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Luis J V Galietta - One of the best experts on this subject based on the ideXlab platform.
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nanomolar affinity small molecule correctors of defective δf508 cftr chloride Channel Gating
Journal of Biological Chemistry, 2003Co-Authors: Hong Yang, Nicoletta Pedemonte, Alessandro Taddei, Anang Shelat, Vadiraj S Gopinath, A Tonghui, Kai Du, Gergely L Lukacs, Chiara Folli, Luis J V GaliettaAbstract:Abstract Deletion of Phe-508 (ΔF508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. ΔF508-CFTR has defects in both Channel Gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective ΔF508-CFTR Cl– Channel Gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing ΔF508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which ΔF508-CFTR was targeted to the plasma membrane by culture at 27 °C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360–480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated ΔF508-CFTR Cl– conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after ΔF508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective ΔF508-CFTR Gating was also demonstrated in human bronchial epithelial cells from a ΔF508 cystic fibrosis subject after 27 °C temperature rescue. In conjunction with correctors of defective ΔF508-CFTR processing, small molecule potentiators of defective ΔF508-CFTR Gating may be useful for therapy of cystic fibrosis caused by the ΔF508 mutation.
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nanomolar affinity small molecule correctors of defective δf508 cftr chloride Channel Gating
Journal of Biological Chemistry, 2003Co-Authors: Hong Yang, Nicoletta Pedemonte, Alessandro Taddei, Vadiraj S Gopinath, A Tonghui, Gergely L Lukacs, Chiara Folli, Anang A Shelat, Kiplin R Guy, Luis J V GaliettaAbstract:Deletion of Phe-508 (Delta F508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. Delta F508-CFTR has defects in both Channel Gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective Delta F508-CFTR Cl- Channel Gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing Delta F508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which Delta F508-CFTR was targeted to the plasma membrane by culture at 27 degrees C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated DeltaF508-CFTR Cl- conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after Delta F508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective Delta F508-CFTR Gating was also demonstrated in human bronchial epithelial cells from a Delta F508 cystic fibrosis subject after 27 degrees C temperature rescue. In conjunction with correctors of defective Delta F508-CFTR processing, small molecule potentiators of defective Delta F508-CFTR Gating may be useful for therapy of cystic fibrosis caused by the Delta F508 mutation.
Nicoletta Pedemonte - One of the best experts on this subject based on the ideXlab platform.
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phenylglycine and sulfonamide correctors of defective δf508 and g551d cystic fibrosis transmembrane conductance regulator chloride Channel Gating
Molecular Pharmacology, 2005Co-Authors: Nicoletta Pedemonte, N D Sonawane, Alessandro Taddei, Jie Hu, Olga Zegarramoran, Yat Fan Suen, Lori I Robins, Christopher W Dicus, Dan Willenbring, Michael H NantzAbstract:Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride Channel cause cystic fibrosis. The F508 mutation produces defects in Channel Gating and cellular processing, whereas the G551D mutation produces primarily a Gating defect. To identify correctors of Gating, 50,000 diverse small molecules were screened at 2.5 M (with forskolin, 20 M) by an iodide uptake assay in epithelial cells coexpressing F508-CFTR and a fluorescent halide indicator (yellow fluorescent protein-H148Q/I152L) after F508-CFTR rescue by 24-h culture at 27°C. Secondary analysis and testing of 1000 structural analogs yielded two novel classes of correctors of defective F508-CFTR Gating (“potentiators”) with nanomolar potency that were active in human F508 and G551D cells. The most potent compound of the phenylglycine class, 2-[(2–1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide, reversibly activated F508CFTR in the presence of forskolin with Ka 70 nM and also activated the CFTR Gating mutants G551D and G1349D with Ka values of 1100 and 40 nM, respectively. The most potent sulfonamide, 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide, had Ka 20 nM for activation of F508-CFTR. In cell-attached patch-clamp experiments, phenylglycine-01 (PG-01) and sulfonamide-01 (SF-01) increased Channel open probability 5-fold by the reduction of interburst closed time. An interesting property of these compounds was their ability to act in synergy with cAMP agonists. Microsome metabolism studies and rat pharmacokinetic analysis suggested significantly more rapid metabolism of PG-01 than SF-03. Phenylglycine and sulfonamide compounds may be useful for monotherapy of cystic fibrosis caused by Gating mutants and possibly for a subset of F508 subjects with significant F508-CFTR plasma-membrane expression.
