In silico and functional characterisation of an ultra-rare CFTR mutation identifies novel lasso motif interactions regulating channel gating

Characterisation of I37R, a novel mutation in the lasso motif of ABC-transporter CFTR, a chloride channel, was conducted by theratyping using CFTR potentiators which increase channel gating activity and correctors which repair protein trafficking defects. I37R-CFTR function was characterised using intestinal current measurements (ICM) in rectal biopsies, forskolin-induced swelling (FIS) in intestinal organoids and short circuit current measurements (Isc) in organoid-derived monolayers from an individual with I37R/F508del CFTR genotype. We demonstrated that the I37R-CFTR mutation results in a residual function defect amenable to treatment with potentiators and type III, but not to type I, correctors. Molecular dynamics of I37R-CFTR using an extended model of the phosphorylated, ATP-bound human CFTR identified an altered lasso motif conformation which results in an unfavourable strengthening of the interactions between the lasso motif, the regulatory (R) domain and the transmembrane domain two (TMD2). In conclusion, structural and functional characterisation of the I37R-CFTR mutation increases understanding of CFTR channel regulation and provides a potential pathway to access CFTR modulator treatments for individuals with CF caused by ultra-rare CFTR mutations.

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KCNE3 Truncation Mutants Reveal a Bipartite Modulation of KCNQ1 K+ Channels

The Journal of General Physiology ◽

10.1085/jgp.200409114 ◽

2004 ◽

Vol 124 (6) ◽

pp. 759-771 ◽

Cited By ~ 29

Author(s):

Steven D. Gage ◽

William R. Kobertz

Keyword(s):

Amino Acids ◽

Transmembrane Domain ◽

Channel Gating ◽

Terminal Region ◽

Type I ◽

Long Qt ◽

Transmembrane Peptides ◽

K Channels ◽

Voltage Gated ◽

La Luna

The five KCNE genes encode a family of type I transmembrane peptides that assemble with KCNQ1 and other voltage-gated K+ channels, resulting in potassium conducting complexes with varied channel-gating properties. It has been recently proposed that a triplet of amino acids within the transmembrane domain of KCNE1 and KCNE3 confers modulation specificity to the peptide, since swapping of these three residues essentially converts the recipient KCNE into the donor (Melman, Y.F., A. Domenech, S. de la Luna, and T.V. McDonald. 2001. J. Biol. Chem. 276:6439–6444). However, these results are in stark contrast with earlier KCNE1 deletion studies, which demonstrated that a COOH-terminal region, highly conserved between KCNE1 and KCNE3, was responsible for KCNE1 modulation of KCNQ1 (Tapper, A.R., and A.L. George. 2000 J. Gen. Physiol. 116:379–389.). To ascertain whether KCNE3 peptides behave similarly to KCNE1, we examined a panel of NH2- and COOH-terminal KCNE3 truncation mutants to directly determine the regions required for assembly with and modulation of KCNQ1 channels. Truncations lacking the majority of their NH2 terminus, COOH terminus, or mutants harboring both truncations gave rise to KCNQ1 channel complexes with basal activation, a hallmark of KCNE3 modulation. These results demonstrate that the KCNE3 transmembrane domain is sufficient for assembly with and modulation of KCNQ1 channels and suggests a bipartite model for KCNQ1 modulation by KCNE1 and KCNE3 subunits. In this model, the KCNE3 transmembrane domain is active in modulation and overrides the COOH terminus' contribution, whereas the KCNE1 transmembrane domain is passive and reveals COOH-terminal modulation of KCNQ1 channels. We furthermore test the validity of this model by using the active KCNE3 transmembrane domain to functionally rescue a nonconducting, yet assembly and trafficking competent, long QT mutation located in the conserved COOH-terminal region of KCNE1.

