ABCMdb

PMID: 21383017

Pedemonte N, Tomati V, Sondo E, Caci E, Millo E, Armirotti A, Damonte G, Zegarra-Moran O, Galietta LJ
Dual activity of aminoarylthiazoles on the trafficking and gating defects of the cystic fibrosis transmembrane conductance regulator chloride channel caused by cystic fibrosis mutations.
J Biol Chem. 2011 Apr 29;286(17):15215-26. Epub 2011 Mar 7., 2011-04-29 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Login to comment 14 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Conversely, class III mutations (like G551D and G1349D) do not impair protein trafficking but severely decrease CFTR channel opening in response to cAMP elevation. Login to comment 21 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In other words, maximal stimulation of mutant CFTR with a cAMP agonist evokes only a fraction (20-40% for F508del, less than 5-10% for G551D and G1349D) of the total activity obtained by also adding a potentiator (10, 11, 17-19). Login to comment 47 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp EXPERIMENTAL PROCEDURES Cell Culture-Fischer rat thyroid (FRT) cells were stably transfected with F508del, G551D, or G1349D-CFTR (12). Login to comment 100 ABCC7 p.Gly1349Asp Patch Clamp Experiments-Single channel analysis was done on FRT cells stably expressing CFTR with G1349D mutation in cell-attached and inside-out configurations. Login to comment 213 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Therefore, we tested corr-2b and EN277I on FRT cells expressing G1349D-CFTR or G551D-CFTR mutant, using the HS-YFP assay and short circuit current recordings (Fig. 5). Login to comment 214 ABCC7 p.Gly1349Asp On G1349D-CFTR cells, long term treatment with AATs or corr-4a did not change QRTOT (Fig. 5A, top panel). Login to comment 216 ABCC7 p.Gly551Asp Similarly, on G551D-CFTR FRT cells, FIGURE4.AnalysisofAATmechanismofaction.A,timecourseofdruguptakeofindicatedcompoundsmeasuredinFRTcells(meansϮS.E.,nϭ3).B,maximal effect obtained for EN277I and genistein (50 ␮M for both compounds) tested as potentiators (30-45 min of incubation) of F508del-CFTR in the presence of forskolin (20 ␮M) in FRT (left panel) and A549 (right panel) cells, normalized to genistein effect (means Ϯ S.E., n ϭ 8). Login to comment 228 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Using the HS-YFP assay, we compared the concentrations of EN277I and genistein that are effective in improving the relative forskolin response of F508del-, G1349D-, and G551D-CFTR mutants (Fig. 5C). Login to comment 230 ABCC7 p.Gly551Asp As expected from previous studies (10, 11, 17), we found that the concentrations of genistein required to activate G551D-CFTR were 5-10 times higher than those effective on the other two mutants (Fig. 5C, left panel). Login to comment 231 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Indeed the Kd values for genistein were: 11.8 Ϯ 1.5 ␮M for F508del; 9.7 Ϯ 0.7 ␮M for G1349D; and 62.4 Ϯ 5.3 ␮M for G551D. Login to comment 232 ABCC7 p.Gly551Asp Although the net potentiating effect of genistein at very high concentrations may be partially diminished by open channel block (34), it has been repeatedly found that G551D responds to genistein and other potentiators with significantly reduced affinity (10, 11, 17). Login to comment 233 ABCC7 p.Gly551Asp This behavior may be interpreted as a direct or allosteric alteration of the binding site for potentiators caused by the G551D mutation. Login to comment 235 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The Kd values were as follows: 19.2 Ϯ 0.8 and 20.1 Ϯ 2.2 ␮M for F508del (correction of trafficking and gating defects, respectively); 15.8 Ϯ 1.1 ␮M for G1349D; and 25.1 Ϯ 1.9 ␮M for G551D. Login to comment 237 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The ability of AATs to correct the gating defect of class 3 mutants after long term treatment was confirmed performing short circuit current recordings on FRT cells expressing