We examined the effects of a dihydropyridine analog, PAK-200, on guinea pig myocardium during experimental ischemia and reperfusion. In isolated ventricular cardiomyocytes, PAK-200 (1 μM) had no ...effect on the basal peak inward and steady-state currents but inhibited the isoprenaline-induced time-independent Cl− current. In the right atria, PAK-200 had no effect on the beating rate and the chronotropic response to isoprenaline. In an ischemia–reperfusion model with coronary-perfused right ventricular tissue, a decrease in contractile force and a rise in tension were observed during a period of 30-min no-flow ischemia. Upon reperfusion, contractile force returned to less than 50% of preischemic values. PAK-200 had no effect on the decline in contractile force during the no-flow ischemia but reduced the rise in resting tension. PAK-200 significantly improved the recovery of contractile force after reperfusion to about 70% of the preischemic value. PAK-200 was also shown to attenuate the decrease in tissue ATP during ischemia. Treatment of ventricular myocytes with an ischemia-mimetic solution resulted in depolarization of the mitochondrial membrane potential and an increase in cytoplasmic and mitochondrial Ca2+ concentrations. PAK-200 significantly delayed these changes. Thus, PAK-200 inhibits the cAMP-activated chloride current in cardiac muscle and may have protective effects against ischemia–reperfusion injury through novel mechanisms.
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 channel's 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.
Ion channels are potentially exploitable as pharmacological targets to treat asthma. This study evaluated the role of KCa3.1 channels, encoded by Kcnn4, in regulating the gene expression of mouse ...airway epithelium and the development of asthma traits. We used the ovalbumin (OVA) challenge as an asthma model in wild type and Kcnn4
mice, performed histological analysis, and measured serum IgE to evaluate asthma traits. We analyzed gene expression of isolated epithelial cells of trachea or bronchi using mRNA sequencing and gene ontology and performed Ussing chamber experiments in mouse trachea to evaluate anion secretion. Gene expression of epithelial cells from mouse airways differed between trachea and bronchi, indicating regional differences in the inflammatory and transepithelial transport properties of proximal and distal airways. We found that Kcnn4 silencing reduced mast cell numbers, mucus, and collagen in the airways, and reduced the amount of epithelial anion secretion in the OVA-challenged animals. Additionally, gene expression was differentially modified in the trachea and bronchi, with Kcnn4 genetic silencing significantly altering the expression of genes involved in the TNF pathway, supporting the potential of KCa3.1 as a therapeutic target for asthma.
Activation of the proteasome pathway is one of the secondary processes of cell damage, which ultimately lead to muscle degeneration and necrosis in Duchenne muscular dystrophy (DMD). In mdx mice, the ...proteasome inhibitor bortezomib up-regulates the membrane expression of members of the dystrophin complex and reduces the inflammatory reaction. However, chronic inhibition of the 26S proteasome may be toxic, as indicated by the systemic side-effects caused by this drug. Therefore, we sought to determine the components of the ubiquitin-proteasome pathway that are specifically activated in human dystrophin-deficient muscles. The analysis of a cohort of patients with genetically determined DMD or Becker muscular dystrophy (BMD) unveiled a selective up-regulation of the ubiquitin ligase tripartite motif-containing protein 32 (TRIM32). The induction of TRIM32 was due to a transcriptional effect and it correlated with disease severity in BMD patients. In contrast, atrogin1 and muscle RING-finger protein-1 (MuRF-1), which are strongly increased in distinct types of muscular atrophy, were not affected by the DMD dystrophic process. Knock-out models showed that TRIM32 is involved in ubiquitination of muscle cytoskeletal proteins as well as of protein inhibitor of activated STAT protein gamma (Piasγ) and N-myc downstream-regulated gene, two inhibitors of satellite cell proliferation and differentiation. Accordingly, we showed that in DMD/BMD muscle tissue, TRIM32 induction was more pronounced in regenerating myofibers rather than in necrotic muscle cells, thus pointing out a role of this protein in the regulation of human myoblast cell fate. This finding highlights TRIM32 as a possible therapeutic target to favor skeletal muscle regeneration in DMD patients.
New Findings
What is the central question of this study?
What is the precise subcellular localization of the epithelial sodium channel (ENaC) in human airway epithelium?
What is the main finding and ...its importance?
ENaC protein has an unexpected localization in the peripheral region of the apical membrane of bronchial epithelial cells, very close to tight junctions. This may be important for the mechanism of Na+ absorption
The epithelial sodium channel (ENaC) has a key role in absorbing fluid across the human airway epithelium. Altered activity of ENaC may perturb the process of mucociliary clearance, thus impairing the innate defence mechanisms against microbial agents. The proteins forming ENaC are present on the apical membrane of the epithelium. However, their precise localization is unknown. In the present study, we used two antibodies recognizing the α and β ENaC subunits. Both antibodies revealed a restricted localization of ENaC in the peripheral region of the apical membrane of cultured bronchial epithelial cells, close to but not overlapping with tight junctions. In contrast, the cystic fibrosis transmembrane conductance regulator chloride channel was more diffusely expressed on the whole apical membrane. Modulation of ENaC activity by aprotinin or elastase resulted in a decrease or increase in the peripheral localization, respectively. Our results suggest that sodium absorption is mainly occurring close to tight junctions where this cation may be rapidly expelled by the Na+/K+ pump present in lateral membranes. This arrangement of channels and pumps may limit Na+ build‐up in other regions of the cells.
