Fischer Rat Thyroid Cells Research Articles

Personalized CFTR Modulator Therapy for G85E and N1303K Homozygous Patients with Cystic Fibrosis.

CFTR modulator therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has been approved for people with CF and at least one F508del allele in Europe. In the US, the ETI label has been expanded to 177 rare CFTR mutations responsive in Fischer rat thyroid cells, including G85E, but not N1303K. However, knowledge on the effect of ETI on G85E or N1303K CFTR function remains limited. In vitro effects of ETI were measured in primary human nasal epithelial cultures (pHNECs) of a G85E homozygous patient and an N1303K homozygous patient. Effects of ETI therapy in vivo in these patients were assessed using clinical outcomes, including multiple breath washout and lung MRI, and the CFTR biomarkers sweat chloride concentration (SCC), nasal potential difference (NPD) and intestinal current measurement (ICM), before and after initiation of ETI. ETI increased CFTR-mediated chloride transport in G85E/G85E and N1303K/N1303K pHNECs. In the G85E/G85E and the N1303K/N1303K patient, we observed an improvement in lung function, SCC, and CFTR function in the respiratory and rectal epithelium after initiation of ETI. The approach of combining preclinical in vitro testing with subsequent in vivo verification can facilitate access to CFTR modulator therapy and enhance precision medicine for patients carrying rare CFTR mutations.

Sesquiterpene lactones improve secretory diarrhea symptoms by inhibiting intestinal Ca2+-activated Cl– channel activities directly and indirectly

Secretory diarrhea caused by bacteria and viruses is usually accompanied by activation of the cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated Cl– channels (CaCCs) in the intestinal epithelium. Inhibition of CFTR and CaCCs activities significantly reduces fluid losses and intestinal motility in diarrheal diseases. For this reason, CFTR and CaCCs are potential targets of therapeutic drug screening. Here, we reported that the sesquiterpene lactones, alantolactone (AL) and isoalantolactone (iAL), significantly inhibited ATP and Eact-induced short-circuit currents in T84, HT-29 and Fischer rat thyroid (FRT) cells expressing transmembrane protein 16A (TMEM16A) in a concentration-dependent manner. AL and iAL also inhibited the CaCC-mediated short-circuit currents induced by carbachol in the mouse colons. Both compounds inhibited forskolin-induced currents in T84 cells but did not significantly affect mouse colons. In vivo studies indicated that AL and iAL attenuated gastrointestinal motility and decreased watery diarrhea in rotavirus-infected neonatal mice. Preliminary mechanism studies showed that AL and iAL inhibited CaCCs at least partially by inhibiting Ca2+ release and basolateral membrane K+ channels activity. These findings suggest a new pharmacological activity of sesquiterpene lactone compounds that might lead to the development of treatments for rotaviral secretory diarrhea.

592 Ribosome profiling reveals distinct translation termination kinetics and efficiency between Fischer rat thyroid and human bronchial epidermal cells

592 Ribosome profiling reveals distinct translation termination kinetics and efficiency between Fischer rat thyroid and human bronchial epidermal cells

682 Cystic fibrosis transmembrane conductance regulator variants in Fischer rat thyroid cells exhibit distinct bicarbonate to chloride conductance ratios that are affected by forskolin and ivacaftor

682 Cystic fibrosis transmembrane conductance regulator variants in Fischer rat thyroid cells exhibit distinct bicarbonate to chloride conductance ratios that are affected by forskolin and ivacaftor

Role of SNARE Proteins in the Insertion of KCa3.1 in the Plasma Membrane of a Polarized Epithelium.

