scholarly journals Convective washout reduces the antidiarrheal efficacy of enterocyte surface–targeted antisecretory drugs

2013 ◽  
Vol 141 (2) ◽  
pp. 261-272 ◽  
Author(s):  
Byung-Ju Jin ◽  
Jay R. Thiagarajah ◽  
A.S. Verkman
Keyword(s):  
Three Dimensional ◽  
Fluid Secretion ◽  
Transmembrane Conductance Regulator ◽  
Inhibitor Concentration ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Transmembrane Conductance ◽  
Convection Diffusion ◽  
Cystic Fibrosis Transmembrane ◽  
Dissociation Constant Kd

Secretory diarrheas such as cholera are a major cause of morbidity and mortality in developing countries. We previously introduced the concept of antisecretory therapy for diarrhea using chloride channel inhibitors targeting the cystic fibrosis transmembrane conductance regulator channel pore on the extracellular surface of enterocytes. However, a concern with this strategy is that rapid fluid secretion could cause convective drug washout that would limit the efficacy of extracellularly targeted inhibitors. Here, we developed a convection–diffusion model of washout in an anatomically accurate three-dimensional model of human intestine comprising cylindrical crypts and villi secreting fluid into a central lumen. Input parameters included initial lumen flow and inhibitor concentration, inhibitor dissociation constant (Kd), crypt/villus secretion, and inhibitor diffusion. We modeled both membrane-impermeant and permeable inhibitors. The model predicted greatly reduced inhibitor efficacy for high crypt fluid secretion as occurs in cholera. We conclude that the antisecretory efficacy of an orally administered membrane-impermeant, surface-targeted inhibitor requires both (a) high inhibitor affinity (low nanomolar Kd) to obtain sufficiently high luminal inhibitor concentration (>100-fold Kd), and (b) sustained high luminal inhibitor concentration or slow inhibitor dissociation compared with oral administration frequency. Efficacy of a surface-targeted permeable inhibitor delivered from the blood requires high inhibitor permeability and blood concentration (relative to Kd).

scholarly journals Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore

2011 ◽  
Vol 138 (2) ◽  
pp. 165-178 ◽  
Author(s):  
Wuyang Wang ◽  
Yassine El Hiani ◽  
Paul Linsdell
Keyword(s):  
Cystic Fibrosis ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Transmembrane Conductance Regulator ◽  
Patch Clamp Recording ◽  
Transmembrane Conductance ◽  
Three Dimensional Structure ◽  
Cystic Fibrosis Transmembrane ◽  
Pore Lining ◽  
Cytoplasmic Side

Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl− channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84–T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.

scholarly journals A Rapid Membrane Potential Assay to Monitor CFTR Function and Inhibition

2013 ◽  
Vol 18 (9) ◽  
pp. 1132-1137 ◽  
Author(s):  
Rangan Maitra ◽  
Perumal Sivashanmugam ◽  
Keith Warner
Keyword(s):  
Cystic Fibrosis ◽  
Membrane Potential ◽  
Genetic Disorder ◽  
Fluid Secretion ◽  
Secretory Diarrhea ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Important Regulator ◽  
Cftr Protein ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is an important regulator of ion transport and fluid secretion in humans. Mutations to CFTR cause cystic fibrosis, which is a common recessive genetic disorder in Caucasians. Involvement of CFTR has been noted in other important diseases, such as secretory diarrhea and polycystic kidney disease. The assays to monitor CFTR function that have been described to date either are complicated or require specialized instrumentation and training for execution. In this report, we describe a rapid FlexStation-based membrane potential assay to monitor CFTR function. In this assay, agonist-mediated activation of CFTR results in membrane depolarization that can be monitored using a fluorescent membrane potential probe. Availability of a simple mix-and-read assay to monitor the function of this important protein might accelerate the discovery of CFTR ligands to study a variety of conditions.

scholarly journals Functional analysis of the C-terminal boundary of the second nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator and structural implications

2002 ◽  
Vol 366 (2) ◽  
pp. 541-548 ◽  
Author(s):  
Martina GENTZSCH ◽  
Andrei ALEKSANDROV ◽  
Luba ALEKSANDROV ◽  
John R. RIORDAN
Keyword(s):  
Cystic Fibrosis ◽  
Cellular Localization ◽  
Three Dimensional ◽  
Large Family ◽  
Nucleotide Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Abc Proteins ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) contains two nucleotide-binding domains (NBDs) or ATP-binding cassettes (ABCs) that characterize a large family of membrane transporters. Although the three-dimensional structures of these domains from several ABC proteins have been determined, this is not the case for CFTR, and hence the domains are defined simply on the basis of sequence alignment. The functional C-terminal boundary of NBD1 of CFTR was located by analysis of chloride channel function [Chan, Csanady, Seto-Young, Nairn and Gadsby (2000) J. Gen. Physiol. 116, 163–180]. However, the boundary between the C-terminal end of NBD2 and sequences further downstream in the whole protein, that are important for its cellular localization and endocytotic turnover, has not been defined. We have now done this by assaying the influence of progressive C-terminal truncations on photolabelling of NBD2 by 8-azido-ATP, which reflects hydrolysis, as well as binding, at that domain, and on NBD2-dependent channel gating itself. The boundary defined in this way is between residues 1420 and 1424, which corresponds to the final β-strand in aligned NBDs whose structures have been determined. Utilization of this information should facilitate the generation of monodisperse NBD2 polypeptides for structural analysis, which until now has not been possible. The established boundary includes within NBD2 a hydrophobic patch of four residues (1413–1416) previously shown to be essential for CFTR maturation and stability [Gentzsch and Riordan (2001) J. Biol. Chem. 276, 1291–1298]. This hydrophobic cluster is conserved in most ABC proteins, and on alignment with ones of known structure constitutes the penultimate β-strand of the domain which is likely to participate in essential structure-stabilizing β-sheet formation.