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phenylglycine and sulfonamide correctors of defective δf508 and g551d cystic fibrosis transmembrane conductance regulator chloride Channel Gating
Molecular Pharmacology, 2005Co-Authors: Nicoletta Pedemonte, N D Sonawane, Alessandro Taddei, Olga Zegarramoran, Yat Fan Suen, Lori I Robins, Christopher W Dicus, Dan Willenbring, Michael H Nantz, Mark J KurthAbstract:Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride Channel cause cystic fibrosis. The delta F508 mutation produces defects in Channel Gating and cellular processing, whereas the G551D mutation produces primarily a Gating defect. To identify correctors of Gating, 50,000 diverse small molecules were screened at 2.5 microM (with forskolin, 20 microM) by an iodide uptake assay in epithelial cells coexpressing delta F508-CFTR and a fluorescent halide indicator (yellow fluorescent protein-H148Q/I152L) after delta F508-CFTR rescue by 24-h culture at 27 degrees C. Secondary analysis and testing of >1000 structural analogs yielded two novel classes of correctors of defective delta F508-CFTR Gating ("potentiators") with nanomolar potency that were active in human delta F508 and G551D cells. The most potent compound of the phenylglycine class, 2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide, reversibly activated delta F508-CFTR in the presence of forskolin with K(a) approximately 70 nM and also activated the CFTR Gating mutants G551D and G1349D with K(a) values of approximately 1100 and 40 nM, respectively. The most potent sulfonamide, 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide, had K(a) approximately 20 nM for activation of delta F508-CFTR. In cell-attached patch-clamp experiments, phenylglycine-01 (PG-01) and sulfonamide-01 (SF-01) increased Channel open probability >5-fold by the reduction of interburst closed time. An interesting property of these compounds was their ability to act in synergy with cAMP agonists. Microsome metabolism studies and rat pharmacokinetic analysis suggested significantly more rapid metabolism of PG-01 than SF-03. Phenylglycine and sulfonamide compounds may be useful for monotherapy of cystic fibrosis caused by Gating mutants and possibly for a subset of delta F508 subjects with significant delta F508-CFTR plasma-membrane expression.
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nanomolar affinity small molecule correctors of defective δf508 cftr chloride Channel Gating
Journal of Biological Chemistry, 2003Co-Authors: Hong Yang, Nicoletta Pedemonte, Alessandro Taddei, Anang Shelat, Vadiraj S Gopinath, A Tonghui, Kai Du, Gergely L Lukacs, Chiara Folli, Luis J V GaliettaAbstract:Abstract Deletion of Phe-508 (ΔF508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. ΔF508-CFTR has defects in both Channel Gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective ΔF508-CFTR Cl– Channel Gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing ΔF508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which ΔF508-CFTR was targeted to the plasma membrane by culture at 27 °C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360–480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated ΔF508-CFTR Cl– conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after ΔF508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective ΔF508-CFTR Gating was also demonstrated in human bronchial epithelial cells from a ΔF508 cystic fibrosis subject after 27 °C temperature rescue. In conjunction with correctors of defective ΔF508-CFTR processing, small molecule potentiators of defective ΔF508-CFTR Gating may be useful for therapy of cystic fibrosis caused by the ΔF508 mutation.
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nanomolar affinity small molecule correctors of defective δf508 cftr chloride Channel Gating
Journal of Biological Chemistry, 2003Co-Authors: Hong Yang, Nicoletta Pedemonte, Alessandro Taddei, Vadiraj S Gopinath, A Tonghui, Gergely L Lukacs, Chiara Folli, Anang A Shelat, Kiplin R Guy, Luis J V GaliettaAbstract:Deletion of Phe-508 (Delta F508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. Delta F508-CFTR has defects in both Channel Gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective Delta F508-CFTR Cl- Channel Gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing Delta F508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which Delta F508-CFTR was targeted to the plasma membrane by culture at 27 degrees C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated DeltaF508-CFTR Cl- conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after Delta F508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective Delta F508-CFTR Gating was also demonstrated in human bronchial epithelial cells from a Delta F508 cystic fibrosis subject after 27 degrees C temperature rescue. In conjunction with correctors of defective Delta F508-CFTR processing, small molecule potentiators of defective Delta F508-CFTR Gating may be useful for therapy of cystic fibrosis caused by the Delta F508 mutation.
