TMEM16A is a plasma membrane protein with voltage- and calcium-dependent chloride channel activity. The role of the various TMEM16A domains in expression and function is poorly known. In a previous ...study, we found that replacing the first ATG of the TMEM16A coding sequence with a nonsense codon (M1X mutation), to force translation from the second ATG localized at position 117, only had minor functional consequences. Therefore, we concluded that this region is dispensable for TMEM16A processing and channel activity. We have now removed the first 116 codons from the TMEM16A coding sequence. Surprisingly, the expression of the resulting mutant, Δ(1–116), resulted in complete loss of activity. We hypothesized that, in the mutant M1X, translation may start at a position before the second ATG, using a non-canonical start codon. Therefore, we placed an HA-epitope at position 89 in the M1X mutant. We found, by western blot analysis, that the HA-epitope can be detected, thus demonstrating that translation starts from an upstream non-ATG codon. We truncated the N-terminus of TMEM16A at different sites while keeping the HA-epitope. We found that stepwise shortening of TMEM16A caused an in parallel stepwise decrease in TMEM16A expression and function. Our results indicate that indeed the N-terminus of TMEM16A is important for its activity. The use of an alternative start codon appears to occur in a naturally-occurring TMEM16A isoform that is particularly expressed in human testis. Future experiments will need to address the role of normal and alternative amino-terminus in TMEM16A structure and function.
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•TMEM16A is a membrane protein with chloride channel activity.•Two ATG codons are present at positions 1 and 117 of the TMEM16A coding sequence.•Deletion of the first ATG, in contrast to removal of the first 116 codons, does not abolish TMEM16A function.•Without the first ATG, TMEM16A translation begins from a non-canonical start codon.
Here, we present a standardized protocol for isolation, maintenance, and polarization of the respiratory epithelial primary cells from patient samples acquired from nasal brushing, polyp specimens, ...or lung explants. This protocol generates a clearly defined polarized layer of epithelial cells on filters, with a good number of ciliated cells and a thin layer of mucus. We detail the steps for samples prepared from patients with cystic fibrosis as well as from subjects without cystic fibrosis.
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•Protocol for isolating and maintaining respiratory epithelial cells from patient samples•Generates polarized cells with ion transport properties on filters•Can be used for electrophysiological measurements using an Ussing system•Applicable for airway surface liquid volume analysis or immunofluorescent staining
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Here, we present a standardized protocol for isolation, maintenance, and polarization of the respiratory epithelial primary cells from patient samples acquired from nasal brushing, polyp specimens, or lung explants. This protocol generates a clearly defined polarized layer of epithelial cells on filters, with a good number of ciliated cells and a thin layer of mucus. We detail the steps for samples prepared from patients with cystic fibrosis as well as from subjects without cystic fibrosis.
The fluid covering the surface of airway epithelia represents a first barrier against pathogens. The chemical and physical properties of the airway surface fluid are controlled by the activity of ion ...channels and transporters. In cystic fibrosis (CF), loss of CFTR chloride channel function causes airway surface dehydration, bacterial infection, and inflammation. We investigated the effects of IL-17A plus TNF-α, 2 cytokines with relevant roles in CF and other chronic lung diseases. Transcriptome analysis revealed a profound change with upregulation of several genes involved in ion transport, antibacterial defense, and neutrophil recruitment. At the functional level, bronchial epithelia treated in vitro with the cytokine combination showed upregulation of ENaC channel, ATP12A proton pump, ADRB2 β-adrenergic receptor, and SLC26A4 anion exchanger. The overall result of IL-17A/TNF-α treatment was hyperviscosity of the airway surface, as demonstrated by fluorescence recovery after photobleaching (FRAP) experiments. Importantly, stimulation with a β-adrenergic agonist switched airway surface to a low-viscosity state in non-CF but not in CF epithelia. Our study suggests that CF lung disease is sustained by a vicious cycle in which epithelia cannot exit from the hyperviscous state, thus perpetuating the proinflammatory airway surface condition.
WOREE syndrome is a rare neurodevelopmental disorder featuring drug-resistant epilepsy and global developmental delay. The disease, caused by biallelic pathogenic variants in the
gene, usually leads ...to severe disability or death within the first years of life. Clinicians have become more confident with the phenotypic picture of WOREE syndrome, allowing earlier clinical diagnosis. We report a boy with a peculiar clinic-radiological pattern supporting the diagnosis of WOREE syndrome.
DNA was extracted from blood samples of the proband and his parents and subjected to Exome Sequencing (ES). Agarose gel electrophoresis, real-time quantitative PCR (Q-PCR), and array-CGH 180K were also performed.