Targeting proteins to a specific membrane is crucial for proper epithelial cell function. KCa3.1, a calcium-activated, intermediate-conductance potassium channel, is targeted to the basolateral membrane (BLM) in epithelial cells. Surprisingly, the mechanism of KCa3.1 membrane targeting is poorly understood. We previously reported that targeting of KCa3.1 to the BLM of epithelial cells is Myosin-Vc-, Rab1-and Rab8-dependent. Here, we examine the role of the SNARE proteins VAMP3, SNAP-23 and syntaxin 4 (STX-4) in the targeting of KCa3.1 to the BLM of Fischer rat thyroid (FRT) epithelial cells. We carried out immunoblot, siRNA and Ussing chamber experiments on FRT cells, stably expressing KCa3.1-BLAP/Bir-A-KDEL, grown as high-resistance monolayers. siRNA-mediated knockdown of VAMP3 reduced BLM expression of KCa3.1 by 57 ± 5% (p ≤ 0.05, n = 5). Measurements of BLM-localized KCa3.1 currents, in Ussing chambers, demonstrated knockdown of VAMP3 reduced KCa3.1 current by 70 ± 4% (p ≤ 0.05, n = 5). Similarly, siRNA knockdown of SNAP-23 reduced the expression of KCa3.1 at the BLM by 56 ± 7% (p ≤ 0.01, n = 6) and reduced KCa3.1 current by 80 ± 11% (p ≤ 0.05, n = 6). Also, knockdown of STX-4 lowered the BLM expression of KCa3.1 by 54 ± 6% (p ≤ 0.05, n = 5) and reduced KCa3.1 current by 78 ± 11% (p ≤ 0.05, n = 5). Finally, co-immunoprecipitation experiments demonstrated associations between KCa3.1, VAMP3, SNAP-23 and STX-4. These data indicate that VAMP3, SNAP-23 and STX-4 are critical for the targeting KCa3.1 to BLM of polarized epithelial cells.

Alteration of Membrane Cholesterol Content Plays a Key Role in Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Channel Activity.

Altered cholesterol homeostasis in cystic fibrosis patients has been reported, although controversy remains. As a major membrane lipid component, cholesterol modulates the function of multiple ion channels by complicated mechanisms. However, whether cholesterol directly modulates cystic fibrosis transmembrane conductance regulator (CFTR) channel function remains unknown. To answer this question, we determined the effects of changing plasma membrane cholesterol levels on CFTR channel function utilizing polarized fischer rat thyroid (FRT) cells and primary human bronchial epithelial (HBE) cells. Treatment with methyl-β-cyclodextrin (MβCD) significantly reduced total cholesterol content in FRT cells, which significantly decreased forskolin (FSK)-mediated activation of both wildtype (WT-) and P67L-CFTR. This effect was also seen in HBE cells expressing WT-CFTR. Cholesterol modification by cholesterol oxidase and cholesterol esterase also distinctly affected activation of CFTR by FSK. In addition, alteration of cholesterol increased the potency of VX-770, a clinically used potentiator of CFTR, when both WT- and P67L-CFTR channels were activated at low FSK concentrations; this likely reflects the apparent shift in the sensitivity of WT-CFTR to FSK after alteration of membrane cholesterol. These results demonstrate that changes in the plasma membrane cholesterol level significantly modulate CFTR channel function and consequently may affect sensitivity to clinical therapeutics in CF patients.

Extracellular phosphate enhances the function of F508del-CFTR rescued by CFTR correctors

Background: The clinical response to cystic fibrosis transmembrane conductance regulator (CFTR) modulators varies between people with cystic fibrosis (CF) of the same genotype, in part through the action of solute carriers encoded by modifier genes. Here, we investigate whether phosphate transport by SLC34A2 modulates the function of F508del-CFTR after its rescue by CFTR correctors.Methods: With Fischer rat thyroid (FRT) cells heterologously expressing wild-type and F508del-CFTR and fully-differentiated CF and non-CF human airway epithelial cells, we studied SLC34A2 expression and the effects of phosphate on CFTR-mediated transepithelial ion transport. F508del-CFTR was trafficked to the plasma membrane by incubation with different CFTR correctors (alone or in combination) or by low temperature.Results: Quantitative RT-PCR demonstrated that both FRT and primary airway epithelial cells express SLC34A2 mRNA and no differences were found between cells expressing wild-type and F508del-CFTR. For both heterologously expressed and native F508del-CFTR rescued by either VX-809 or C18, the magnitude of CFTR-mediated Cl− currents was dependent on the presence of extracellular phosphate. However, this effect of phosphate was not detected with wild-type and low temperature-rescued F508del-CFTR Cl− currents. Importantly, the modulatory effect of phosphate was observed in native CF airway cells exposed to VX-445, VX-661 and VX-770 (Trikafta) and was dependent on the presence of both sodium and phosphate.Conclusions: Extracellular phosphate modulates the magnitude of CFTR-mediated Cl− currents after F508del-CFTR rescue by clinically-approved CFTR correctors. This effect likely involves electrogenic phosphate transport by SLC34A2. It might contribute to inter-individual variability in the clinical response to CFTR correctors.