Cerebellar hypoplasia and dysplasia encompass a group of clinically and genetically heterogeneous disorders frequently associated with neurodevelopmental impairment. The Neuron Navigator 2 (
NAV2
) ...gene (MIM: 607,026) encodes a member of the Neuron Navigator protein family, widely expressed within the central nervous system (CNS), and particularly abundant in the developing cerebellum. Evidence across different species supports a pivotal function of NAV2 in cytoskeletal dynamics and neurite outgrowth. Specifically, deficiency of
Nav2
in mice leads to cerebellar hypoplasia with abnormal foliation due to impaired axonal outgrowth. However, little is known about the involvement of the
NAV2
gene in human disease phenotypes. In this study, we identified a female affected with neurodevelopmental impairment and a complex brain and cardiac malformations in which clinical exome sequencing led to the identification of
NAV2
biallelic truncating variants. Through protein expression analysis and cell migration assay in patient-derived fibroblasts, we provide evidence linking NAV2 deficiency to cellular migration deficits. In model organisms, the overall CNS histopathology of the
Nav2
hypomorphic mouse revealed developmental anomalies including cerebellar hypoplasia and dysplasia, corpus callosum hypo-dysgenesis, and agenesis of the olfactory bulbs. Lastly, we show that the
NAV2
ortholog in
Drosophila
,
sickie
(
sick
) is widely expressed in the fly brain, and
sick
mutants are mostly lethal with surviving escapers showing neurobehavioral phenotypes. In summary, our results unveil a novel human neurodevelopmental disorder due to genetic loss of
NAV2
, highlighting a critical conserved role of the
NAV2
gene in brain and cerebellar development across species.
F508del, the most frequent mutation causing cystic fibrosis (CF), results in mistrafficking and premature degradation of the CFTR chloride channel. Small molecules named correctors may rescue ...F508del-CFTR and therefore represent promising drugs to target the basic defect in CF. We screened a carefully designed chemical library to find F508del-CFTR correctors. The initial active compound resulting from the primary screening underwent extensive chemical optimization. The final compound, ARN23765, showed an extremely high potency in bronchial epithelial cells from F508del homozygous patients, with an EC
of 38 picomolar, which is more than 5000-fold lower compared to presently available corrector drugs. ARN23765 also showed high efficacy, synergy with other types of correctors, and compatibility with chronic VX-770 potentiator. Besides being a promising drug, particularly suited for drug combinations, ARN23765 represents a high-affinity probe for CFTR structure-function studies.
Abstract Background In CF patients, the defective ion transport causes a simultaneous reduction of fluid, Cl− and HCO3− secretion. We aimed to demonstrate that the resulting altered properties of ...mucus can be recovered using lumacaftor, a CFTR corrector. Methods The micro-rheology of non-CF and CF mucus was analysed using Multiple Particle Tracking. Results The diffusion coefficient of nano-beads imbedded in mucus from CF human bronchial epithelium was lower than in non-CF mucus, and the elastic and viscous moduli were higher. We found that 25% correction of F508del-CFTR mutation with lumacaftor was enough to improve significantly CF mucus properties. Surprisingly, also incubation with amiloride, a compound that reduces fluid absorption but might not change the secretion of HCO3− towards the airway surface fluid, improved CF mucus properties. Conclusion CF mucus properties can be recovered by either improving the hydration of the airways or recovering Cl− and HCO3− secretion across the mutated protein treated with a corrector compound.
Pathogenic variants in PEX-genes can affect peroxisome assembly and function and cause Zellweger spectrum disorders (ZSDs), characterized by variable phenotypes in terms of disease severity, age of ...onset and clinical presentations. So far, defects in at least 15 PEX-genes have been implicated in Mendelian diseases, but in some of the ultra-rare ZSD subtypes genotype-phenotype correlations and disease mechanisms remain elusive. We report five families carrying biallelic variants in PEX13. The identified variants were initially evaluated by using a combination of computational approaches. Immunofluorescence and complementation studies on patient-derived fibroblasts were performed in two patients to investigate the cellular impact of the identified mutations. Three out of five families carried a recurrent p.Arg294Trp non-synonymous variant. Individuals affected with PEX13-related ZSD presented heterogeneous clinical features, including hypotonia, developmental regression, hearing/vision impairment, progressive spasticity and brain leukodystrophy. Computational predictions highlighted the involvement of the Arg294 residue in PEX13 homodimerization, and the analysis of blind docking predicted that the p.Arg294Trp variant alters the formation of dimers, impairing the stability of the PEX13/PEX14 translocation module. Studies on muscle tissues and patient-derived fibroblasts revealed biochemical alterations of mitochondrial function and identified mislocalized mitochondria and a reduced number of peroxisomes with abnormal PEX13 concentration. This study expands the phenotypic and mutational spectrum of PEX13-related ZSDs and also highlight a variety of disease mechanisms contributing to PEX13-related clinical phenotypes, including the emerging contribution of secondary mitochondrial dysfunction to the pathophysiology of ZSDs.
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.
► Two isoforms of the TMEM16A chloride channel were analyzed by functional assays. ► Both isoforms are activated by calcium and transport anions. ► The shorter isoform, TMEM16A(0), is devoid of voltage-dependence. ► Alternative splicing modifies the properties of TMEM16A-associated ion channels.
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