to 10 mM and osmolarity was adjusted adding sucrose or replacing NaCl by Na-gluconate. Additionally, 140 NaCl was replaced by equimolar NMDG-Cl. Intracellular solutions contained (in mM): 130 CsCl, 10 HEPES, 10 HEDTA, pH 7.2, and various amounts of CaCl2, as calculated with the program WinMAXC , to obtain calculated free Ca2+ concentrations in the range between 1 and 240 μM. The zero Ca2+ solution contained 130 CsCl, 10 HEPES, 2 EGTA, 2 MgCl2, pH 7.3, resulting in an estimated free [Ca2+] in the low picomolar range. If not otherwise specified, the standard intracellular solution contained 3 µM free Ca2+ (with 3.209 mM CaCl2 added). All chemicals were purchased from Sigma-Aldrich-Merck (Milano, Italy).
The standard IV stimulation protocol consisted of voltage steps of 300 ms duration ranging from − 100 to + 180 mV or to + 140 mV (with 20-mV increments), followed by a 175-ms tail pulse to − 80 mV, from a holding potential of 0 mV. Current amplitudes were evaluated at the end of the test pulse, between 280 and 300 ms. Other stimulation protocols are given in the figure legends.
Current activation was evaluated from the first potential step at which time-dependent currents were observed (V threshold). Instantaneous current amplitudes, measured in the time interval between 5 and 10 ms after the onset of the test pulse, were subtracted from the current amplitudes determined at the end of the test pulse (in the time interval between 275 and 300 ms). V threshold was defined as the first potential step at which the difference current amplitude exceeded the noise level (standard deviation calculated at the end of the test pulse) of the current traces by more than two times.
To estimate reversal potentials (V rev), cells were subjected to an activating 200-ms prepulse to + 140 mV, followed by hyperpolarizing steps with 10-mV increments. Tail currents were fitted to single-exponential functions to extrapolate the tail current value at each voltage step. Tail current values were plotted as a function of the applied membrane potential, and V rev was estimated from a linear fit in a ± 40-mV interval around V rev.
Data analysis and figures were made using Ana (freely available at http://users.ge.ibf.cnr.it/pusch/programs-mik.htm) and Igor Pro software (Wavemetrics, Lake Oswego, OR, USA). For the sake of clarity, the capacitative transients of some current traces were trimmed in the figures.
Phospholipid scrambling assay
Transiently transfected HEK293 cells were seeded in glass-bottom petri dishes and tested for scramblase activity after 48–72 h (TMEM16E898-EGFP, TMEM16F-EGFP) or 36–48 h (TMEM16E 898 T498I -EGFP). Cells were washed in a buffer solution (140 mM NaCl, 2.5 or 5 mM CaCl2, 10 mM HEPES, pH 7.4) and incubated with Cy3-conjugated annexin-V (Enzo Life Sciences), at a dilution of 1:100–200, in the absence or presence of the Ca2+-ionophore A23187 (5–10 μM; Sigma-Aldrich-Merck) in cold solution for 5 min. Ionophore solution was prepared freshly from a 1-mM stock solution (in DMSO) stored at − 20 °C.
In live cell experiments, fresh buffer solution was added to the petri dish after the incubation period. Alternatively, cells were washed with buffer solution and fixed for 5 min at room temperature by adding formalin solution (10%). After further washing, cells were observed by fluorescence microscopy using a Leica TCS-SL confocal microscope (see “Confocal fluorescence microscopy”).
Ca2+-dependent activation of scrambling activity was further assessed by measuring annexin-V binding during patch-clamp experiments in HEK293 expressing TMEM16E898-EGFP and in non-transfected control cells. Cells were bathed in standard extracellular solution (in mM: 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, pH 7.4) supplemented with Alexa Fluor 555-conjugated annexin-V (Life Technologies Italia), and scramblase activity was stimulated using standard intracellular solution containing 3 μM free Ca2+. To avoid photobleaching, fresh annexin-V was added after cell selection. Alexa Fluor 555 was excited at 555 nm using a polychromatic light selector equipped with a Xenon lamp (Polychrome V, Till Photonics) and fluorescence signals were acquired using an Imago CCD camera (TILL Photonics) mounted on a Zeiss Axiovert 200 inverted microscope equipped with a 100× oil objective (1.3 numerical aperture). After the whole-cell configuration was achieved, time-lapse imaging was performed at 1-min intervals synchronously with voltage-clamp recordings (IV protocols in 5-min intervals).
TILLvisION imaging software (TILL Photonics) was used for data acquisition, while ImageJ (NIH, Bethesda, MD, ) and Igor Pro for data analysis. Data are shown as the integrated fluorescence intensity of a region of interest that included the whole cell, normalized to the average of the maximal fluorescence change observed in transfected HEK cells within a 25-min acquisition period. The baseline fluorescence intensity measured at the beginning of the recording period was subtracted for each cell.
Data are presented as mean ± sem, with n indicating the number of cells. Normality of the data was assessed using the Shapiro–Wilk test. Statistical significance was determined using paired t test or Mann–Whitney U test. P values < 0.05 were considered significant.
We sincerely thank Dr. Kei Tobiumi (Hiroshima University), Dr. Luis Galietta (Istituto Giannina Gaslini, Genova) and Dr. Lily Jan (University of California, San Francisco) for the generous gift of DNA constructs. Special thanks to Francesca Quartino for technical assistance and to all IBF colleagues for sharing instrumentation and for insightful scientific discussions. This study was supported by Grants from Telethon (Exploratory Project GEP15078) and Fondazione Compagnia di San Paolo, Torino (2013.0922) to AB. The authors declare no competing financial interests.
AB and JSS conceived the project, AB, EDZ, AG and JSS designed the experiments, AB, EDZ, AG performed experiments and analyzed data, AB and JSS wrote the manuscript, EDZ and AG reviewed the manuscript.
Compliance with ethical standards
Conflict of interest
None of the authors have any competing interests.
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