A small molecule inhibitor of the chloride channel TMEM16A blocks vascular smooth muscle contraction and lowers blood pressure in spontaneously hypertensive rats

Hypertension is a major cause of cardiovascular morbidity and mortality, despite the availability of antihypertensive drugs with different targets and mechanisms of action. Here, we provide evidence that pharmacological inhibition of TMEM16A (ANO1), a calcium-activated chloride channel expressed in vascular smooth muscle cells, blocks calcium-activated chloride currents and contraction in vascular smooth muscle invitro and decreases blood pressure in spontaneously hypertensive rats. The acylaminocycloalkylthiophene TMinh-23 fully inhibited calcium-activated TMEM16A chloride current with nanomolar potency in Fischer rat thyroid cells expressing TMEM16A, and in primary cultures of rat vascular smooth muscle cells. TMinh-23 reduced vasoconstriction caused by the thromboxane mimetic U46619 in mesenteric resistance arteries of wild-type and spontaneously hypertensive rats, with a greater inhibition in spontaneously hypertensive rats. Blood pressure measurements by tail-cuff and telemetry showed up to a 45-mmHg reduction in systolic blood pressure lasting for four-six hours in spontaneously hypertensive rats after a single dose of TMinh-23. A minimal effect on blood pressure was seen in wild-type rats or mice treated with TMinh-23. Five-day twice daily treatment of spontaneously hypertensive rats with TMinh-23 produced sustained reductions of 20-25 mmHg in daily mean systolic and diastolic blood pressure. TMinh-23 action was reversible, with blood pressure returning to baseline in spontaneously hypertensive rats by three days after treatment discontinuation. Thus, our studies provide validation for TMEM16A as a target for antihypertensive therapy and demonstrate the efficacy of TMinh-23 as an antihypertensive with a novel mechanism of action.

Novel Correctors and Potentiators Enhance Translational Readthrough in CFTR Nonsense Mutations.

Premature-termination codons (PTCs) in CFTR (cystic fibrosis [CF] transmembrane conductance regulator) result in nonfunctional CFTR protein and are the proximate cause of ∼11% of CF-causing alleles, for which no treatments exist. The CFTR corrector lumacaftor and the potentiator ivacaftor improve CFTR function with terminal PTC mutations and enhance the effect of readthrough agents. Novel correctors GLPG2222 (corrector 1 [C1]), GLPG3221 (corrector 2 [C2]), and potentiator GLPG1837 compare favorably with lumacaftor and ivacaftor in vitro. Here, we evaluated the effect of correctors C1a and C2a (derivatives of C1 and C2) and GLPG1837 alone or in combination with the readthrough compound G418 on CFTR function using heterologous Fischer rat thyroid (FRT) cells, the genetically engineered human bronchial epithelial (HBE) 16HBE14o- cell lines, and primary human cells with PTC mutations. In FRT lines pretreated with G418, GLPG1837 elicited dose-dependent increases in CFTR activity that exceeded those from ivacaftor in FRT-W1282X and FRT-R1162X cells. A three-mechanism strategy consisting of G418, GLPG1837, and two correctors (C1a + C2a) yielded the greatest functional improvements in FRT and 16HBE14o- PTC variants, noting that correction and potentiation without readthrough was sufficient to stimulate CFTR activity for W1282X cells. GLPG1837 + C1a + C2a restored substantial function in G542X/F508del HBE cells and restored even more function for W1282X/F508del cells, largely because of the corrector/potentiator effect, with no additional benefit from G418. In G542X/R553X or R1162X/R1162X organoids, enhanced forskolin-induced swelling was observed with G418 + GLPG1837 + C1a + C2a, although GLPG1837 + C1a + C2a alone was sufficient to improve forskolin-induced swelling in W1282X/W1282X organoids. Combination of CFTR correctors, potentiators, and readthrough compounds augments the functional repair of CFTR nonsense mutations, indicating the potential for novel correctors and potentiators to restore function to truncated W1282X CFTR.