Alessandro Taddei - One of the best experts on this subject based on the ideXlab platform.
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phenylglycine and sulfonamide correctors of defective δf508 and g551d cystic fibrosis transmembrane conductance regulator chloride Channel Gating
Molecular Pharmacology, 2005Co-Authors: Nicoletta Pedemonte, N D Sonawane, Alessandro Taddei, Jie Hu, Olga Zegarramoran, Yat Fan Suen, Lori I Robins, Christopher W Dicus, Dan Willenbring, Michael H NantzAbstract:Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride Channel cause cystic fibrosis. The F508 mutation produces defects in Channel Gating and cellular processing, whereas the G551D mutation produces primarily a Gating defect. To identify correctors of Gating, 50,000 diverse small molecules were screened at 2.5 M (with forskolin, 20 M) by an iodide uptake assay in epithelial cells coexpressing F508-CFTR and a fluorescent halide indicator (yellow fluorescent protein-H148Q/I152L) after F508-CFTR rescue by 24-h culture at 27°C. Secondary analysis and testing of 1000 structural analogs yielded two novel classes of correctors of defective F508-CFTR Gating (“potentiators”) with nanomolar potency that were active in human F508 and G551D cells. The most potent compound of the phenylglycine class, 2-[(2–1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide, reversibly activated F508CFTR in the presence of forskolin with Ka 70 nM and also activated the CFTR Gating mutants G551D and G1349D with Ka values of 1100 and 40 nM, respectively. The most potent sulfonamide, 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide, had Ka 20 nM for activation of F508-CFTR. In cell-attached patch-clamp experiments, phenylglycine-01 (PG-01) and sulfonamide-01 (SF-01) increased Channel open probability 5-fold by the reduction of interburst closed time. An interesting property of these compounds was their ability to act in synergy with cAMP agonists. Microsome metabolism studies and rat pharmacokinetic analysis suggested significantly more rapid metabolism of PG-01 than SF-03. Phenylglycine and sulfonamide compounds may be useful for monotherapy of cystic fibrosis caused by Gating mutants and possibly for a subset of F508 subjects with significant F508-CFTR plasma-membrane expression.
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phenylglycine and sulfonamide correctors of defective δf508 and g551d cystic fibrosis transmembrane conductance regulator chloride Channel Gating
Molecular Pharmacology, 2005Co-Authors: Nicoletta Pedemonte, N D Sonawane, Alessandro Taddei, Olga Zegarramoran, Yat Fan Suen, Lori I Robins, Christopher W Dicus, Dan Willenbring, Michael H Nantz, Mark J KurthAbstract:Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride Channel cause cystic fibrosis. The delta F508 mutation produces defects in Channel Gating and cellular processing, whereas the G551D mutation produces primarily a Gating defect. To identify correctors of Gating, 50,000 diverse small molecules were screened at 2.5 microM (with forskolin, 20 microM) by an iodide uptake assay in epithelial cells coexpressing delta F508-CFTR and a fluorescent halide indicator (yellow fluorescent protein-H148Q/I152L) after delta F508-CFTR rescue by 24-h culture at 27 degrees C. Secondary analysis and testing of >1000 structural analogs yielded two novel classes of correctors of defective delta F508-CFTR Gating ("potentiators") with nanomolar potency that were active in human delta F508 and G551D cells. The most potent compound of the phenylglycine class, 2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide, reversibly activated delta F508-CFTR in the presence of forskolin with K(a) approximately 70 nM and also activated the CFTR Gating mutants G551D and G1349D with K(a) values of approximately 1100 and 40 nM, respectively. The most potent sulfonamide, 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide, had K(a) approximately 20 nM for activation of delta F508-CFTR. In cell-attached patch-clamp experiments, phenylglycine-01 (PG-01) and sulfonamide-01 (SF-01) increased Channel open probability >5-fold by the reduction of interburst closed time. An interesting property of these compounds was their ability to act in synergy with cAMP agonists. Microsome metabolism studies and rat pharmacokinetic analysis suggested significantly more rapid metabolism of PG-01 than SF-03. Phenylglycine and sulfonamide compounds may be useful for monotherapy of cystic fibrosis caused by Gating mutants and possibly for a subset of delta F508 subjects with significant delta F508-CFTR plasma-membrane expression.
