Inhibition of CFTR channels by a peptide toxin of scorpion venom

2004 ◽  
Vol 287 (5) ◽  
pp. C1328-C1341 ◽  
Author(s):  
Matthew D. Fuller ◽  
Zhi-Ren Zhang ◽  
Guiying Cui ◽  
Julia Kubanek ◽  
Nael A. McCarty
Keyword(s):  
Cystic Fibrosis ◽  
Single Channel ◽  
Genetic Disorders ◽  
Scorpion Venom ◽  
Burst Duration ◽  
Open Probability ◽  
Independent Manner ◽  
Peptide Toxin ◽  
Peptide Toxins ◽  
Block Mechanism

Peptide toxins have been valuable probes in efforts to identify amino acid residues that line the permeation pathway of cation-selective channels. However, no peptide toxins have been identified that interact with known anion-selective channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR channels are expressed in epithelial cells and are associated with several genetic disorders, including cystic fibrosis and polycystic kidney disease. Several organic inhibitors have been used to investigate the structure of the Cl− permeation pathway in CFTR. However, investigations of the wider cytoplasmic vestibule have been hindered by the lack of a high-affinity blocker that interacts with residues in this area. In this study we show that venom of the scorpion Leiurus quinquestriatus hebraeus reversibly inhibits CFTR, in a voltage-independent manner, by decreasing single-channel mean burst duration and open probability only when applied to the cytoplasmic surface of phosphorylated channels. Venom was able to decrease burst duration and open probability even when CFTR channels were locked open by treatment with either vanadate or adenosine 5′-(β,γ-imido)triphosphate, and block was strengthened on reduction of extracellular Cl− concentration, suggesting inhibition by a pore-block mechanism. Venom had no effect on ATP-dependent macroscopic opening rate in channels studied by inside-out macropatches. Interestingly, the inhibitory activity was abolished by proteinase treatment. We conclude that a peptide toxin contained in the scorpion venom inhibits CFTR channels by a pore-block mechanism; these experiments provide the first step toward isolation of the active component, which would be highly valuable as a probe for CFTR structure and function.

scholarly journals Mutations within the P-Loop of Kir6.2 Modulate the Intraburst Kinetics of the Atp-Sensitive Potassium Channel

2001 ◽  
Vol 118 (4) ◽  
pp. 341-353 ◽  
Author(s):  
Peter Proks ◽  
Charlotte E. Capener ◽  
Phillippa Jones ◽  
Frances M. Ashcroft
Keyword(s):  
Katp Channel ◽  
Single Channel ◽  
Katp Channels ◽  
Burst Duration ◽  
Open Probability ◽  
Closed Intervals ◽  
Open Time ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Kinetics Of

The ATP-sensitive potassium (KATP) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 KATP channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (τo) and the short closed time (τC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer τo, shorter τC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the KATP channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings.

Mechanism of sodium hyperabsorption in cultured cystic fibrosis nasal epithelium: a patch-clamp study

1994 ◽  
Vol 266 (4) ◽  
pp. C1061-C1068 ◽  
Author(s):  
T. C. Chinet ◽  
J. M. Fullton ◽  
J. R. Yankaskas ◽  
R. C. Boucher ◽  
M. J. Stutts
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Patch Clamp ◽  
Single Channel ◽  
Apical Membrane ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Nasal Epithelial Cells ◽  
Na Permeability

Transepithelial Na+ absorption is increased two to three times in cystic fibrosis (CF) compared with normal (NL) airway epithelia. This increase has been associated with a higher Na+ permeability of the apical membrane of airway epithelial cells. Because Na+ absorption is electrogenic and abolished by amiloride, Na+ channels are thought to dominate the apical membrane Na+ permeability. Three Na+ channel-related mechanisms may explain the increase in apical Na+ permeability in CF cells: increased number of channels, increased single-channel conductance, and increased single-channel open probability. We compared the properties of Na(+)-permeable channels in the apical membrane of confluent preparations of human NL and CF nasal epithelial cells cultured on permeable supports. Na(+)-permeable channels were studied using the patch-clamp technique in the excised inside-out and cell-attached configurations. The same types of Na(+)-permeable channels were recorded in CF and NL cells. In excised patches, nonselective (Na+/K+) cation channels were recorded, and no differences between CF and NL were found in the properties, incidence, single-channel conductance, and single-channel open probability. In cell-attached patches, channels with a higher Na+ vs. K+ selectivity dominated. There was no difference between CF and NL cells in the incidence (18.8 vs. 21.4%, respectively) and conductance (17.2 +/- 2.8 vs. 21.4 +/- 1.5 pS, respectively) of Na(+)-permeable channels. However, the open probability was higher in CF cells compared with NL cells (30.0 +/- 3.4%, n = 6, vs. 15.0 +/- 3.9%, n = 13; P < 0.05). We conclude that, in CF nasal epithelial cells, the increase in Na+ permeability of the apical membrane results from an increase in the open probability of Na(+)-permeable channels in the apical membrane.

