ABCMdb

PMID: 11600430

Derand R, Bulteau-Pignoux L, Mettey Y, Zegarra-Moran O, Howell LD, Randak C, Galietta LJ, Cohn JA, Norez C, Romio L, Vierfond JM, Joffre M, Becq F
Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation.
Am J Physiol Cell Physiol. 2001 Nov;281(5):C1657-66., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Gly551Asp 281:C1657-C1666, 2001.Am J Physiol Cell Physiol Becq Caroline Norez, Leila Romio, Jean-Michel Vierfond, Michel Joffre and Frédéric L. Daniel Howell, Christoph Randak, Luis J. V. Galietta, Jonathan A. Cohn, Renaud Dérand, Laurence Bulteau-Pignoux, Yvette Mettey, Olga Zegarra-Moran, regulation by ATPase activity and phosphorylation Activation of G551D CFTR channel with MPB-91: You might find this additional info useful... 25 articles, 15 of which can be accessed free at:This article cites http://ajpcell.physiology.org/content/281/5/C1657.full.html#ref-list-1 3 other HighWire hosted articlesThis article has been cited by [PDF][Full Text][Abstract] , September 27, 2002; 277 (39): 35999-36004.J. Login to comment 2 ABCC7 p.Gly551Asp Renaud Dérand, Laurence Bulteau-Pignoux and Frédéric Becq the Inhibitory Genistein Binding Site Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel and Abolishes The Cystic Fibrosis Mutation G551D Alters the Non-Michaelis-Menten Behavior of the [PDF][Full Text][Abstract] , November 13, 2007; 37 (6): 631-639.AJRCMB Lusky, Frederic Becq, Pierre Boulanger, Joseph Zabner and Saw-See Hong Ophelia Granio, Caroline Norez, Katherine J. Login to comment 11 ABCC7 p.Gly551Asp It is published 12 timesAJP - Cell Physiology Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation RENAUD DE´ RAND,1 LAURENCE BULTEAU-PIGNOUX,1 YVETTE METTEY,2 OLGA ZEGARRA-MORAN,3 L. DANIEL HOWELL,4 CHRISTOPH RANDAK,5 LUIS J. V. GALIETTA,3 JONATHAN A. COHN,4 CAROLINE NOREZ,1 LEILA ROMIO,3 JEAN-MICHEL VIERFOND,2 MICHEL JOFFRE,1 AND FRE´ DE´ RIC BECQ1 1 Laboratoire de Physiologie des Re´gulations Cellulaires, Unite´ Mixte de Recherche 6558, 86022 Poitiers, and 2 Laboratoire de Chimie Organique, Faculte´ de Me´decine et de Pharmacie, 86005 Poitiers, France; 3 Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Genoa 16148, Italy; 4 Durham Veterans Affairs and Duke University Medical Center, Durham, North Carolina; and 5 Kinderklinik im Dr. von Haunerschen Kinderspital, Ludwig-Maximilians-Universita¨t, Munich, Germany Received 19 April 2001; accepted in final form 16 July 2001 De´rand, Renaud, Laurence Bulteau-Pignoux, Yvette Mettey, Olga Zegarra-Moran, L. Daniel Howell, Christoph Randak, Luis J. V. Galietta, Jonathan A. Cohn, Caroline Norez, Leila Romio, Jean-Michel Vierfond, Michel Joffre, and Fre´de´ric Becq. Login to comment 12 ABCC7 p.Gly551Asp Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation. Login to comment 13 ABCC7 p.Gly551Asp Am J Physiol Cell Physiol 281: C1657-C1666, 2001.-We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. Login to comment 14 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (EC50 ϭ 85 ␮M) and activated CFTR in Calu-3 cells (EC50 ϭ 47 ␮M). Login to comment 15 ABCC7 p.Gly551Asp MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. Login to comment 22 ABCC7 p.Gly551Asp The main mutations are a deletion of the phenylalanine at position 508 (⌬F508, class II mutation), which represents ϳ66% of the mutated chromosomes, and a glycine-to-aspartic acid missense mutation at codon 551 (G551D, class III mutation) with a frequency of 2-5% (33). Login to comment 23 ABCC7 p.Gly551Asp Both mutations are located in the first nucleotide binding domain (NBD1) (21), but, whereas ⌬F508-CFTR is not correctly addressed toward the membrane (8, 13), the processing, maturation, and assigned apical location of G551D CFTR are not affected (30, 33). Login to comment 24 ABCC7 p.Gly551Asp However, in cells expressing the G551D mutant, chloride transport cannot be activated by cAMP-elevating agents (1, 11, 33). Login to comment 27 ABCC7 p.Gly551Asp We have further performed a structure-activity study and identified 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91), a compound able to restore chloride secretion in G551D and wild-type CFTR-expressing cells. Login to comment 41 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Chinese hamster ovary (CHO) cells stably transfected with pNUT vector alone (CHO pNUT) or containing wild-type CFTR [CFTR(ϩ) CHO], G551D CFTR (G551D CHO), or 10SA CFTR (10SA CHO) were provided by J. R. Riordan and X.