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P2Y11, a purinergic receptor acting via cAMP, mediates secretion by pancreatic duct epithelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2001.280.5.g795 ◽

2001 ◽

Vol 280 (5) ◽

pp. G795-G804 ◽

Cited By ~ 39

Author(s):

T. D. Nguyen ◽

S. Meichle ◽

U. S. Kim ◽

T. Wong ◽

M. W. Moody

Keyword(s):

Epithelial Cells ◽

Pancreatic Duct ◽

Purinergic Receptor ◽

Short Circuit ◽

Short Circuit Current ◽

Exocrine Secretion ◽

Type I ◽

Acetoxymethyl Ester ◽

Ussing Chambers ◽

Receptor Blockers

Pancreatic duct epithelial cells (PDEC) mediate the exocrine secretion of fluid and electrolytes. We previously reported that ATP and UTP interact with P2Y2 receptors on nontransformed canine PDEC to increase intracellular free Ca2+ concentration ([Ca2+]i) and stimulate Ca2+-activated Cl− and K+channels. We now report that ATP interacts with additional purinergic receptors to increase cAMP and activate Cl−channels. ATP, 2-methylthio-ATP, and ATP-γ-S stimulated a 4- to 10-fold cAMP increase with EC50 of 10–100 μM. Neither UTP nor adenosine stimulated a cAMP increase, excluding a role for P2Y2 or P1 receptors. Although UTP stimulated an125I− efflux that was fully inhibited by 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), ATP stimulated a partially resistant efflux, suggesting activation of additional Cl−conductances through P2Y2-independent and Ca2+-independent pathways. In Ussing chambers, increased cAMP stimulated a much larger short-circuit current ( I sc) increase from basolaterally permeabilized PDEC monolayers than increased [Ca2+]i. Luminal ATP and UTP and serosal UTP stimulated a small Ca2+-type I sc increase, whereas serosal ATP stimulated a large cAMP-type I scresponse. Serosal ATP effect was inhibited by P2 receptor blockers and unaffected by BAPTA-AM, supporting ATP activation of Cl−conductances through P2 receptors and a Ca2+-independent pathway. RT-PCR confirmed the presence of P2Y11 receptor mRNA, the only P2Y receptor acting via cAMP.

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Characterization of PKA isoforms and kinase-dependent activation of chloride secretion in T84 cells

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.2.c562 ◽

1998 ◽

Vol 275 (2) ◽

pp. C562-C570 ◽

Cited By ~ 20

Author(s):

A. K. Singh ◽

K. Taskén ◽

W. Walker ◽

R. A. Frizzell ◽

S. C. Watkins ◽

...

Keyword(s):

Epithelial Cells ◽

Short Circuit ◽

Short Circuit Current ◽

Chloride Secretion ◽

Immunogold Electron Microscopy ◽

Apical Plasma Membrane ◽

Epithelial Cell Line ◽

Type I ◽

Type Ii ◽

T84 Cells

Chloride exit across the apical membranes of secretory epithelial cells is acutely regulated by the cAMP-mediated second messenger cascade. To better understand the regulation of transepithelial chloride secretion, we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms present in the human colonic epithelial cell line T84. Our results show that both type I and type II PKA are present in T84 cells. Immunoprecipitation of 8-azido-[32P]cAMP-labeled cell lysates revealed that the major regulatory subunits of PKA were RIα and RIIα. In addition, immunogold electron microscopy showed that RIIα labeling was found on membranes of the trans Golgi network and on apical plasma membrane. In contrast, RIα was randomly distributed throughout the cytoplasm, with no discernible membrane association. Northern blot analysis of T84 RNA revealed that Cα was the predominantly expressed catalytic subunit. Short-circuit current measurements were performed in the presence of combinations of site-selective cAMP analog pairs to preferentially activate either PKA type I or PKA type II in intact T84 cell monolayers. Maximal levels of chloride secretion (∼100 μA/cm2) were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase chloride secretion. Thus activation of either type I or type II PKA is able to maximally stimulate chloride secretion in T84 colonic epithelial cells.

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Characterization of mouse alveolar epithelial cell monolayers

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00021.2009 ◽

2009 ◽

Vol 296 (6) ◽

pp. L1051-L1058 ◽

Cited By ~ 49

Author(s):

Lucas DeMaio ◽

Wanru Tseng ◽

Zerlinde Balverde ◽

Juan R. Alvarez ◽

Kwang-Jin Kim ◽

...