scholarly journals Catalyst-like modulation of transition states for CFTR channel opening and closing: New stimulation strategy exploits nonequilibrium gating

2014 ◽  
Vol 143 (2) ◽  
pp. 269-287 ◽  
Author(s):  
László Csanády ◽  
Beáta Töröcsik
Keyword(s):  
Cystic Fibrosis ◽  
Ion Channels ◽  
Single Channel ◽  
Transition States ◽  
Chloride Ion ◽  
Open Probability ◽  
Gating Mechanism ◽  
Channel Opening ◽  
The Stability ◽  
Opening And Closing

Cystic fibrosis transmembrane conductance regulator (CFTR) is the chloride ion channel mutated in cystic fibrosis (CF) patients. It is an ATP-binding cassette protein, and its resulting cyclic nonequilibrium gating mechanism sets it apart from most other ion channels. The most common CF mutation (ΔF508) impairs folding of CFTR but also channel gating, reducing open probability (Po). This gating defect must be addressed to effectively treat CF. Combining single-channel and macroscopic current measurements in inside-out patches, we show here that the two effects of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) on CFTR, pore block and gating stimulation, are independent, suggesting action at distinct sites. Furthermore, detailed kinetic analysis revealed that NPPB potently increases Po, also of ΔF508 CFTR, by affecting the stability of gating transition states. This finding is unexpected, because for most ion channels, which gate at equilibrium, altering transition-state stabilities has no effect on Po; rather, agonists usually stimulate by stabilizing open states. Our results highlight how for CFTR, because of its unique cyclic mechanism, gating transition states determine Po and offer strategic targets for potentiator compounds to achieve maximal efficacy.

Zinc Potentiates Neuronal Nicotinic Receptors by Increasing Burst Duration

2008 ◽  
Vol 99 (2) ◽  
pp. 999-1007 ◽  
Author(s):  
Bernard Hsiao ◽  
Karla B. Mihalak ◽  
Karl L. Magleby ◽  
Charles W. Luetje
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Large Fraction ◽  
Burst Duration ◽  
Dependent Manner ◽  
Neuronal Nicotinic Acetylcholine Receptors ◽  
Open Probability ◽  
Neuronal Nicotinic Receptors ◽  
Channel Current

Micromolar zinc potentiates neuronal nicotinic acetylcholine receptors (nAChRs) in a subtype-dependent manner. Zinc potentiates receptor function even at saturating agonist concentrations, without altering the receptor desensitization rate. Potentiation could occur through an increase in the number of available receptors, an increase in single-channel current amplitude, or an increase in single-channel open probability. To distinguish among these possibilities, we examined rat neuronal nAChRs expressed in Xenopus oocytes. Blockade of a large fraction of ACh activated α4β4 or α4β2 receptors by the open channel blocker hexamethonium failed to change the extent of potentiation by zinc, suggesting that zinc does not change the number of available receptors. The single-channel amplitudes of ACh (1 μM) activated α4β4 receptors in outside-out patches were similar in the absence and the presence of 100 μM zinc (3.0 ± 0.1 and 2.9 ± 0.1 pA, respectively). To determine the effect of zinc on single-channel open probability, we examined α4β4 receptors in cell-attached patches. The open probability at 100 nM ACh (0.011 ± 0.002) was increased 4.5-fold by 100 μM zinc (0.050 ± 0.008), accounting for most of the potentiation observed at the whole cell level. The increase in open probability was due to an increase in burst duration, which increased from 207 ± 38 ms in the absence of zinc to 830 ± 189 ms in the presence of zinc. Our results suggest that potentiation of neuronal nAChRs by zinc is due to a stabilization of the bursting states of the receptor.