-B. Chang (Scottsdale, AZ) (6, 31). Login to comment 57 ABCC7 p.Gly551Asp Ussing chamber experiments in G551D Fisher rat thyroid cells. Login to comment 75 ABCC7 p.Gly1208Leu A second fusion protein (MBP/NBD2) of the maltose-binding protein (MBP) and the human CFTR protein sequence from Gly-1208 to Leu-1399 (numbering according to Ref. 25), enclosing the whole predicted second nucleotide binding domain of CFTR (NBD2), was expressed, purified, and characterized in Escherichia coli (23, 24); all enzymatic assays were performed as described in Refs. 23 and 24. Login to comment 93 ABCC7 p.Gly551Asp We found it necessary to study the additive stimulatory effect of forskolin and MPB-91 in CFTR(ϩ) CHO cells, since the effect on G551D CFTR had been tested in the same cell line. Login to comment 107 ABCC7 p.Gly551Asp C1659MPB-91 STIMULATES G551D CFTR CHANNEL ACTIVITY AJP-Cell Physiol • VOL 281 • NOVEMBER 2001 • www.ajpcell.org 1.11 Ϯ 0.14 min-1 , n ϭ 8), even in the presence of forskolin (not shown). Login to comment 119 ABCC7 p.Gly551Asp MPB-91 but not MPB-07 is an activator of G551D CFTR. Login to comment 120 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Because little is known about the pharmacology of G551D CFTR, we determined the effects of MPB-07 and MPB-91 on the activity of G551D expressed in CHO cells using iodide efflux and whole cell patch-clamp methods. Login to comment 121 ABCC7 p.Gly551Asp Control experiments confirmed that forskolin indeed failed to stimulate iodide efflux from G551D-CFTR-expressing cells (peak rate-to-basal: 0.10 Ϯ 0.01 min-1 , n ϭ 14; 10 ␮M forskolin: 0.10 Ϯ 0.01 min-1 , n ϭ 14). Login to comment 122 ABCC7 p.Gly551Asp We found that MPB-07 (250 ␮M) either alone (peak rate: 0.09 Ϯ 0.01 min-1 , n ϭ 4) or in combination with 10 ␮M forskolin (peak rate: 0.09 Ϯ 0.01 min-1 , n ϭ 4) failed to stimulate the iodide efflux in G551D cells. Login to comment 134 ABCC7 p.Gly551Asp These results indicate that the MPB-91-stimulated iodide efflux and whole cell chloride current in G551D cells are due to CFTR activation. Login to comment 135 ABCC7 p.Gly551Asp Figure 4 shows short-circuit current measurements on FRT cells transfected with G551D CFTR. Login to comment 137 ABCC7 p.Gly551Asp However, subsequent stimulation with MPB-91 produced a dose-dependent increase of transepithelial conductance in G551D CFTR cells (Fig. 4). Login to comment 142 ABCC7 p.Gly551Asp The activation of G551D CFTR by MPB-91 occurred with an EC50 of 84.8 Ϯ 9.3 ␮M (n ϭ 3). Login to comment 145 ABCC7 p.Gly551Asp Whole cell CFTR current activated by MPB-91 in CHO G551D cells. Login to comment 150 ABCC7 p.Gly551Asp Transepithelial conductance (G) response to MPB-91 on G551D CFTR-transfected FRT cells. Login to comment 151 ABCC7 p.Gly551Asp A: response of a G551D CFTR monolayer to 500 ␮M 8-(4-chlorophenylthio)adenosine-3Ј,5Ј-cyclic monophosphate (CPT-cAMP) followed by increasing concentrations of MPB-91 (in ␮M) in the apical solution. Login to comment 155 ABCC7 p.Gly551Asp C1661MPB-91 STIMULATES G551D CFTR CHANNEL ACTIVITY AJP-Cell Physiol • VOL 281 • NOVEMBER 2001 • www.ajpcell.org both NBD1 and NBD2 ATPase activities. Login to comment 156 ABCC7 p.Gly551Asp First, wild-type and G551D NBD1/R/GST ATPase activities were measured at their respective Michaelis-Menten constant (Km) values (70 and 250 ␮M; Ref. 14) and at 1 mM ATP. Login to comment 157 ABCC7 p.Gly551Asp G551D NBD1 has a significantly reduced ATPase activity compared with wild-type NBD1 (Fig. 5A) as expected from previous studies (14, 19). Login to comment 158 ABCC7 p.Gly551Asp MPB-91 does not affect the ATPase activity of wild-type or G551D NBD1 at any ATP and MPB-91 concentrations tested. Login to comment 164 ABCC7 p.Gly551Asp To determine whether wild-type and G551D CFTR activation by MPB-91 involved phosphorylation of the channel, the effects of two kinase inhibitors were investigated. Login to comment 173 ABCC7 p.Gly551Asp Wild-type (WT) NBD1/R/GST (50 ng) and G551D NBD1/R/GST (50 ng) were