Keyword(s):

Epithelial Cell ◽

Transport Properties ◽

Alveolar Epithelial Cell ◽

Short Circuit ◽

Short Circuit Current ◽

Type I ◽

Cell Marker ◽

Laminin 5 ◽

Alveolar Epithelial ◽

Primary Mouse

We investigated the influence of extracellular matrix on transport properties of mouse alveolar epithelial cell (AEC) monolayers (MAECM) and transdifferentiation of isolated mouse alveolar epithelial type II (AT2) cells into an alveolar epithelial type I (AT1) cell-like phenotype. Primary mouse AT2 cells plated on laminin 5-coated polycarbonate filters formed monolayers with transepithelial resistance ( RT) and equivalent short-circuit current ( IEQ) of 1.8 kΩ·cm2 and 5.3 μA/cm2, respectively, after 8 days in culture. Amiloride (10 μM), ouabain (0.1 mM), and pimozide (10 μM) decreased MAECM IEQ to 40%, 10%, and 65% of its initial value, respectively. Sequential addition of pimozide and amiloride, in either order, revealed that their inhibitory effects are additive, suggesting that cyclic nucleotide-gated channels contribute to amiloride-insensitive active ion transport across MAECM. Ussing chamber measurements of unidirectional ion fluxes across MAECM under short-circuit conditions indicated that net absorption of Na+ in the apical-to-basolateral direction is comparable to net ion flux calculated from the observed short-circuit current: 0.38 and 0.33 μeq·cm−2·h−1, respectively. Between days 1 and 9 in culture, AEC demonstrated increased expression of aquaporin-5 protein, an AT1 cell marker, and decreased expression of pro-surfactant protein-C protein, an AT2 cell marker, consistent with transition to an AT1 cell-like phenotype. These results demonstrate that AT1 cell-like MAECM grown on laminin 5-coated polycarbonate filters exhibit active and passive transport properties that likely reflect the properties of intact mouse alveolar epithelium. This mouse in vitro model will enhance the study of AEC derived from mutant strains of mice and help define important structure-function correlations.

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A Precision Medicine Approach to Optimize Modulator Therapy for Rare CFTR Folding Mutants

Journal of Personalized Medicine ◽

10.3390/jpm11070643 ◽

2021 ◽

Vol 11 (7) ◽

pp. 643

Author(s):

Guido Veit ◽

Tony Velkov ◽

Haijin Xu ◽

Nathalie Vadeboncoeur ◽

Lara Bilodeau ◽

...

Keyword(s):

Precision Medicine ◽

Drug Exposure ◽

Financial Burden ◽

Short Circuit ◽

Short Circuit Current ◽

Bronchial Epithelial Cell ◽

Epithelial Cell Line ◽

Type I ◽

Missense Mutations ◽

Combination Drug

Trikafta, a triple-combination drug, consisting of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor) and the gating potentiator VX-770 (ivacaftor) provided unprecedented clinical benefits for patients with the most common cystic fibrosis (CF) mutation, F508del. Trikafta indications were recently expanded to additional 177 mutations in the CF transmembrane conductance regulator (CFTR). To minimize life-long pharmacological and financial burden of drug administration, if possible, we determined the necessary and sufficient modulator combination that can achieve maximal benefit in preclinical setting for selected mutants. To this end, the biochemical and functional rescue of single corrector-responsive rare mutants were investigated in a bronchial epithelial cell line and patient-derived human primary nasal epithelia (HNE), respectively. The plasma membrane density of P67L-, L206W- or S549R-CFTR corrected by VX-661 or other type I correctors was moderately increased by VX-445. Short-circuit current measurements of HNE, however, uncovered that correction comparable to Trikafta was achieved for S549R-CFTR by VX-661 + VX-770 and for P67L- and L206W-CFTR by the VX-661 + VX-445 combination. Thus, introduction of a third modulator may not provide additional benefit for patients with a subset of rare CFTR missense mutations. These results also underscore that HNE, as a precision medicine model, enable the optimization of mutation-specific modulator combinations to maximize their efficacy and minimize life-long drug exposure of CF patients.