scholarly journals Potentiation of the cystic fibrosis transmembrane conductance regulator Cl− channel by ivacaftor is temperature independent

2018 ◽  
Vol 315 (5) ◽  
pp. L846-L857 ◽  
Author(s):  
Yiting Wang ◽  
Zhiwei Cai ◽  
Martin Gosling ◽  
David N. Sheppard
Keyword(s):  
Cystic Fibrosis ◽  
Temperature Dependence ◽  
Single Channel ◽  
Channel Gating ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Cystic Fibrosis Transmembrane

Ivacaftor is the first drug to target directly defects in the cystic fibrosis transmembrane conductance regulator (CFTR), which causes cystic fibrosis (CF). To understand better how ivacaftor potentiates CFTR channel gating, here we investigated the effects of temperature on its action. As a control, we studied the benzimidazolone UCCF-853, which potentiates CFTR by a different mechanism. Using the patch-clamp technique and cells expressing recombinant CFTR, we studied the single-channel behavior of wild-type and F508del-CFTR, the most common CF mutation. Raising the temperature of the intracellular solution from 23 to 37°C increased the frequency but reduced the duration of wild-type and F508del-CFTR channel openings. Although the open probability ( Po) of wild-type CFTR increased progressively as temperature was elevated, the relationship between Po and temperature for F508del-CFTR was bell-shaped with a maximum Po at ~30°C. For wild-type CFTR and to a greatly reduced extent F508del-CFTR, the temperature dependence of channel gating was asymmetric with the opening rate demonstrating greater temperature sensitivity than the closing rate. At all temperatures tested, ivacaftor and UCCF-853 potentiated wild-type and F508del-CFTR. Strikingly, ivacaftor but not UCCF-853 abolished the asymmetric temperature dependence of CFTR channel gating. At all temperatures tested, Po values of wild-type CFTR in the presence of ivacaftor were approximately double those of F508del-CFTR, which were equivalent to or greater than those of wild-type CFTR at 37°C in the absence of the drug. We conclude that the principal effect of ivacaftor is to promote channel opening to abolish the temperature dependence of CFTR channel gating.

scholarly journals A common mechanism for CFTR potentiators

2017 ◽  
Vol 149 (12) ◽  
pp. 1105-1118 ◽  
Author(s):  
Han-I Yeh ◽  
Yoshiro Sohma ◽  
Katja Conrath ◽  
Tzyh-Chang Hwang
Keyword(s):  
Cystic Fibrosis ◽  
Atp Hydrolysis ◽  
Single Channel ◽  
Underlying Mechanism ◽  
Common Mechanism ◽  
Loss Of Function ◽  
Open Probability ◽  
High Throughput Drug Screening ◽  
State Dependent ◽  
Opening And Closing

Cystic fibrosis (CF) is a channelopathy caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a phosphorylation-activated and adenosine triphosphate (ATP)–gated chloride channel. In the past few years, high-throughput drug screening has successfully realized the first US Food and Drug Administration–approved therapy for CF, called ivacaftor (or VX-770). A more recent CFTR potentiator, GLPG1837 (N-(3-carbamoyl-5,5,7,7-tetramethyl-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-1H-pyrazole-3-carboxamide), has been shown to exhibit a higher efficacy than ivacaftor for the G551D mutation, yet the underlying mechanism of GLPG1837 remains unclear. Here we find that despite their differences in potency and efficacy, GLPG1837 and VX-770 potentiate CFTR gating in a remarkably similar manner. Specifically, they share similar effects on single-channel kinetics of wild-type CFTR. Their actions are independent of nucleotide-binding domain (NBD) dimerization and ATP hydrolysis, critical steps controlling CFTR’s gate opening and closing, respectively. By applying the two reagents together, we provide evidence that GLPG1837 and VX-770 likely compete for the same site, whereas GLPG1837 and the high-affinity ATP analogue 2′-deoxy-N6-(2-phenylethyl)-adenosine-5′-O-triphosphate (dPATP) work synergistically through two different sites. We also find that the apparent affinity for GLPG1837 is dependent on the open probability of the channel, suggesting a state-dependent binding of the drug to CFTR (higher binding affinity for the open state than the closed state), which is consistent with the classic mechanism for allosteric modulation. We propose a simple four-state kinetic model featuring an energetic coupling between CFTR gating and potentiator binding to explain our experimental results.