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nanomolar affinity small molecule correctors of defective δf508 cftr chloride Channel Gating
Journal of Biological Chemistry, 2003Co-Authors: Hong Yang, Nicoletta Pedemonte, Alessandro Taddei, Anang Shelat, Vadiraj S Gopinath, A Tonghui, Kai Du, Gergely L Lukacs, Chiara Folli, Luis J V GaliettaAbstract:Abstract Deletion of Phe-508 (ΔF508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. ΔF508-CFTR has defects in both Channel Gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective ΔF508-CFTR Cl– Channel Gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing ΔF508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which ΔF508-CFTR was targeted to the plasma membrane by culture at 27 °C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360–480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated ΔF508-CFTR Cl– conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after ΔF508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective ΔF508-CFTR Gating was also demonstrated in human bronchial epithelial cells from a ΔF508 cystic fibrosis subject after 27 °C temperature rescue. In conjunction with correctors of defective ΔF508-CFTR processing, small molecule potentiators of defective ΔF508-CFTR Gating may be useful for therapy of cystic fibrosis caused by the ΔF508 mutation.
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nanomolar affinity small molecule correctors of defective δf508 cftr chloride Channel Gating
Journal of Biological Chemistry, 2003Co-Authors: Hong Yang, Nicoletta Pedemonte, Alessandro Taddei, Vadiraj S Gopinath, A Tonghui, Gergely L Lukacs, Chiara Folli, Anang A Shelat, Kiplin R Guy, Luis J V GaliettaAbstract:Deletion of Phe-508 (Delta F508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. Delta F508-CFTR has defects in both Channel Gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective Delta F508-CFTR Cl- Channel Gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing Delta F508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which Delta F508-CFTR was targeted to the plasma membrane by culture at 27 degrees C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated DeltaF508-CFTR Cl- conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after Delta F508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective Delta F508-CFTR Gating was also demonstrated in human bronchial epithelial cells from a Delta F508 cystic fibrosis subject after 27 degrees C temperature rescue. In conjunction with correctors of defective Delta F508-CFTR processing, small molecule potentiators of defective Delta F508-CFTR Gating may be useful for therapy of cystic fibrosis caused by the Delta F508 mutation.
Manoj K Patel - One of the best experts on this subject based on the ideXlab platform.
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temporal lobe epilepsy induces intrinsic alterations in na Channel Gating in layer ii medial entorhinal cortex neurons
Neurobiology of Disease, 2011Co-Authors: Nicholas J Hargus, Ellen C Merrick, Aradhya Nigam, Christopher L Kalmar, Aparna R Baheti, Edward H Bertram, Manoj K PatelAbstract:Temporal lobe epilepsy (TLE) is the most common form of adult epilepsy involving the limbic structures of the temporal lobe. Layer II neurons of the entorhinal cortex (EC) form the major excitatory input into the hippocampus via the perforant path and consist of non-stellate and stellate neurons. These neurons are spared and hyper-excitable in TLE. The basis for the hyper-excitability is likely multifactorial and may include alterations in intrinsic properties. In a rat model of TLE, medial EC (mEC) non-stellate and stellate neurons had significantly higher action potential (AP) firing frequencies than in control. The increase remained in the presence of synaptic blockers, suggesting intrinsic mechanisms. Since sodium (Na) Channels play a critical role in AP generation and conduction we sought to determine if Na Channel Gating parameters and expression levels were altered in TLE. Na Channel currents recorded from isolated mEC TLE neurons revealed increased Na Channel conductances, depolarizing shifts in inactivation parameters and larger persistent (INaP) and resurgent (INaR) Na currents. Immunofluorescence experiments revealed increased staining of Nav1.6 within the axon initial segment and Nav1.2 within the cell bodies of mEC TLE neurons. These studies provide support for additional intrinsic alterations within mEC layer II neurons in TLE and implicate alterations in Na Channel activity and expression, in part, for establishing the profound increase in intrinsic membrane excitability of mEC layer II neurons in TLE. These intrinsic changes, together with changes in the synaptic network, could support seizure activity in TLE.