2,3′,4,4′,5-Pentachlorobiphenyl Induced Thyrocyte Autophagy by Promoting Calcium Influx via Store-Operated Ca2+ Entry

PCB118, a 2,3',4,4',5-pentachlorobiphenyl, has been shown to destroy thyroidal ultrastructure and induce thyrocyte autophagy. Previously, we reported that PCB118 promoted autophagosome formation in vivo and in vitro, but more details remain to be revealed. To explore the underlying mechanism by which PCB118 regulates thyrocyte autophagy, Fischer rat thyroid cell line-5 (FRTL-5) cells were exposed to different doses of PCB118 at 0, 0.25, 2.5, and 25 nM for 0-48 h. Western blot analysis of autophagy-related proteins P62, BECLIN1, and LC3 demonstrated that PCB118 induced autophagy formation in dose- and time-dependent manner. Moreover, laser scanning confocal microscopy and flow cytometry showed PCB118 treatment led to time- and dose-dependent increase in intracellular calcium concentration ([Ca2+]i). Additionally, PCB118 promoted store-operated Ca2+ entry (SOCE) channel followed by significant increase of ORAI1 and STIM1 protein levels. Onthe other hand, PCB118 induced thyroidal autophagy via class III β-tubulin (TUBB3)/death-associated protein kinase 2(DAPK2)/myosin regulatory light chain (MRLC)/autophagy-related 9A (ATG9A) pathway in FRTL-5 cells. Pretreatment with SOCE inhibitor SKF96365 reduced cytosolic Ca2+, ORAI1, STIM1, and BECLIN1 levels as well as LC3 II/LC3 I ratio, while increased P62 expression. SKF96365 also inhibited TUBB3/DAPK2/MRLC/ATG9A pathway in FRTL-5 cells treated by PCB118. Our results provide evidence that PCB118 may induce thyroidal autophagy through TUBB3-related signaling pathway, and these effects are likely to be regulated by calcium influx via SOCE channel.

Sorting Nexins 1, 2, 3 and 17 Regulate Epithelial Sodium Channel Trafficking

The epithelial sodium channel (ENaC) facilitates sodium absorption of polarized epithelia and therefore, is required for regulation of salt and water homeostasis. ENaC’s apical membrane population is strictly controlled, with loss of this control leading to hyper‐ or hypotensive disorders such as Liddle’s Syndrome, or Pseudohypoaldosteronism type 1, respectively. Retromer and retriever are conserved endosome‐localized protein trafficking complexes that mediate recycling of membrane proteins back to the cell surface either directly, via recycling endosomes or via the trans‐Golgi network. Both retromer and retriever associate with accessory proteins including sorting nexins (SNX) proteins which play a role in the establishment of a tubulation complex leading to the formation of endosomal tubule‐shaped extensions which contain the cargo selected for recycling. We hypothesized that retromer and/or retriever are required for ENaC recycling. Using three epithelial cell lines (Fischer rat thyroid (FRT), mouse cortical collecting duct (mCCD) or human embryonic kidney cells (HEK293), we found that siRNA knockdown of retromer and retriever associated SNX proteins reduced ENaC amiloride‐sensitive short circuit current (Isc‐Amil). SNX1 and SNX2 are reported to be essential for cargo recycling through retromer. Individually, knockdown of SNX1 (n=6) or SNX2 (n=8) reduced Isc‐Amil by 38±9% and 39±8% compared with control FRT epithelia, respectively. Double‐knockdown of those SNXs reduced Isc‐Amil by 47±1% (n=6) compared with control FRT epithelia. SNX3 is known to bind directly with the retromer complex. Knockdown of SNX3 decreased Isc‐Amil by 47±5% FRT (n=9) and 35±4% in mCCD (n=9) epithelia compared with control epithelia. Additionally, it is known that SNX17 mediates cargo retrieval via the retriever complex. Knockdown of SNX17 lowered Isc‐Amil by 56±1% (n=9) in FRT and 35±6% (n=9) in mCCD epithelia compared with control epithelia. With cell surface biotinylation experiments and using FRT cells, knockdown of SNX3 and SNX17 reduced the ENaC cell surface population by 68±25% (n=3) and 77±17% (n=3), respectively, thus, attributing to the reduced Isc‐Amil. Furthermore, co‐immunoprecipitation experiments of αβHAγENaC overexpressed in HEK293 cells, demonstrated a protein‐protein interaction between retriever associated SNX17 and ENaC (n=3) suggesting SNX17 may act as a cargo binding protein between ENaC and the retriever complex. However, there was no evidence for an interaction between SNX3 and ENaC (n=3). Together, our findings suggest that SNX protein complexes play a role in ENaC recycling to the plasma membrane in polarized epithelia. Additionally, these data suggest that both the retromer and retriever recycling complexes may aid in the trafficking of ENaC to the plasma membrane.Support or Funding InformationThis work was supported by a grant from the New Zealand Lottery Health Board. Further support was provided by the School of Biomedical Sciences and the Department of Physiology of the University of Otago.