ES detected a pathogenic stop variant (c.790C > T, p.Arg264*) in one allele of
in the proband and his unaffected mother. A 180K array-CGH analysis revealed a 84,828-bp (g.chr16:78,360,803-78,445,630) deletion encompassing exon 6. The Q-PCR product showed that the proband and his father harbored the same deleted fragment, fusing exons 5 and 7 of
.
Genetic testing remains crucial in establishing the definitive diagnosis of WOREE syndrome and allows prenatal interventions/parental counseling. However, our findings suggest that targeted Next Generation Sequencing-based testing may occasionally show technical pitfalls, prompting further genetic investigation in selected cases with high clinical suspicion.
Calcium-activated anion secretion is expected to ameliorate cystic fibrosis, a genetic disease that carries an anion secretory defect in exocrine tissues. Human patients and animal models of the ...disease that present a mild intestinal phenotype have been postulated to bear a compensatory calcium-activated anion secretion in the intestine. TMEM16A is calcium-activated anion channel whose presence in the intestinal epithelium is contradictory. We aim to test the functional expression of TMEM16A using animal models with
and/or
intestinal silencing. Expression of TMEM16A was studied in a wild type and intestinal
knockout mice by mRNA-seq, mass-spectrometry, q-PCR, Western blotting and immunolocalization. Calcium-activated anion secretion was recorded in the ileum and proximal colon of these animals including intestinal
knockout and double mutants with dual
and
intestinal ablation. Mucus homeostasis was studied by immune-analysis of Mucin-2 (Muc2) and survival curves were recorded.
transcript was found in intestine. Nevertheless, protein was barely detected in colon samples. Electrophysiological measurements demonstrated that the intestinal deletion of
did not change calcium-activated anion secretion induced by carbachol or ATP in ileum and proximal colon. Muc2 architecture was not altered by
silencing as was observed when
was deleted from mouse intestine.
silencing neither affected animal survival nor modified the lethality observed in the intestinal
-null mouse. Our results demonstrate that TMEM16A function in the murine intestine is not related to electrogenic calcium-activated anion transport and does not affect mucus homeostasis and survival of animals.
Abstract Background Cell origin of aldosterone-producing adenomas, a major cause of hypertension, is unknown. A less common subtype of these adenomas, composed of cells resembling zona glomerulosa, ...have mutations in genes ATP1A1 and CACNA1D . To understand whether the adenomas originate from zona glomerulosa, we carried out a microarray analysis comparing transcriptomes of zona glomerulosa, zona fasciculata, and tumour in human adrenal tissue, and investigated the functional role of genes upregulated in the zona glomerulosa. Methods Using a microarray analysis (Affymetrix, High Wycombe, UK), we compared transcriptomes of zona glomerulosa, zona fasciculata, and tumour obtained by laser capture microdissection of 14 patients with aldestosterone adenomas and seven with phaeochromocytoma. One of the most zona glomerulosa-selective genes was ANO4 , a member of the anoctamin family. Subcellular localisation was observed by immunofluorescence microscopy of transfected HEK293 cells. Yellow fluorescent protein-based assay was performed to detect ANO4 activity as a calcium-activated chloride channel. H295R cells were transfected by ANO4 to measure aldosterone and CYP11B2 expression. Findings Microarray analysis revealed 28 genes that were at least five times overexpressed in zona glomerulosa compared with zona fasciculata. ANO4 was 19·9 times higher in zona glomerulosa than in zona fasciculata (p=6·6 × 10−24 ). Haemagglutinin-tagged ANO4 was localised to the plasma membrane of transfected HEK293 cells. In response to increased intracellular calcium, ANO4-transfected cells triggered a lower flow of iodide than did other anoctamins. ANO4 overexpression in H295R cells increased aldosterone secretion from mean 0·9 pmol/μg protein (SE 0·2) to 1·1 (0·1), whereas CYP11B2 mRNA expression increased five times. Interpretation We show that ANO4 is one of the most highly expressed genes in zona glomerulosa of the human adrenal gland. When overexpressed in vitro, it increases aldosterone production. Funding British Heart Foundation.
Key points
Eact is a putative pharmacological activator of TMEM16A.
Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous ...TMEM16A expression.
Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca2+‐permeable TRPV4 channel.
In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types.
Intracellular Ca2+ elevation by TRPV4 stimulation leads to CFTR channel activation.