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temporal lobe epilepsy induces intrinsic alterations in na Channel Gating in layer ii medial entorhinal cortex neurons
Neurobiology of Disease, 2011Co-Authors: Nicholas J Hargus, Ellen C Merrick, Aradhya Nigam, Christopher L Kalmar, Aparna R Baheti, Edward H Bertram, Manoj K PatelAbstract:Temporal lobe epilepsy (TLE) is the most common form of adult epilepsy involving the limbic structures of the temporal lobe. Layer II neurons of the entorhinal cortex (EC) form the major excitatory input into the hippocampus via the perforant path and consist of non-stellate and stellate neurons. These neurons are spared and hyper-excitable in TLE. The basis for the hyper-excitability is likely multifactorial and may include alterations in intrinsic properties. In a rat model of TLE, medial EC (mEC) non-stellate and stellate neurons had significantly higher action potential (AP) firing frequencies than in control. The increase remained in the presence of synaptic blockers, suggesting intrinsic mechanisms. Since sodium (Na) Channels play a critical role in AP generation and conduction we sought to determine if Na Channel Gating parameters and expression levels were altered in TLE. Na Channel currents recorded from isolated mEC TLE neurons revealed increased Na Channel conductances, depolarizing shifts in inactivation parameters and larger persistent (INaP) and resurgent (INaR) Na currents. Immunofluorescence experiments revealed increased staining of Nav1.6 within the axon initial segment and Nav1.2 within the cell bodies of mEC TLE neurons. These studies provide support for additional intrinsic alterations within mEC layer II neurons in TLE and implicate alterations in Na Channel activity and expression, in part, for establishing the profound increase in intrinsic membrane excitability of mEC layer II neurons in TLE. These intrinsic changes, together with changes in the synaptic network, could support seizure activity in TLE.
Hong Yang - One of the best experts on this subject based on the ideXlab platform.
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nanomolar affinity small molecule correctors of defective δf508 cftr chloride Channel Gating
Journal of Biological Chemistry, 2003Co-Authors: Hong Yang, Nicoletta Pedemonte, Alessandro Taddei, Anang Shelat, Vadiraj S Gopinath, A Tonghui, Kai Du, Gergely L Lukacs, Chiara Folli, Luis J V GaliettaAbstract:Abstract Deletion of Phe-508 (ΔF508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. ΔF508-CFTR has defects in both Channel Gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective ΔF508-CFTR Cl– Channel Gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing ΔF508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which ΔF508-CFTR was targeted to the plasma membrane by culture at 27 °C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360–480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated ΔF508-CFTR Cl– conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after ΔF508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective ΔF508-CFTR Gating was also demonstrated in human bronchial epithelial cells from a ΔF508 cystic fibrosis subject after 27 °C temperature rescue. In conjunction with correctors of defective ΔF508-CFTR processing, small molecule potentiators of defective ΔF508-CFTR Gating may be useful for therapy of cystic fibrosis caused by the ΔF508 mutation.
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nanomolar affinity small molecule correctors of defective δf508 cftr chloride Channel Gating
Journal of Biological Chemistry, 2003Co-Authors: Hong Yang, Nicoletta Pedemonte, Alessandro Taddei, Vadiraj S Gopinath, A Tonghui, Gergely L Lukacs, Chiara Folli, Anang A Shelat, Kiplin R Guy, Luis J V GaliettaAbstract:Deletion of Phe-508 (Delta F508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. Delta F508-CFTR has defects in both Channel Gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective Delta F508-CFTR Cl- Channel Gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing Delta F508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which Delta F508-CFTR was targeted to the plasma membrane by culture at 27 degrees C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated DeltaF508-CFTR Cl- conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after Delta F508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective Delta F508-CFTR Gating was also demonstrated in human bronchial epithelial cells from a Delta F508 cystic fibrosis subject after 27 degrees C temperature rescue. In conjunction with correctors of defective Delta F508-CFTR processing, small molecule potentiators of defective Delta F508-CFTR Gating may be useful for therapy of cystic fibrosis caused by the Delta F508 mutation.