Identification of a region of strong discrimination in the pore of CFTR

2001 ◽  
Vol 281 (4) ◽  
pp. L852-L867 ◽  
Author(s):  
Nael A. McCarty ◽  
Zhi-Ren Zhang
Keyword(s):  
Cystic Fibrosis ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Structural Determinants ◽  
Anion Selectivity ◽  
Relative Conductance ◽  
Pore Domain ◽  
Cystic Fibrosis Transmembrane

The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl− channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl−and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl− and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl− and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.

scholarly journals CFTR structure, stability, function and regulation

2019 ◽  
Vol 400 (10) ◽  
pp. 1359-1370 ◽  
Author(s):  
Xin Meng ◽  
Jack Clews ◽  
Anca D. Ciuta ◽  
Eleanor R. Martin ◽  
Robert C. Ford
Keyword(s):  
Cystic Fibrosis ◽  
Regulatory Region ◽  
Three Dimensional ◽  
Structure Stability ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Experimental Conditions ◽  
Interacting Protein ◽  
European Origin ◽  
Cystic Fibrosis Transmembrane

Abstract Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette family of proteins because it has evolved into a channel. Mutations in CFTR cause cystic fibrosis, the most common genetic disease in people of European origin. The F508del mutation is found in about 90% of patients and here we present data that suggest its main effect is on CFTR stability rather than on the three-dimensional (3D) folded state. A survey of recent cryo-electron microscopy studies was carried out and this highlighted differences in terms of CFTR conformation despite similarities in experimental conditions. We further studied CFTR structure under various phosphorylation states and with the CFTR-interacting protein NHERF1. The coexistence of outward-facing and inward-facing conformations under a range of experimental conditions was suggested from these data. These results are discussed in terms of structural models for channel gating, and favour the model where the mostly disordered regulatory-region of the protein acts as a channel plug.

scholarly journals Hypercapnia modulates cAMP signalling and cystic fibrosis transmembrane conductance regulator-dependent anion and fluid secretion in airway epithelia

2015 ◽  
Vol 594 (6) ◽  
pp. 1643-1661 ◽  
Author(s):  
Mark J. Turner ◽  
Vinciane Saint-Criq ◽  
Waseema Patel ◽  
Salam H. Ibrahim ◽  
Bernard Verdon ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Fluid Secretion ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Airway Epithelia ◽  
Camp Signalling ◽  
Cystic Fibrosis Transmembrane

Ion transport across the jejunum in normal and cystic fibrosis mice

1995 ◽  
Vol 268 (3) ◽  
pp. G505-G513 ◽  
Author(s):  
B. R. Grubb
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Targeted Disruption ◽  
Cl Secretion ◽  
Ion Substitution ◽  
Cystic Fibrosis Transmembrane

Cystic fibrosis (CF) mice created by targeted disruption of the murine cystic fibrosis transmembrane conductance regulator gene lack adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion and exhibit marked intestinal complications secondary to inadequate fluid secretion. The basal short-circuit current (Isc) in the normal murine jejuna [43.2 +/- 5.9 microA.cm-2, n = 10 (mean +/- SE)] exhibits marked spontaneous n = 10 (mean +/- SE)] exhibits marked spontaneous oscillations (amplitude = 47.9 microA.cm-2, n = 18), which were completely absent in the CF jejunum. Treatment of normal jejuna with the neuronal blocker tetrodotoxin completely eliminated the oscillations and decreased the Isc to levels not significantly different from the low basal Isc (5.4 +/- 2.8 microA.cm-2, n = 16) exhibited by CF tissue. Ion substitution studies revealed basal Isc in normal jejuna to be due primarily to Cl- secretion but these tissues appeared to be capable of HCO3- secretion as well. In contrast, CF jejuna spontaneously secreted neither Cl- nor HCO3-, which may indicate that CF jejuna have a defect in the ability to secrete both of these anions. Apical glucose elicited an electrogenic absorption of Na+ of identical magnitude in normal and CF jejuna. Without apical glucose, CF jejuna exhibited a very small Isc response to forskolin (delta 2.2 +/- 0.67 microA.cm-2, n = 10). However, in the presence of apical glucose, forskolin elicited an eightfold greater Isc response in the CF tissue (delta 17.2 +/- 4.8 microA.cm-2, n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)

Sign in / Sign up

Export Citation Format

Share Document