TMEM16A is a Ca2+‐activated Cl− channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short‐circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca2+ imaging and RNA interference, we found that Eact is actually an activator of the Ca2+‐permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4 stimulation induced activation of the CFTR Cl− channel. Our study reveals the existence of separate Ca2+ signalling pathways linked to different Cl− secretory processes.
Key points
Eact is a putative pharmacological activator of TMEM16A.
Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression.
Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca2+‐permeable TRPV4 channel.
In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types.
Intracellular Ca2+ elevation by TRPV4 stimulation leads to CFTR channel activation.
Key points
TMEM16F is a membrane protein with possible dual function as an ion channel and a phospholipid scramblase.
The properties of ion channels associated with TMEM16F and the link between ion ...channel and scramblase activity are a matter of debate.
We studied the properties of four isoforms of TMEM16F generated by alternative splicing.
Upregulation of three TMEM16F isoforms or silencing of endogenous TMEM16F increased and decreased, respectively, both scramblase and channel activities.
Introduction of an activating mutation in TMEM16F sequence caused a marked increase in phosphatidylserine scrambling and in ion transport indicating direct involvement of the protein in both functions.
TMEM16F, also known as ANO6, is a membrane protein that has been associated with phospholipid scramblase and ion channel activity. However, the characteristics of TMEM16F‐dependent channels, particularly the ion selectivity, are a matter of debate. Furthermore, the direct involvement of TMEM16F in phospholipid scrambling has been questioned. We studied the properties of different TMEM16F variants generated by alternative splicing. Using whole‐cell patch‐clamp recordings, we found that V1, V2 and V5 variants generated membrane currents activated by very high (micromolar) intracellular Ca2+ concentrations and positive membrane potentials. These variants showed different degrees of Ca2+ sensitivity and kinetics of activation but similar ion permeability, characterized by a slight selectivity for Cl− over Na+. A fourth variant (V3) showing a unique carboxy‐terminus was devoid of activity, in agreement with its intracellular localization. We also measured scramblase activity using the binding of annexin V to detect phosphatidylserine on the cell surface. V1, V2 and V5 variants were associated with calcium‐dependent phosphatidylserine externalization. Interestingly, introduction of an activating mutation, D409G, produced a marked increase in the apparent Ca2+ sensitivity of TMEM16F‐dependent channels. In parallel, this mutation also enhanced the extent of phosphatidylserine externalization that occurred even under resting conditions. These results support the conclusion that TMEM16F proteins are directly involved in dual activity, as a phospholipid scramblase and as an ion channel.
Key Points
•
Chloride channels are important for proper hydration of the airway surface.
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TMEM16A protein is an important component of calcium‐activated chloride channels.
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Interleukin‐4, a ...cytokine that induces mucous cell metaplasia, also upregulates calcium‐dependent chloride secretion in human bronchial epithelial cells.
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In bronchial epithelial cells treated with interleukin‐4, we found that TMEM16A protein becomes highly expressed in goblet but not in ciliated cells.
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Upregulation of TMEM16A by interleukin‐4 may be important for secretion and proper expansion of mucins.
The TMEM16A protein has a potential role as a Ca2+‐activated Cl− channel (CaCC) in airway epithelia where it may be important in the homeostasis of the airway surface fluid. We investigated the function and expression of TMEM16A in primary human bronchial epithelial cells and in a bronchial cell line (CFBE41o–). Under resting conditions, TMEM16A protein expression was relatively low. However, TMEM16A silencing with short‐interfering RNAs caused a marked inhibition of CaCC activity, thus demonstrating that a low TMEM16A expression is sufficient to support Ca2+‐dependent Cl− transport. Following treatment for 24–72 h with interleukin‐4 (IL‐4), a cytokine that induces mucous cell metaplasia, TMEM16A protein expression was strongly increased in approximately 50% of primary bronchial epithelial cells, with a specific localization in the apical membrane. IL‐4 treatment also increased the percentage of cells expressing MUC5AC, a marker of goblet cells. Interestingly, MUC5AC was detected specifically in cells expressing TMEM16A. In particular, MUC5AC was found in 15 and 60% of TMEM16A‐positive cells when epithelia were treated with IL‐4 for 24 or 72 h, respectively. In contrast, ciliated cells showed expression of the cystic fibrosis transmembrane conductance regulator Cl− channel but not of TMEM16A. Our results indicate that TMEM16A protein is responsible for CaCC activity in airway epithelial cells, particularly in cells treated with IL‐4, and that TMEM16A upregulation by IL‐4 appears as an early event of goblet cell differentiation. These findings suggest that TMEM16A expression is particularly required under conditions of mucus hypersecretion to ensure adequate secretion of electrolytes and water.
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