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Contribution of CFTR to apical-basolateral fluid transport in cultured human alveolar epithelial type II cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00178.2005 ◽

2006 ◽

Vol 290 (2) ◽

pp. L242-L249 ◽

Cited By ~ 109

Author(s):

Xiaohui Fang ◽

Yuanlin Song ◽

Jan Hirsch ◽

Luis J. V. Galietta ◽

Nicoletta Pedemonte ◽

...

Keyword(s):

Epithelial Cells ◽

Fluid Transport ◽

Cultured Cells ◽

Short Circuit ◽

Short Circuit Current ◽

Airway Epithelial Cells ◽

Potential Contribution ◽

Type I ◽

Type Ii ◽

Alveolar Epithelial

Previous studies in intact lung suggest that CFTR may play a role in cAMP-regulated fluid transport from the distal air spaces of the lung. However, the potential contribution of different epithelial cells (alveolar epithelial type I, type II, or bronchial epithelial cells) to CFTR-regulated fluid transport is unknown. In this study we determined whether the CFTR gene is expressed in human lung alveolar epithelial type II (AT II) cells and whether the CFTR chloride channel contributes to cAMP-regulated fluid transport in cultured human AT II cells. Human AT II cells were isolated and cultured on collagen I-coated Transwell membranes for 120–144 h with an air-liquid interface. The cultured cells retained typical AT II-like features based on morphologic studies. Net basal fluid transport was 0.9 ± 0.1 μl·cm−2·h−1 and increased to 1.35 ± 0.11 μl·cm−2·h−1 (mean ± SE, n = 18, P < 0.05) by stimulation with cAMP agonists. The CFTR inhibitor, CFTRinh-172, inhibited cAMP stimulated but not basal fluid transport. In short-circuit current ( Isc) studies with an apical-to-basolateral transepithelial Cl− gradient, apical application of CFTRinh-172 reversed the forskolin-induced decrease in Isc. Real time RT-PCR demonstrated CFTR transcript expression in human AT II cells at a level similar to that in airway epithelial cells. We conclude that CFTR is expressed in cultured human AT II cells and may contribute to cAMP-regulated apical-basolateral fluid transport.

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Assessment of mineralocorticoid activity in the rabbit colon

AJP Renal Physiology ◽

10.1152/ajprenal.1984.246.4.f437 ◽

1984 ◽

Vol 246 (4) ◽

pp. F437-F446 ◽

Cited By ~ 1

Author(s):

D. Marver

Keyword(s):

Binding Sites ◽

Citrate Synthase ◽

Short Circuit ◽

Short Circuit Current ◽

Distal Colon ◽

Type I ◽

Type Ii ◽

Target Segment ◽

D Values ◽

Mineralocorticoid Activity

Analyses of [3H]corticosteroid binding sites in distal colon indicated high-affinity binding sites or receptors for both [3H]aldosterone (Type I, Kd = 6.5 X 10(-9) M) and [3H]dexamethasone (Type II, Kd = 5.5 X 10(-8) M). The relative affinity of dexamethasone (D) was 1/20 that of aldosterone (A) for Type I sites and the affinity of A for Type II sites was 1/50 that of D at 37 degrees C. Citrate synthase (CS) activity was assayed and found to be reduced in enterocytes harvested from adrenalectomized (ADX) vs. normal colon segments (0.24 vs. 0.44 U/mg protein, P less than 0.025). Aldosterone (10 micrograms/kg body wt) increased CS at 2 h to a level intermediate between normal and ADX animals and thus not significantly different from either group, but was significantly increased over ADX + D values. Transmural potential difference was increased by 10(-8) M A but not by 10(-8) M D. Since both steroids enhanced short-circuit current at this concentration, the dichotomy of the glucocorticoid vs. mineralocorticoid results can be best explained by the pronounced effect of D on resistance (R) across the tissue (R at 4 h + D was 50% that of paired controls). These findings would suggest that the rabbit distal colon is a target segment for both mineralocorticoids and glucocorticoids. Furthermore, as in the kidney, the two steroids may play coordinated but, perhaps in some way, unique roles in the regulation of transport.

