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Striated muscle contraction is regulated by the translocation of troponin-tropomyosin strands over the thin filament surface. Relaxation relies partly on highly-favorable, conformation-dependent electrostatic contacts between actin and tropomyosin, which position tropomyosin such that it impedes actomyosin associations. Impaired relaxation and hypercontractile properties are hallmarks of various muscle disorders. The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation lies near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here, we investigate M305L actin in vivo, in vitro, and in silico to resolve emergent pathological properties and disease mechanisms. Our data suggest the mutation reduces actin flexibility and distorts the actin-tropomyosin electrostatic energy landscape that, in muscle, result in aberrant contractile inhibition and excessive force. Thus, actin flexibility may be required to establish and maintain interfacial contacts with tropomyosin as well as facilitate its movement over distinct actin surface features and is, therefore, likely necessary for proper regulation of contraction.
The genetic basis of brain tumor development is poorly understood. Here, leukocyte DNA of 21 patients from 15 families with ≥ 2 glioma cases each was analyzed by whole-genome or targeted sequencing. As a result, we identified two families with rare germline variants, p.(A592T) or p.(A817V), in the E-cadherin gene CDH1 that co-segregate with the tumor phenotype, consisting primarily of oligodendrogliomas, WHO grade II/III, IDH-mutant, 1p/19q-codeleted (ODs). Rare CDH1 variants, previously shown to predispose to gastric and breast cancer, were significantly overrepresented in these glioma families (13.3%) versus controls (1.7%). In 68 individuals from 28 gastric cancer families with pathogenic CDH1 germline variants, brain tumors, including a pituitary adenoma, were observed in three cases (4.4%), a significantly higher prevalence than in the general population (0.2%). Furthermore, rare CDH1 variants were identified in tumor DNA of 6/99 (6%) ODs. CDH1 expression was detected in undifferentiated and differentiating oligodendroglial cells isolated from rat brain. Functional studies using CRISPR/Cas9-mediated knock-in or stably transfected cell models demonstrated that the identified CDH1 germline variants affect cell membrane expression, cell migration and aggregation. E-cadherin ectodomain containing variant p.(A592T) had an increased intramolecular flexibility in a molecular dynamics simulation model. E-cadherin harboring intracellular variant p.(A817V) showed reduced β-catenin binding resulting in increased cytosolic and nuclear β-catenin levels reverted by treatment with the MAPK interacting serine/threonine kinase 1 inhibitor CGP 57380. Our data provide evidence for a role of deactivating CDH1 variants in the risk and tumorigenesis of neuroepithelial and epithelial brain tumors, particularly ODs, possibly via WNT/β-catenin signaling.
The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.