Alteration of Membrane Cholesterol Content Plays a Key Role in Regulation of CFTR Channel Activity

CFTR has recently been shown to exist in two distinct populations in the plasma membrane of primary human bronchial epithelial (HBE) cells: one population is diffusely distributed and the other is in cholesterol-dependent lipid rafts. Cholesterol homeostasis has been found to be altered in Cystic Fibrosis patients, although some controversy remains. For example, epithelial cells expressing F508del-CFTR exhibit increased cholesterol; however, neutrophils derived from CF patients show reduced cholesterol levels in the plasma membrane. As a major membrane lipid component, cholesterol modulates the function of multiple ion channels by complicated mechanisms, including direct cholesterol-channel protein interactions, indirect changes in physical properties of the membrane, and more. Thus far, whether cholesterol directly modulates CFTR channel function remains unknown. To answer this question, we determined the effects of changing plasma membrane cholesterol levels on CFTR channel function utilizing polarized Fischer Rat Thyroid (FRT) cells and HBE cells. We found that cholesterol depletion with 5 mM methyl-β-cyclodextrin (MβCD) significantly reduced total cholesterol in FRT cells. This cholesterol reduction by MβCD significantly decreased sensitivity of WT- and P67L-CFTR forskolin (FSK)-mediated activation. This is also seen in HBE cells expressing WT-CFTR. Cholesterol modification by cholesterol oxidase and cholesterol esterase also distinctly affected activation of P67L-CFTR by FSK. In addition, alteration of cholesterol increased efficacy of VX-770, a clinically-used potentiator of CFTR, when both WT- and P67L-CFTR channels were activated at low FSK concentrations, but not at high FSK concentrations. This change in efficacy of VX-770-mediated potentiation likely reflects the apparent shift in the sensitivity of WT-CFTR to FSK after alteration of membrane cholesterol. These results demonstrate that changes in the cholesterol level of the plasma membrane significantly modulate CFTR channel function and consequently affect potentiator efficiency in CF patients.

TRPV4 and purinergic receptor signalling pathways are separately linked in airway epithelia to CFTR and TMEM16A chloride channels.

Eact is a putative pharmacological activator of TMEM16A. Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression. Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel. In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types. Intracellular Ca2+ elevation by TRPV4stimulation leads to CFTR channel activation. TMEM16A is a Ca2+ -activated Cl- channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short-circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca2+ imaging and RNA interference, we found that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4stimulation induced activation of the CFTR Cl- channel. Our study reveals the existence of separate Ca2+ signalling pathways linked to different Cl- secretory processes.

Plumbagin Prevents Secretory Diarrhea by Inhibiting CaCC and CFTR Channel Activities.

Secretory diarrhea, which primarily originates through intestinal pathogens and viruses, is a health burden in many regions worldwide. Enterocyte Cl− channels, as the final step in enterotoxin-induced fluid secretion, constitute an attractive class of targets for diarrhea therapy. Chloride channel inhibitors have become a new class of candidates for antisecretion and anti-intestinal motility agents. In the present study, we identified plumbagin as a transmembrane protein 16A (TMEM16A) inhibitor in a cell-based fluorescence-quenching assay, and the IC50 value was ∼12.46 µM. Short-circuit current measurements showed that plumbagin reversibly inhibited the Eact-induced Cl− current on the apical side of TMEM16A-transfected Fischer rat thyroid (FRT) cells with no significant effect on cytoplasmic Ca2+ signaling. Notably, plumbagin also inhibited the activity of intestinal epithelial calcium-activated chloride channel (CaCC) and cystic fibrosis transmembrane conductance regulator (CFTR) in both HT-29 cells and mouse colons, but had no effects on the activity of the Na+-K+ ATPase or K+ channels. In in vivo experiments, the administration of plumbagin reduced both Escherichia coli heat-stable enterotoxin (STa)- and cholera toxin (CT)-induced intestinal fluid secretion. In neonatal mouse models of CT- and rotavirus infection-induced diarrhea, 0.4 µg plumbagin inhibited secretory diarrhea by >40% and 50%, respectively, without affecting intestinal epithelial integrity or the rotaviral infection. In addition, plumbagin exerted inhibitory effects on the vasoactive intestinal peptide (VIP)-, prostaglandin E2 (PGE2)-, and 5-hydroxytryptamine (5-HT)-stimulated Cl− currents. In the evaluations of intestinal motility, plumbagin significantly delayed intestinal motility and inhibited intestinal smooth muscle contractility without an evident impact on contractive frequency. Collectively, our results indicate that plumbagin inhibits both Ca2+- and cAMP-activated Cl− channels, accounting for the mechanisms of plumbagin inhibition of chloride secretion and intestinal motility. Thus, plumbagin can be a lead compound in the treatment of CT-induced, Traveler’s, and rotaviral diarrhea, as well as other types of secretory diarrhea that result from excessive intestinal fluid secretion and increased intestinal peristalsis.

Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma

Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma

Assessing cell-specific effects of genetic variations using tRNA microarrays

BackgroundBy definition, effect of synonymous single-nucleotide variants (SNVs) on protein folding and function are neutral, as they alter the codon and not the encoded amino acid. Recent examples indicate tissue-specific and transfer RNA (tRNA)-dependent effects of some genetic variations arguing against neutrality of synonymous SNVs for protein biogenesis.ResultsWe performed systematic analysis of tRNA abunandance across in various models used in cystic fibrosis (CF) research and drug development, including Fischer rat thyroid (FRT) cells, patient-derived primary human bronchial epithelia (HBE) from lung biopsies, primary human nasal epithelia (HNE) from nasal curettage, intestinal organoids, and airway progenitor-directed differentiation of human induced pluripotent stem cells (iPSCs). These were compared to an immortalized CF bronchial cell model (CFBE41o−) and two widely used laboratory cell lines, HeLa and HEK293. We discovered that specific synonymous SNVs exhibited differential effects which correlated with variable concentrations of cognate tRNAs.ConclusionsOur results highlight ways in which the presence of synonymous SNVs may alter local kinetics of mRNA translation; and thus, impact protein biogenesis and function. This effect is likely to influence results from mechansistic analysis and/or drug screeining efforts, and establishes importance of cereful model system selection based on genetic variation profile.

Human CFTR Channel Function is Regulated by Cholesterol

Cholesterol, a major membrane lipid component, has been known to modulate the function of multiple ion channels by specific lipid-protein interactions, by physical property changes of the membrane, or by modification of regulatory proteins associated with the channels in signaling complexes. Previous studies show that epithelial cells expressing the most common mutant version of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), F508del-CFTR, exhibit increased cholesterol content at the plasma membrane compared to wild type control cells; however, neutrophils derived from CF patients show reduced cholesterol levels in the plasma membrane. But whether cholesterol directly modulates CFTR channel function remains unknown. To answer this question, we combine heterologous expression in oocytes with more physiologically relevant Ussing chamber recordings utilizing polarized Fischer Rat Thyroid (FRT) cells to determine the effects of changing plasma membrane cholesterol levels on CFTR channel function. Here, we report that cholesterol depletion with methyl-β-cyclodextrin (MβCD) or cholesterol oxidase (CO) has no macroscopic effect on the magnitude of CFTR-mediated whole-oocyte currents. However, depletion of cholesterol increased the effect of VX-770, a clinically-used potentiator of CFTR, when channels were activated at high PKA concentrations, but did not change the effect of VX-770 when channels were activated at low PKA concentrations. This change in efficacy of VX-770-mediated potentiation likely reflects the apparent shift in the sensitivity of WT-CFTR to PKA after depletion of membrane cholesterol. In FRT cells, P67L-CFTR also exhibited changes in phosphorylation-dependent activation after depletion of plasma membrane cholesterol. Sensitivity of WT-CFTR to block by GlyH-101 was significantly stronger after plasma membrane cholesterol depletion. These results demonstrate that changes in the cholesterol level of the plasma membrane significantly modulate multiple CFTR channel functions. (Support: CF Fdn. MCCART17G0, NIH T32 GM008602, NIH F31 HL143863-01).