scholarly journals G551D and G1349D, Two CF-associated Mutations in the Signature Sequences of CFTR, Exhibit Distinct Gating Defects

2007 ◽  
Vol 129 (4) ◽  
pp. 285-298 ◽  
Author(s):  
Silvia G. Bompadre ◽  
Yoshiro Sohma ◽  
Min Li ◽  
Tzyh-Chang Hwang
Keyword(s):  
Cystic Fibrosis ◽  
Single Channel ◽  
Clinical Phenotype ◽  
Binding Pocket ◽  
The Other ◽  
Atp Binding ◽  
Open Probability ◽  
Physiological Importance ◽  
Gating Model ◽  
Signature Sequence

Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). CFTR is a chloride channel that is regulated by phosphorylation and gated by ATP binding and hydrolysis at its nucleotide binding domains (NBDs). G551D-CFTR, the third most common CF-associated mutation, has been characterized as having a lower open probability (Po) than wild-type (WT) channels. Patients carrying the G551D mutation present a severe clinical phenotype. On the other hand, G1349D, also a mutant with gating dysfunction, is associated with a milder clinical phenotype. Residues G551 and G1349 are located at equivalent positions in the highly conserved signature sequence of each NBD. The physiological importance of these residues lies in the fact that the signature sequence of one NBD and the Walker A and B motifs from the other NBD form the ATP-binding pocket (ABP1 and ABP2, named after the location of the Walker A motif) once the two NBDs dimerize. Our studies show distinct gating characteristics for these mutants. The G551D mutation completely eliminates the ability of ATP to increase the channel activity, and the observed activity is ∼100-fold smaller than WT-CFTR. G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg2+]. The low activity of G551D-CFTR likely represents the rare ATP-independent gating events seen with WT channels long after the removal of ATP. G1349D-CFTR maintains ATP dependence, albeit with a Po ∼10-fold lower than WT. Interestingly, compared to WT results, the ATP dose–response relationship of G1349D-CFTR is less steep and shows a higher apparent affinity for ATP. G1349D data could be well described by a gating model that predicts that binding of ATP at ABP1 hinders channel opening. Thus, our data provide a quantitative explanation at the single-channel level for different phenotypes presented by patients carrying these two mutations. In addition, these results support the idea that CFTR's two ABPs play distinct functional roles in gating.

Role of actin in regulation of epithelial sodium channels by CFTR

1997 ◽  
Vol 272 (4) ◽  
pp. C1077-C1086 ◽  
Author(s):  
I. I. Ismailov ◽  
B. K. Berdiev ◽  
V. G. Shlyonsky ◽  
C. M. Fuller ◽  
A. G. Prat ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Actin Filaments ◽  
Single Channel ◽  
Na Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Open Probability ◽  
Airway Epithelia ◽  
Alpha Beta ◽  
Actin Protein

Cystic fibrosis (CF) airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that actin plays a role in the regulation of a cloned epithelial Na+ channel (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR). We found that immunopurified bovine tracheal CFTR coreconstituted into a planar lipid bilayer with alpha,beta,gamma-rat ENaC (rENaC) decreased single-channel open probability (Po) of rENaC in the presence of actin by over 60%, a significantly greater effect than was observed in the absence of actin (approximately 20%). In the presence of actin, protein kinase A plus ATP activated both CFTR and rENaC, but CFTR was activated in a sustained manner, whereas the activation of rENaC was transitory. ATP alone could also activate ENaC transiently in the presence ofactin but had no effect on CFTR. Stabilizing short actin filaments at a fixed length with gelsolin (at a ratio to actin of 2:1) produced a sustained activation of alpha,beta,gamma-rENaC in both the presence or absence of CFTR. Gelsolin alone (i.e., in the absence of actin) had no effect on the conductance or Po of either CFTR or rENaC. We have also found that short actin filaments produced their modulatory action on alpha-rENaC independent of the presence of the beta- or gamma-rENaC subunits. In contrast, CFTR did not affect any properties of the channel formed by alpha-rENaC alone, i.e., in the absence of beta- or gamma-rENaC. These results indicate that CFTR can directly downregulate single Na+ channel activity, which may account for the observed differences between Na+ transport in normal and CF-affected airway epithelia. Moreover, the presence of actin confers an enhanced modulatory ability of CFTR on Na+ channels.