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Ca2+-mediated higher-order assembly of b0,+AT-rBAT is a key step for system b0,+ biogenesis and cystinuria

10.1101/2021.05.06.443019 ◽

2021 ◽

Author(s):

Yongchan Lee ◽

Pattama Wiriyasermkul ◽

Satomi Moriyama ◽

Deryck J Mills ◽

Werner Kühlbrandt ◽

...

Keyword(s):

Protein Trafficking ◽

Genetic Disorder ◽

Higher Order ◽

Type I ◽

Cationic Amino Acid ◽

Catalytic Inactivation ◽

Trafficking Defects ◽

Structural Insights ◽

Function Cell ◽

Dibasic Amino Acids

Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, which causes recurrent kidney stones and occasionally severe kidney failure. Mutations of the two responsible proteins, rBAT and b0,+AT, which comprise system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Although recent structural insights into human b0,+AT-rBAT suggested a model for transport-inactivating mutations, the mechanisms by which type I mutations trigger trafficking deficiencies are not well understood. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+-mediated higher-order assembly of system b0,+ is the key to its trafficking on the cell surface. We show that Ca2+ stabilizes the interface between two rBAT molecules to mediate super-dimerization, and this in turn facilitates the N-glycan maturation of system b0,+. A common cystinuria mutant T216M and mutations that disrupt the Ca2+ site in rBAT cause the loss of higher-order assemblies, resulting in protein trafficking deficiency. Mutations at the super-dimer interface reproduce the mis-trafficking phenotype, demonstrating that super-dimerization is essential for cellular function. Cell-based transport assays confirmed the importance of the Ca2+ site and super-dimerization, and additionally suggested which residues are involved in cationic amino acid recognition. Taken together, our results provide the molecular basis of type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and trafficking diseases in general.

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Aldosterone receptor occupancy and sodium transport in the urinary bladder of Bufo marinus

AJP Cell Physiology ◽

10.1152/ajpcell.1978.235.3.c90 ◽

1978 ◽

Vol 235 (3) ◽

pp. C90-C96 ◽

Cited By ~ 28

Author(s):

N. Farman ◽

M. Kusch ◽

I. S. Edelman

Keyword(s):

Urinary Bladder ◽

Short Circuit ◽

High Capacity ◽

Receptor Occupancy ◽

Short Circuit Current ◽

Bufo Marinus ◽

Type I ◽

Maximal Increase ◽

Aldosterone Receptor ◽

The Relationship

The concentration dependence of binding of [3H]aldosterone to cytoplasmic and nuclear receptors was evaluated in urinary bladder epithelial cells of Colombian toads. One class of specific sites (sensitive to displacement by excess aldosterone) was detected in the cytosol. However, two classes of specific nuclear [3H]aldosterone binding sites were evident. In the nucleus, high-affinity (Kd = 2.7 X 10(-9) M), low-capacity (N = 15 X 10(-14) mol/mg DNA) sites (type I) were completely saturated at approximately 4 X 10(-8) M aldosterone, a concentration which gave a maximal increase in short-circuit current (SCC). Occupancy of low-affinity (Kd = 4.6 X 10(-7) M), high-capacity sites (N = 150 X 10(-14) mol/mg DNA) (type II) occurred at higher steroid concentrations and did not correlate with further increase in SCC. Binding parameters of Colombian and Dominican variants of Bufo marinus were compared. In both variants, the SCC increase elicited by aldosterone correlated with accumulation of type I complexes in the nucleus, but the relationship was markedly nonlinear. Various alternatives were considered as the basis for a curvilinear dependence of the increment in Na+ transport on abundance of nuclear type I complexes.

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