ABCMdb

PMID: 16604470

Melin P, Norez C, Callebaut I, Becq F
The glycine residues G551 and G1349 within the ATP-binding cassette signature motifs play critical roles in the activation and inhibition of cystic fibrosis transmembrane conductance regulator channels by phloxine B.
J Membr Biol. 2005 Dec;208(3):203-12. Epub 2006 Apr 7., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. Login to comment 4 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 ± 1.13 lM) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 ± 1.01 lM) but not activated by phloxine B. Finally, the double mutant G551D/ G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Login to comment 7 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Key words: Cystic fibrosis - CFTR - ABC signature - G551D - G1349D - Phloxine B - Genistein - Non-Michaelis-Menten Introduction The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel belongs to the ATP-binding cassette (ABC) transporters, a large evolutionarily conserved family of integral membrane proteins that catalyze the active transport of a variety of solutes across biological membranes coupled to hydrolysis of nucleotides as a source of energy (Riordan et al., 1989; Vankeerberghen, Cuppens & Cassima, 2002). Login to comment 22 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Individual delF508, G551D and G1349D mutations were created by site-directed mutagenesis as previously described (Melin et al., 2004). Login to comment 23 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Double mutant G551D/G1349D (named 2GD-CFTR) was derived from pEGFP-G551D-CFTR. Login to comment 61 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Results PROPERTIES OF G1349D-CFTR CHLORIDE CURRENT Individual delF508, G551D and G1349D and double G551D/G1349D (named 2GD-CFTR) mutations were created by site-directed mutagenesis and inserted into the pEGFP-C1-CFTR mammalian expression vector, allowing fusion of GFP to the N terminus of CFTR as previously described (Melin et al., 2004). Login to comment 64 ABCC7 p.Gly1349Asp The basal whole-cell current (mean current density of 14 ± 4 pA/pF at +40 mV, n = 3) was not significantly affected by the expression of G1349D-CFTR (mean current density of 12 ± 3 pA/pF at +40 mV, n = 7; Fig. 2A and D). Login to comment 65 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The class III mutation G1349D, like G551D, impaired activation of CFTR channels by the adenylate cyclase activator forskolin (Fsk). Login to comment 66 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The corresponding mean current densities are 11 ± 4 pA/pF for G551D-CFTR and 9 ± 4 pA/pF for G1349D-CFTR at +40 mV in the presence of 10 lM Fsk (n = 3). Login to comment 67 ABCC7 p.Gly1349Asp Using iodide efflux experiments, we tested increasing concentrations of Fsk from 0.1 to 100 lM but failed to stimulate any iodide efflux in G1349D-CFTR-expressing cells (n = 4 for each concentration, not shown). Login to comment 70 ABCC7 p.Gly1349Asp The cyclic adenosine monophosphate (cAMP)-activated G1349D-CFTR Cl) current was completely inhibited by 100 lM glibenclamide (Fig. 2C and D) (Sheppard & Welsh, 1993). Login to comment 71 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Using iodide efflux experiments, we determined the concentration dependence of the effect of glibenclamide on G1349D-CFTR and G551D/ G1349D (noted 2GD) in cells activated by 10 lM Fsk + 100 lM IBMX. Login to comment 72 ABCC7 p.Gly1349Asp We calculated IC50 values of 8.3 ± 1.06 lM for G1349D-CFTR (n = 4; black symbols, Fig. 2E) and 5.5 ± 1.13 lM for 2GD-CFTR (n = 4; open symbols, Fig. 2E). Login to comment 75 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp We explored the effect of PhlxB on the Cl) channel activity of the following CFTR mutants: delF508, G551D, G1349D and 2GD. Login to comment 87 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp EFFECTS OF PHLXB ON G551D, G1349D AND G551D/G1349D CHANNELS In the next series of experiments, we examined the consequence of the glycine-to-aspartic acid mutations in NBD1 and NBD2 on the response to PhlxB in the presence of Fsk. Login to comment 88 ABCC7 p.Gly551Asp A classical sigmoidal concentration-response relationship describes the effect of PhlxB on G551D (Fig. 3B, black triangles). Login to comment 89 ABCC7 p.Gly551Asp These results indicate a complete loss of inhibition at high PhlxB concentrations (>10 lM) of G551D-CFTR compared to wt-CFTR (see Fig. 3A). Login to comment 90 ABCC7 p.Gly551Asp The calculated Ks = 2 ± 1.13 lM (n = 4) shows that PhlxB is a potent activator of G551D channels (Table 1). Login to comment 91 ABCC7 p.Gly1349Asp Then, we examined the response to PhlxB of G1349D channels also in the presence of Fsk. Login to comment 92 ABCC7 p.Gly551Asp Contrary to the previous results with the NBD1 mutation G551D, we Fig. 1. Login to comment 98 ABCC7 p.Gly1349Asp found no evidence for stimulation of G1349D channels by increasing concentrations of PhlxB despite the presence of Fsk (Fig. 