10 research outputs found
Low Temperature and Chemical Rescue Affect Molecular Proximity of F508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Epithelial Sodium Channel (ENaC)
An imbalance of chloride and sodium ion transport in several epithelia is a feature of cystic fibrosis (CF), an inherited disease that is a consequence of mutations in the cftr gene. The cftr gene codes for a Cl(-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Some mutations in this gene cause the balance between Cl(-) secretion and Na(+) absorption to be disturbed in the airways; Cl(-) secretion is impaired, whereas Na(+) absorption is elevated. Enhanced Na(+) absorption through the epithelial sodium channel (ENaC) is attributed to the failure of mutated CFTR to restrict ENaC-mediated Na(+) transport. The mechanism of this regulation is controversial. Recently, we have found evidence for a close association of wild type (WT) CFTR and WT ENaC, further underscoring the role of ENaC along with CFTR in the pathophysiology of CF airway disease. In this study, we have examined the association of ENaC subunits with mutated ΔF508-CFTR, the most common mutation in CF. Deletion of phenylalanine at position 508 (ΔF508) prevents proper processing and targeting of CFTR to the plasma membrane. When ΔF508-CFTR and ENaC subunits were co-expressed in HEK293T cells, we found that individual ENaC subunits could be co-immunoprecipitated with ΔF508-CFTR, much like WT CFTR. However, when we evaluated the ΔF508-CFTR and ENaC association using fluorescence resonance energy transfer (FRET), FRET efficiencies were not significantly different from negative controls, suggesting that ΔF508-CFTR and ENaC are not in close proximity to each other under basal conditions. However, with partial correction of ΔF508-CFTR misprocessing by low temperature and chemical rescue, leading to surface expression as assessed by total internal reflection fluorescence (TIRF) microscopy, we observed a positive FRET signal. Our findings suggest that the ΔF508 mutation alters the close association of CFTR and ENaC
Regions in the carboxy terminus of α-bENaC involved in gating and functional effects of actin
Subunit Stoichiometry of a Core Conduction Element in a Cloned Epithelial Amiloride-Sensitive Na+ Channel
The molecular composition of a core conduction element formed by the α-subunit of cloned epithelial Na+ channels (ENaC) was studied in planar lipid bilayers. Two pairs of in vitro translated proteins were employed in combinatorial experiments: 1) wild-type (WT) and an N-terminally truncated αΔN-rENaC that displays accelerated kinetics (τo = 32 ± 13 ms, τc = 42 ± 11 ms), as compared with the WT channel (τc1 = 18 ± 8 ms, τc2 = 252 ± 31 ms, and τo = 157 ± 43 ms); and 2) WT and an amiloride binding mutant, αΔ278–283-rENaC. The channels that formed in a αWT:αΔN mixture fell into two groups: one with τo and τc that corresponded to those exhibited by the αΔN-rENaC alone, and another with a double-exponentially distributed closed time and a single-exponentially distributed open time that corresponded to the αWT-rENaC alone. Five channel subtypes with distinct sensitivities to amiloride were found in a 1αWT:1αΔ278–283 protein mixture. Statistical analyses of the distributions of channel phenotypes observed for either set of the WT:mutant combinations suggest a tetrameric organization of α-subunits as a minimal model for the core conduction element in ENaCs
Long-Read Sequencing Improves the Detection of Structural Variations Impacting Complex Non-Coding Elements of the Genome
The advent of long-read sequencing offers a new assessment method of detecting genomic structural variation (SV) in numerous rare genetic diseases. For autism spectrum disorders (ASD) cases where pathogenic variants fail to be found in the protein-coding genic regions along chromosomes, we proposed a scalable workflow to characterize the risk factor of SVs impacting non-coding elements of the genome. We applied whole-genome sequencing on an Emirati family having three children with ASD using long and short-read sequencing technology. A series of analytical pipelines were established to identify a set of SVs with high sensitivity and specificity. At 15-fold coverage, we observed that long-read sequencing technology (987 variants) detected a significantly higher number of SVs when compared to variants detected using short-read technology (509 variants) (p-value < 1.1020 × 10−57). Further comparison showed 97.9% of long-read sequencing variants were spanning within the 1–100 kb size range (p-value < 9.080 × 10−67) and impacting over 5000 genes. Moreover, long-read variants detected 604 non-coding RNAs (p-value < 9.02 × 10−9), comprising 58% microRNA, 31.9% lncRNA, and 9.1% snoRNA. Even at low coverage, long-read sequencing has shown to be a reliable technology in detecting SVs impacting complex elements of the genome
Single-cell transcriptome identifies molecular subtype of autism spectrum disorder impacted by de novo loss-of-function variants regulating glial cells
Abstract
Background
In recent years, several hundred autism spectrum disorder (ASD) implicated genes have been discovered impacting a wide range of molecular pathways. However, the molecular underpinning of ASD, particularly from the point of view of ‘brain to behaviour’ pathogenic mechanisms, remains largely unknown.
Methods
We undertook a study to investigate patterns of spatiotemporal and cell type expression of ASD-implicated genes by integrating large-scale brain single-cell transcriptomes (> million cells) and de novo loss-of-function (LOF) ASD variants (impacting 852 genes from 40,122 cases).
Results
We identified multiple single-cell clusters from three distinct developmental human brain regions (anterior cingulate cortex, middle temporal gyrus and primary visual cortex) that evidenced high evolutionary constraint through enrichment for brain critical exons and high pLI genes. These clusters also showed significant enrichment with ASD loss-of-function variant genes (p < 5.23 × 10–11) that are transcriptionally highly active in prenatal brain regions (visual cortex and dorsolateral prefrontal cortex). Mapping ASD de novo LOF variant genes into large-scale human and mouse brain single-cell transcriptome analysis demonstrate enrichment of such genes into neuronal subtypes and are also enriched for subtype of non-neuronal glial cell types (astrocyte, p < 6.40 × 10–11, oligodendrocyte, p < 1.31 × 10–09).
Conclusion
Among the ASD genes enriched with pathogenic de novo LOF variants (i.e. KANK1, PLXNB1), a subgroup has restricted transcriptional regulation in non-neuronal cell types that are evolutionarily conserved. This association strongly suggests the involvement of subtype of non-neuronal glial cells in the pathogenesis of ASD and the need to explore other biological pathways for this disorder
The Serine Protease Trypsin Cleaves C Termini of β- and γ-Subunits of Epithelial Na+ Channels
Psalmotoxin-1 Docking to Human Acid-sensing Ion Channel-1*
Acid-sensing ion channel-1 (ASIC-1) is a proton-gated ion channel implicated in nociception and neuronal death during ischemia. Recently the first crystal structure of a chicken ASIC was obtained. Expanding upon this work, homology models of the human ASICs were constructed and evaluated. Energy-minimized structures were tested for validity by in silico docking of the models to psalmotoxin-1, which potently inhibits ASIC-1 and not other members of the family. The data are consistent with prior radioligand binding and functional assays while also explaining the selectivity of PcTX-1 for homomeric hASIC-1a. Binding energy calculations suggest that the toxin and channel create a complex that is more stable than the channel alone. The binding is dominated by the coulombic contributions, which account for why the toxin-channel interaction is not observed at low pH. The computational data were experimentally verified with single channel and whole-cell electrophysiological studies. These validated models should allow for the rational design of specific and potent peptidomimetic compounds that may be useful for the treatment of pain or ischemic stroke