G1349D- or G551D-CFTR (Fig. 5, D and E). Login to comment 238 ABCC7 p.Gly551Asp CFTR activity in untreated cells was very small or even undetectable (in the case of G551D), despite the use of a maximal forskolin concentration. Login to comment 241 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp A and B, the bar graphs show the total CFTR activity elicited by forskolin (20 ␮M) plus genistein (50 ␮M for G1349D and 200 ␮M for G551D) (QRTOT, top panels) and the relative activity stimulated by forskolin alone (QRFSK/QRTOT, bottom panels) in FRT cells expressing G1349D-CFTR (A) or G551D-CFTR (B) treated for 24h at 37 °C with the indicated compounds (means Ϯ S.E., n ϭ 8). Login to comment 244 ABCC7 p.Gly551Asp C, comparison between dose-response relationships of genistein (left panel) and EN277I (right panel) in correcting F508del-CFTR trafficking defect (F, for EN277I only) or in increasing the relative forskolin response of F508del-CFTR (E), G1349-CFTR (Ⅺ), or G551D-CFTR (‚). Login to comment 247 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp D and E, representative traces of chloride current measured in Ussing chambers on G1349D-CFTR (D) and G551D-CFTR (E) FRT epithelia incubated for 24 h at 37 °C in the absence or presence of EN277I (30 ␮M) or corr-2b (50 ␮M). Login to comment 248 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The concentrations were: forskolin, 20 ␮M; genistein, 50 ␮M for G1349D and 200 ␮M for G551D; CFTRinh-172 (inh-172), 10 ␮M. Login to comment 250 ABCC7 p.Gly1349Asp Incubation of G1349D cells for 24 h with EN277I or corr-2b did not change the absolute ITOT value, as compared with control condition but enhanced the percentage of the total current activated by forskolin alone, with IFSK/ITOT increasing from Ͻ10% to ϳ50%, as shown in Fig. 5D (control, IFSK ϭ 8.0 Ϯ 1.6 ␮A; corr-2b, IFSK ϭ 35.5 Ϯ 1.5 ␮A, p Ͻ 0.01; EN277I, IFSK ϭ 31.5 Ϯ 1.8 ␮A, p Ͻ 0.01; control, ITOT ϭ 70 Ϯ 6 ␮A; corr-2b, ITOT ϭ 69 Ϯ 8 ␮A, not significant; EN277I, ITOT ϭ 72 Ϯ 7 ␮A, not significant). Login to comment 251 ABCC7 p.Gly551Asp The effect of AATs on G551D cells was qualitatively similar, although the increase in IFSK/ITOT was smaller (from Ͻ5% to ϳ20-30%; see Fig. 5E) (control, IFSK ϭ 3.9 Ϯ 0.5 ␮A; corr-2b, IFSK ϭ 29 Ϯ 3 ␮A, p Ͻ 0.01; EN277I, IFSK ϭ 22 Ϯ 4 ␮A, p Ͻ 0.01; control, ITOT ϭ 79 Ϯ 4 ␮A; corr-2b, ITOT ϭ 80 Ϯ 8 ␮A, not significant; EN277I, ITOT ϭ 82 Ϯ 9 ␮A, not significant). Login to comment 252 ABCC7 p.Gly1349Asp The activity of EN277I on G1349D-CFTR expressed in FRT cells was examined in patch clamp experiments, in cell-attached and in inside-out patch configurations following acute or chronic exposure. Login to comment 260 ABCC7 p.Gly1349Asp We examined also the effect of chronic treatment of G1349D-CFTR cells with EN277I (Fig. 7). Login to comment 262 ABCC7 p.Gly1349Asp Cells treated with the vehicle alone showed low G1349D-CFTR activity, with rare openings separated by long channel closings. Login to comment 269 ABCC7 p.Gly1349Asp Patch clamp analysis of the acute effect of compound EN277I on G1349D-CFTR. Login to comment 295 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp We have also tested AATs on pure class III mutants, namely G1349D and G551D, the latter being the one with the most severe gating defect. Login to comment 296 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Interestingly, we found that dual acting AATs also enhance the fraction of G1349D and G551D activity that is evoked by cAMP, in the absence of the potentiator. Login to comment 297 ABCC7 p.Gly1349Asp The extent of gating enhancement is larger for G1349D relative to the other mutant. Login to comment 309 ABCC7 p.Gly551Asp Interestingly, this site may be different from that of classical potentiators because it shows no difference in affinity between G551D and the other mutants. Login to comment 318 ABCC7 p.Gly1349Asp Patch clamp analysis of the chronic effect of compound EN277I on G1349D-CFTR. Login to comment