tested for ATPase activity at their respective Km values (70 and 250 ␮M) and at 1 mM ATP. Login to comment 185 ABCC7 p.Gly551Asp C1662 MPB-91 STIMULATES G551D CFTR CHANNEL ACTIVITY AJP-Cell Physiol • VOL 281 • NOVEMBER 2001 • www.ajpcell.org onAugust,2011ajpcell.physiology.orgDownloadedfrom 91-stimulated iodide efflux in Calu-3 cells (P Ͼ 0.05, for both inhibitors). Login to comment 187 ABCC7 p.Gly551Asp Finally, experiments were performed on G551D CFTR cells exposed to forskolin (10 ␮M) and MPB-91 (250 ␮M). Login to comment 193 ABCC7 p.Gly551Asp C and D: MPB-91-mediated iodide efflux in Calu-3 (C) and in G551D CHO cells (D). Login to comment 196 ABCC7 p.Gly551Asp C1663MPB-91 STIMULATES G551D CFTR CHANNEL ACTIVITY AJP-Cell Physiol • VOL 281 • NOVEMBER 2001 • www.ajpcell.org kolin ϩ MPB-91 (P Ͻ 0.01, n ϭ 8, Fig. 6D). Login to comment 204 ABCC7 p.Gly551Asp Indeed, we found that MPB-07, which is able to activate wild-type CFTR (2), is not effective as a G551D CFTR activator. Login to comment 205 ABCC7 p.Gly551Asp Modification of the chemical structure using SAR identified an important position within the MPB skeleton, leading to MPB-91, a compound that kept its ability to activate wild-type CFTR but more importantly acquired the property to activate G551D CFTR. Login to comment 208 ABCC7 p.Gly551Asp We demonstrated that MPB-91 restores CFTR-mediated chloride secretion in cells expressing G551D CFTR. Login to comment 209 ABCC7 p.Gly551Asp The inhibitory profile of G551D CFTR channel activity is similar to that described for wild-type CFTR (13, 26). Login to comment 214 ABCC7 p.Gly551Asp Because ATP hydrolysis at NBD1 has been linked to CFTR activation, we initially hypothesized that MPB-91 could modulate wild-type ATPase and/or could restore normal G551D ATPase function (e.g., by reducing the Km value for ATP). Login to comment 215 ABCC7 p.Gly551Asp To study this, we first measured the ATPase activity of wild-type NBD1 in the presence of MPB-91. Our results clearly demonstrated that MPB-91 did not compete with ATP binding, did not modulate ATPase activity, and had no significant effect on reduced ATPase activity of G551D NBD1. Login to comment 216 ABCC7 p.Gly551Asp We then concluded that the mutation G551D interferes with the mechanism of channel opening that is dependent of ATP hydrolysis at NBD1. Login to comment 217 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Because the compound MPB-91 was found to be able to activate wild-type CFTR and G551D CFTR without directly affecting NBD1 ATPase activity, these observations strongly suggest that an alternative pathway of activation exists that bypasses the defective ATPase activity of G551D CFTR proteins. Login to comment 223 ABCC7 p.Gly551Asp For example, these two drugs are both able to activate wild-type and G551D CFTR, but probably through two distinct mechanisms. Login to comment 232 ABCC7 p.Gly551Asp One major difference currently observed between wild-type and G551D CFTR is the role of phosphorylation. Login to comment 234 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Although G551D CFTR could be normally phosphorylated (11) and despite the fact that phosphorylation by itself is not sufficient to activate G551D CFTR (11, 33), it is required for CFTR to be opened by MPB-91 and other compounds like genistein (17). Login to comment 237 ABCC7 p.Gly551Asp In addition, MPB-91 does not alter the cAMP levels in both Calu-3 and G551D CHO cells (T. Me´taye´, R. De´rand, L. Bulteau, and F. Becq, unpublished data). Login to comment 240 ABCC7 p.Gly551Asp In G551D CHO cells we found, however, that MPB-91 alone is not sufficient to stimulate the chloride current. Login to comment 242 ABCC7 p.Gly551Asp However, once phosphorylated, G551D CFTR can be activated by MPB-91. Login to comment 244 ABCC7 p.Gly551Asp These results suggest that phosphorylation of G551D CFTR may act only as a switch, making the mutated protein responsive or not to MPB-91. Login to comment 248 ABCC7 p.Gly551Asp In conclusion, in this report we have presented the potential interest of MPB-91 as a putative drug in CF, since it not only modulates wild-type CFTR but also activates CFTR having the disease-causing mutation G551D. Login to comment