Case report: a child with cystic fibrosis and phenylketonuria

2020 ◽  
Vol 1 (2) ◽  
pp. 38-44
Author(s):  
Irina V. Vakhlova ◽  
Anastasia D. Kazachina ◽  
Olga A. Beglyanina
Keyword(s):  
Cystic Fibrosis ◽  
Case Report ◽  
Clinical Practice ◽  
Neonatal Screening ◽  
Genetic Disorders ◽  
Epileptiform Activity ◽  
Autism Spectrum ◽  
Speech Development ◽  
Inherited Disorders ◽  
Congenital Diseases

Background. In the international clinical practice there have been occasional reports of phenylketonuria (PKU) and cystic fibrosis (CF) found simultaneously in the same patient. Both PKU and CF are the inherited disorders characterized by autosomal recessive type of inheritance. Currently the combination of two or more inherited disorders in one patient is considered to be a clinical rarity.Case description. This is a clinical case of two genetic disorders, CF and PKU, combined in a 5-year old patient who had been followed up since birth. Owing to implementation of neonatal screening for inherited and congenital diseases into clinical practice, during the first month of life the infant was diagnosed with CF (diagnostically significant elevation of immunoreactive trypsin [IRT] at the initial [163.2 ng/mL] and repeat testing on day 21 of life [138.7 ng/mL]) and PKU (phenylalanine [PA] level 15.9 mg/dL). Both disorders have been confirmed by genetic tests, i.e., homozygous DelF508 mutation was found in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and P281L mutation in the phenylalanine hydroxylase (PAH) gene was also present in homozygous state. Child’s parents strictly adhered to dietary and treatment recommendations. By the age of 5 years the child developed symptoms of neurological disorder and disorder of the respiratory system, cognitive impairment and delay in speech development, subclinical epileptiform activity with high risk of epilepsy, and chronic inflammation of the respiratory tract.Conclusion. This case report demonstrates the important role of neonatal screening in early diagnosis and timely start of therapy, and underscores the importance of continuous medication in such genetic disorders as CF and PKU. On the whole, such approach brings about a relative preservation of functioning of the most affected organs and systems. By the age of 5 years the child does not form bronchiectases, shows no signs of chronic hypoxia, nutritional deficiency or pronounced neurologic deficit, and is at low risk for the development of autism spectrum disorder. At the same time, the larger scale and longer-term observations are required in order to make the unequivocal conclusions about the prognosis of these diseases under conditions of modern-day medical follow-up.

Chemical modification of Ca(2+)-activated potassium channels of GH3 anterior pituitary cells

1992 ◽  
Vol 263 (5) ◽  
pp. C1081-C1087 ◽  
Author(s):  
A. M. Frace ◽  
D. C. Eaton
Keyword(s):  
Single Channel ◽  
Gh3 Cells ◽  
Disulfonic Acid ◽  
State Transitions ◽  
Trinitrobenzene Sulfonic Acid ◽  
Voltage Sensitivity ◽  
Pituitary Cells ◽  
Amino Groups ◽  
Open Probability ◽  
Long Duration

The effects of amino group specific reagents were examined on single, large-conductance, Ca(2+)-activated, K+ channels in excised membrane patches from GH3 cells. The reagents used include trinitrobenzene sulfonic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and its 4-acetamido derivative, and sulfophenyl-isothiocyanate. These reagents react covalently with peptide terminal amino groups and the epsilon amino groups of lysine residues, thereby removing positive charge. Internal application of 0.1-1.0 mM reagent to inside-out patches irreversibly increases channel open probability. Single-channel conductance and voltage sensitivity are not affected by modification. Analysis of channel openings and closures shows that the increase in open probability is predominantly due to the loss of long-duration closures of the channel; however, the lengths of long-duration openings are increased. After the modification in the presence of Ca2+ was performed, the channel open probability remains large, regardless of the internal Ca2+ concentration. Transitions among several open and closed states of the modified channel are present in the absence of Ca2+, suggesting that many state transitions are not directly dependent on Ca2+ binding or dissociation.

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