Proteomic Analysis of Zn Depletion/Repletion in the Hormone-Secreting Thyroid Follicular Cell Line FRTL-5.

Zinc deficiency predisposes to a wide spectrum of chronic diseases. The human Zn proteome was predicted to represent about 10% of the total human proteome, reflecting the broad array of metabolic functions in which this micronutrient is known to participate. In the thyroid, Zn was reported to regulate cellular homeostasis, with a yet elusive mechanism. The Fischer Rat Thyroid Cell Line FRTL-5 cell model, derived from a Fischer rat thyroid and displaying a follicular cell phenotype, was used to investigate a possible causal relationship between intracellular Zn levels and thyroid function. A proteomic approach was applied to compare proteins expressed in Zn deficiency, obtained by treating cells with the Zn-specific chelator N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylene-diamine (TPEN), with Zn repleted cells. Quantitative proteomic analysis of whole cell protein extracts was performed using stable isotope dimethyl labelling coupled to nano-ultra performance liquid chromatography-mass spectrometry (UPLC-MS). TPEN treatment led to almost undetectable intracellular Zn, while decreasing thyroglobulin secretion. Subsequent addition of ZnSO4 fully reversed these phenotypes. Comparative proteomic analysis of Zn depleted/repleted cells identified 108 proteins modulated by either treatment. Biological process enrichment analysis identified functions involved in calcium release and the regulation of translation as the most strongly regulated processes in Zn depleted cells.

SLC26A3 inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation.

SLC26A3 (downregulated in adenoma; DRA) is a Cl-/anion exchanger expressed in the luminal membrane of intestinal epithelial cells, where it facilitates electroneutral NaCl absorption. SLC26A3 loss of function in humans or mice causes chloride-losing diarrhea. Here, we identified slc26a3 inhibitors in a screen of 50,000 synthetic small molecules done in Fischer rat thyroid (FRT) cells coexpressing slc26a3 and a genetically encoded halide sensor. Structure-activity relationship studies were done on the most potent inhibitor classes identified in the screen: 4,8-dimethylcoumarins and acetamide-thioimidazoles. The dimethylcoumarin DRAinh-A250 fully and reversibly inhibited slc26a3-mediated Cl- exchange with HCO3-, I-, and thiocyanate (SCN-), with an IC50 of ~0.2 μM. DRAinh-A250 did not inhibit the homologous anion exchangers slc26a4 (pendrin) or slc26a6 (PAT-1), nor did it alter activity of other related proteins or intestinal ion channels. In mice, intraluminal DRAinh-A250 blocked fluid absorption in closed colonic loops but not in jejunal loops, while the NHE3 (SLC9A3) inhibitor tenapanor blocked absorption only in the jejunum. Oral DRAinh-A250 and tenapanor comparably reduced signs of constipation in loperamide-treated mice, with additive effects found on coadministration. DRAinh-A250 was also effective in loperamide-treated cystic fibrosis mice. These studies support a major role of slc26a3 in colonic fluid absorption and suggest the therapeutic utility of SLC26A3 inhibition in constipation.

Residual function of cystic fibrosis mutants predicts response to small molecule CFTR modulators.

Treatment of individuals with cystic fibrosis (CF) has been transformed by small molecule therapies that target select pathogenic variants in the CF transmembrane conductance regulator (CFTR). To expand treatment eligibility, we stably expressed 43 rare missense CFTR variants associated with moderate CF from a single site in the genome of human CF bronchial epithelial (CFBE41o-) cells. The magnitude of drug response was highly correlated with residual CFTR function for the potentiator ivacaftor, the corrector lumacaftor, and ivacaftor-lumacaftor combination therapy. Response of a second set of 16 variants expressed stably in Fischer rat thyroid (FRT) cells showed nearly identical correlations. Subsets of variants were identified that demonstrated statistically significantly higher responses to specific treatments. Furthermore, nearly all variants studied in CFBE cells (40 of 43) and FRT cells (13 of 16) demonstrated greater response to ivacaftor-lumacaftor combination therapy than either modulator alone. Together, these variants represent 87% of individuals in the CFTR2 database with at least 1 missense variant. Thus, our results indicate that most individuals with CF carrying missense variants are (a) likely to respond modestly to currently available modulator therapy, while a small fraction will have pronounced responses, and (b) likely to derive the greatest benefit from combination therapy.