Elvira Sondo

TMEM16A, A Membrane Protein Associated with Calcium-Dependent Chloride Channel Activity

Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.

Thiocyanate Transport in Resting and IL-4-Stimulated Human Bronchial Epithelial Cells: Role of Pendrin and Anion Channels

SCN− (thiocyanate) is an important physiological anion involved in innate defense of mucosal surfaces. SCN− is oxidized by H2O2, a reaction catalyzed by lactoperoxidase, to produce OSCN− (hypothiocyanite), a molecule with antimicrobial activity. Given the importance of the availability of SCN− in the airway surface fluid, we studied transepithelial SCN− transport in the human bronchial epithelium. We found evidence for at least three mechanisms for basolateral to apical SCN− flux. cAMP and Ca2+ regulatory pathways controlled SCN− transport through cystic fibrosis transmembrane conductance regulator and Ca2+-activated Cl− channels, respectively, the latter mechanism being significantly increased by treatment with IL-4. Stimulation with IL-4 also induced the strong up-regulation of an electroneutral SCN−/Cl− exchange. Global gene expression analysis with microarrays and functional studies indicated pendrin (SLC26A4) as the protein responsible for this SCN− transport. Measurements of H2O2 production at the apical surface of bronchial cells indicated that the extent of SCN− transport is important to modulate the conversion of this oxidant molecule by the lactoperoxidase system. Our studies indicate that the human bronchial epithelium expresses various SCN− transport mechanisms under resting and stimulated conditions. Defects in SCN− transport in the airways may be responsible for susceptibility to infections and/or decreased ability to scavenge oxidants.

Influence of cell background on pharmacological rescue of mutant CFTR

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs the maturation and gating of the CFTR protein. Such defects may be corrected in vitro by pharmacological modulators named as correctors and potentiators, respectively. We have evaluated a panel of correctors and potentiators derived from various sources to assess potency, efficacy, and mechanism of action. For this purpose, we have used functional and biochemical assays on two different cell expression systems, Fischer rat thyroid (FRT) and A549 cells. The order of potency and efficacy of potentiators was similar in the two cell types considered, with phenylglycine PG-01 and isoxazole UCCF-152 being the most potent and least potent, respectively. Most potentiators were also effective on two mutations, G551D and G1349D, that cause a purely gating defect. In contrast, corrector effect was strongly affected by cell background, with the extreme case of many compounds working in one cell type only. Our findings are in favor of a direct action of potentiators on CFTR, possibly at a common binding site. In contrast, most correctors seem to work indirectly with various mechanisms of action. Combinations of correctors acting at different levels may lead to additive F508del-CFTR rescue.

The anoctamin family: TMEM16A and TMEM16B as calcium‐activated chloride channels

The Ca2+‐activated Cl− channels (CaCCs) are involved in a variety of physiological functions, such as transepithelial anion transport, smooth muscle contraction and olfaction. Recently, the question of the molecular identity of CaCCs has apparently been resolved with the identification of TMEM16A protein (also known as anoctamin‐1). Expression of TMEM16A is associated with the appearance of Ca2+‐ and voltage‐dependent Cl− currents with properties similar to those of native CaCCs. The putative structure of TMEM16A consists of eight transmembrane domains, with both the amino‐ and the carboxy‐terminus protruding in the cytosol. TMEM16A is also characterized by the existence of different protein variants generated by alternative splicing. A close paralogue of TMEM16A, TMEM16B (anoctamin‐2), is also associated with CaCC activity, although with different properties. The TMEM16B‐dependent channels require higher intracellular Ca2+ concentrations and have faster activation and deactivation kinetics. Expression of other anoctamins is devoid of detectable channel activity. These proteins, such as TMEM16F (anoctamin‐6), may have different functions.

Dual Activity of Aminoarylthiazoles on the Trafficking and Gating Defects of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Caused by Cystic Fibrosis Mutations

A large fraction of mutations causing cystic fibrosis impair the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by causing reduced channel activity (gating defect) and/or impaired exit from the endoplasmic reticulum (trafficking defect). Such defects need to be treated with separate pharmacological compounds termed potentiators and correctors, respectively. Here, we report the characterization of aminoarylthiazoles (AATs) as compounds having dual activity. Cells expressing mutant CFTR were studied with functional assays (fluorescence-based halide transport and short circuit current measurements) to assess the effect of acute and chronic treatment with compounds. We found that AATs are effective on F508del, the most frequent cystic fibrosis mutation, which is associated with both a gating and a trafficking defect. AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Evaluation of a panel of AAT analogs identified EN277I as the most effective compound. Incubation of cells expressing mutant CFTR with EN277I caused a strong stimulation of channel activity as demonstrated by single channel recordings. Compounds with dual activity such as AATs may be useful for the development of effective drugs for the treatment of cystic fibrosis.

Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies.

Ion channel and lipid scramblase activity associated with expression of TMEM16F/ANO6 isoforms.

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.

TMEM16A-TMEM16B chimaeras to investigate the structure-function relationship of calcium-activated chloride channels.

TMEM16A and TMEM16B proteins are CaCCs (Ca2+-activated Cl- channels) with eight putative transmembrane segments. As shown previously, expression of TMEM16B generates CaCCs characterized by a 10-fold lower Ca2+ affinity and by faster activation and deactivation kinetics with respect to TMEM16A. To investigate the basis of the different properties, we generated chimaeric proteins in which different domains of the TMEM16A protein were replaced by the equivalent domains of TMEM16B. Replacement of the N-terminus, TMD (transmembrane domain) 1-2, the first intracellular loop and TMD3-4 did not change the channels properties. Instead, replacement of intracellular loop 3 decreased the apparent Ca2+ affinity by nearly 8-fold with respect to wild-type TMEM16A. In contrast, the membrane currents derived from chimaeras containing TMD7-8 or the C-terminus of TMEM16B showed higher activation and deactivation rates without a change in Ca2+ sensitivity. Significantly accelerated kinetics were also found when the entire C-terminus of the TMEM16A protein (77 amino acid residues) was deleted. Our findings indicate that the third intracellular loop of TMEM16A and TMEM16B is the site involved in Ca2+-sensitivity, whereas the C-terminal part, including TMD7-8, affect the rate of transition between the open and the closed state.

A minimal isoform of the TMEM16A protein associated with chloride channel activity

TMEM16A protein, also known as anoctamin-1, has been recently identified as an essential component of Ca2+-activated Cl− channels. We previously reported the existence of different TMEM16A isoforms generated by alternative splicing. In the present study, we have determined the functional properties of a minimal TMEM16A protein. This isoform, called TMEM16A(0), has a significantly shortened amino-terminus and lacks three alternative segments localized in the intracellular regions of the protein (total length: 840 amino acids). TMEM16A(0) expression is associated with Ca2+-activated Cl− channel activity as measured by three different functional assays based on the halide-sensitive yellow fluorescent protein, short-circuit current recordings, and patch-clamp technique. However, compared to a longer isoform, TMEM16(abc) (total length: 982 amino acids), TMEM16A(0) completely lacks voltage-dependent activation. Furthermore, TMEM16A(0) and TMEM16A(abc) have similar but not identical responses to extracellular anion replacement, thus suggesting a difference in ion selectivity and conductance. Our results indicate that TMEM16A(0) has the basic domains required for anion transport and Ca2+-sensitivity. However, the absence of alternative segments, which are present in more complex isoforms of TMEM16A, modifies the channel gating and ion transport ability.

RESCUE OF THE MUTANT CFTR CHLORIDE CHANNEL BY PHARMACOLOGICAL CORRECTORS AND LOW TEMPERATURE ANALYZED BY GENE EXPRESSION PROFILING

The F508del mutation, the most frequent in cystic fibrosis (CF), impairs the maturation of the CFTR chloride channel. The F508del defect can be partially overcome at low temperature (27°C) or with pharmacological correctors. However, the efficacy of correctors on the mutant protein appears to be dependent on the cell expression system. We have used a bronchial epithelial cell line, CFBE41o-, to determine the efficacy of various known treatments and to discover new correctors. Compared with other cell types, CFBE41o- shows the largest response to low temperature and the lowest one to correctors such as corr-4a and VRT-325. A screening of a small-molecule library identified 9-aminoacridine and ciclopirox, which were significantly more effective than corr-4a and VRT-325. Analysis with microarrays revealed that 9-aminoacridine, ciclopirox, and low temperature, in contrast to corr-4a, cause a profound change in cell transcriptome. These data suggest that 9-aminoacridine and ciclopirox act on F508del-CFTR maturation as proteostasis regulators, a mechanism already proposed for the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). However, we found that 9-aminoacridine, ciclopirox, and SAHA, in contrast to corr-4a, VRT-325, and low temperature, do not increase chloride secretion in primary bronchial epithelial cells from CF patients. These conflicting data appeared to be correlated with different gene expression signatures generated by these treatments in the cell line and in primary bronchial epithelial cells. Our results suggest that F508del-CFTR correctors acting by altering the cell transcriptome may be particularly active in heterologous expression systems but markedly less effective in native epithelial cells.