Sphingomyelinase C (SMase) inhibits CFTR chloride channel activity in multiple cell systems, an effect that could exacerbate disease in CF and COPD patients. The mechanism by which sphingomyelin catalysis inhibits CFTR is not known but evidence suggests that it occurs independently of CFTR’s regulatory “R” domain. In this study we utilized the Xenopus oocyte expression system to shed light on how CFTR channel activity is reduced by SMase. We found that the pathway leading to inhibition is not membrane delimited and that inhibited CFTR channels remain at the cell membrane, indicative of a novel silencing mechanism. Consistent with an effect on CFTR gating behavior, we found that altering gating kinetics influenced the sensitivity to inhibition by SMase. Specifically, increasing channel activity by introducing the mutation K1250A or pretreating with the CFTR potentiator VX-770 (Ivacaftor) imparted resistance to inhibition. In primary bronchial epithelial cells, we found that basolateral, but not apical, application of SMase leads to a redistribution of sphingomyelin and a reduction in forskolin- and VX-770-stimulated currents. Taken together, these data suggest that SMase inhibits CFTR channel function by locking channels into a closed state and that endogenous CFTR in HBEs is affected by SMase activity.

Cystic Fibrosis (CF) is the most common life-shortening genetic disease among Caucasians, resulting from mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). While work to understand this protein has resulted in new treatment strategies, it is important to emphasize that CFTR exists within a complex lipid bilayera concept largely overlooked when performing structural and functional studies. In this review we discuss cellular lipid imbalances in CF, mechanisms by which lipids affect membrane protein activity, and the specific impact of detergents and lipids on CFTR function. C ystic Fibrosis (CF) affects more than 70,000 individuals worldwide. Currently, lung failure is the leading cause of death, although many patients suffer from pancreatic insufficiency, wherein digestive enzymes are not delivered from the pancreas to the gastrointestinal tract 1. In 1989, the gene and protein associated with CF were identified and named the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) 2,3. CFTR is a member of the ATP-Binding Cassette Transporter superfamily (ABCC7). However, whereas all other ABC transporters use ATP to power an enzymatic function, CFTR is the only ABC transporter that functions primarily as an ion channel. Specifically, CFTR conducts chloride and bicarbonate. CFTR's domain architecture includes two transmembrane domains (each with six transmembrane helices (TMs)), two nucleotide binding domains, and a regulatory R-region (Fig. 1). Opening of the CFTR channel requires phosphorylation of the R-region by protein kinase A (PKA) and binding and subsequent hydrolysis of ATP at the nucleotide binding domains 4. Interestingly, the entirety of CFTR is sensitive to mutation, with some 2000 genetic variants having been described. However, F508del-CFTR is by far the most common variant, with ã 70% allele frequency in U.S. patients (https://www.cftr2.org/). Even the ultimate effect of these mutations on CFTR is complex, which is why seven different classes of mutations have been described (Table 1) 5,6. For a subset of these mutations, small-molecule therapeutics exist that increase the activity of CFTR. The first FDA-approved drug, VX-770 (Ivacaftor, KALYDECO ®) 7 , is a gating potentiator that helps certain CFTR mutants conduct more chloride upon activation by sub-maximal phosphorylation 8. The second drug, VX-809 (Lumacaftor), is a trafficking corrector that helps certain CFTR mutants reach the cell membrane 9. Lumacaftor is only ever administered in

Purpose Pediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5 , which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABA A R). We are advancing a therapeutic approach for group 3 based on GABA A R modulation using benzodiazepine-derivatives. Methods We performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional ‘druggable’ GABA A R in group 3 cells. Results Analysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABA A R subunits α5, β3 and γ2 and 3. There are ~ 1000 functional α5-GABA A Rs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 10 9 ions/s. Benzodiazepines, designed to prefer α5-GABA A R, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine (‘KRM-II-08’) binds to the α5-GABA A R (0.8 µM EC 50 ) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization. Conclusion GABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABA A R is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis. Electronic supplementary material The online version of this article (10.1007/s11060-019-03115-0) contains supplementary material, which is available to authorized users.

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The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel whose dysfunction causes cystic fibrosis (CF). The loss of CFTR function in pulmonary epithelial cells causes surface dehydration, mucus build‐up, inflammation, and bacterial infections that lead to lung failure. Little has been done to evaluate the effects of lipid perturbation on CFTR activity, despite CFTR residing in the plasma membrane. This work focuses on the acute effects of sphingomyelinase (SMase), a bacterial virulence factor secreted by CF relevant airway bacteria which degrades sphingomyelin into ceramide and phosphocholine, on the electrical circuitry of pulmonary epithelial monolayers. We report that basolateral SMase decreases CFTR‐mediated transepithelial anion secretion in both primary bronchial and tracheal epithelial cells from explant tissue, with current CFTR modulators unable to rescue this effect. Focusing on primary cells, we took a holistic ion homeostasis approach to determine a cause for reduced anion secretion following SMase treatment. Using impedance analysis, we determined that basolateral SMase inhibits apical and basolateral conductance in non‐CF primary cells without affecting paracellular permeability. In CF primary airway cells, correction with clinically relevant CFTR modulators did not prevent SMase‐mediated inhibition of CFTR currents. Furthermore, SMase was found to inhibit only apical conductance in these cells. Future work should determine the mechanism for SMase‐mediated inhibition of CFTR currents, and further explore the clinical relevance of SMase and sphingolipid imbalances.

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