3B, black squares). Login to comment 99 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The double mutant, carrying G551D and G1349D mutations, is also refractory to PhlxB stimulation (Fig. 3B, open squares). Login to comment 101 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp EFFECT OF PHLXB WHEN G1349D-, G551DAND 2GD-CFTR-EXPRESSING CELLS ARE STIMULATED BY FSK/IBMX Because the mutant G1349D can be activated by Fsk/IBMX but not by Fsk/PhlxB, we took advantage of this difference to investigate whether PhlxB could nevertheless inhibit the mutant channel after its activation by Fsk/IBMX, like the wt-CFTR channels. Login to comment 102 ABCC7 p.Gly1349Asp In the following experiments, we performed whole-cell patch-clamp experiments on COS-7 cells expressing G1349D-CFTR stimulated by Fsk/ IBMX and subsequently challenged by application of 10 lM PhlxB into the bath. Login to comment 106 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp After stable activation of the G1349D-CFTR current, addition of 10 lM PhlxB (in the continuous presence of Fsk/IBMX) inhibited G1349D-CFTR currents (Fig. 4A and B). Login to comment 108 ABCC7 p.Gly1349Asp Further application of 100 lM glibenclamide in the extracellular bath had no additional inhibitory effect (Fig. 4A), confirming that the G1349D-CFTR current was totally inhibited by PhlxB. Login to comment 110 ABCC7 p.Gly551Asp Then, similar experiments were performed with G551D-CFTR-expressing cells. Login to comment 111 ABCC7 p.Gly551Asp As expected from iodide efflux experiments, a significant increase of Cl) current was recorded in G551D-expressing cells stimulated by 10 lM Fsk + 100 lM IBMX (Fig. 5A and B). Login to comment 112 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp However, contrary to G1349D-CFTR, PhlxB did not inhibit G551D-CFTR currents after stable activation of G551D (compare left and right whole-cell 0 100 200 300 400 -1000 0 1000 2000 3000 I(pA) Time (ms) 0 100 200 300 400 -1000 0 1000 2000 3000 I(pA) Time (ms) 0 100 200 300 400 -1000 0 1000 2000 3000I(pA) Time (ms) Fsk + IBMXbasal Fsk + IBMX + Glib -100-80 -60 -40 -20 20 40 60 80 100 -1000 1000 2000 basal Fsk+IBMX +Glib Vm (mV) I (pA) -6 -5 -4 0 25 50 75 100 2GD G1349D Log [glibenclamide] (M) %ofactivation ns ns ns ns ns A B D E C Fig. 2. Login to comment 113 ABCC7 p.Gly1349Asp Activation by cAMP cocktail and inhibition by glibenclamide of G1349D-CFTR channels. Login to comment 117 ABCC7 p.Gly1349Asp Vm, membrane voltage; (E) Concentration- responses curves of glibenclamide on COS-7 cells transfected with GFP-G1349D-CFTR and 2GD after maximal activation with cAMP cocktail. Login to comment 120 ABCC7 p.Gly1349Asp IC50 values calculated in these conditions are for G1349D channels, 8.3 ± 1.06 lM, and for 2GD channels, 5.55 ± 1.13 lM. Login to comment 123 ABCC7 p.Gly551Asp PhlxB did not potentiate G551D-CFTR currents due to maximal stimulation of the current by Fsk/IBMX (Fig. 5A and C). Login to comment 124 ABCC7 p.Gly551Asp Further application of 100 lM glibenclamide in the extracellular bath fully inhibited the currents (Fig. 5A and C), confirming that G551D-CFTR is refractory to inhibition only by PhlxB but not by glibenclamide. Login to comment 126 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp We also noted two differences between G551D and G1349D-CFTR channels stimulated by Fsk+IBMX. Login to comment 127 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp First, the time to maximal current stimulation with G551D-CFTR, 120 ± 9 s (n = 4), was significantly faster (P < 0.01) than that for G1349D (194 ± 8 s, n = 4). Login to comment 128 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Second, the current density measured at +40 mV was 26 ± 5 pA/pF (n = 6) for G551D construct, a value approximately threefold smaller (P < 0.001) than the current density measured for G1349D, 88 ± 13 pA/pF (n = 4). Login to comment 129 ABCC7 p.Gly1349Asp Finally, we determined the concentration-dependent inhibitory effects of PhlxB and genistein on G1349D and 2GD-CFTR channels after stimulation by Fsk/IBMX (Fig. 6). Login to comment 131 ABCC7 p.Gly1349Asp We calculated the Ki for PhlxB on G1349D (40 ± 1.01 lM, n = 4 for each concentration; Fig. 6A, black symbols). Login to comment 133 ABCC7 p.Gly1349Asp With genistein, the calculated Ki was 121 ± 1.14 lM for G1349D channels (n = 4 for each concentration; Fig. 6B, black symbols). Login to comment 142 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In this configuration, each nucleotide is sandwiched between the two NBDs and each nucleotide-binding site is formed by the Walker A, Walker B, Q-loop and wt-CFTR delF508 (24h/27˚C) -7 -6 -5 -4 0.00 0.05 0.10 0.15 0.20 Log [PhlxB] (M) kpeak-kbasal(min-1 ) ** ns ns ** * -7 -6 -5 -4 0.00 0.05 0.10 0.15 0.20 G551D G1349D 2GD Log [PhlxB] (M) kpeak-kbasal(min-1 ) A B Fig. 3. Login to comment 147 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp (B) Concentration-response curves of PhlxB on COS-7 cells transiently transfected with GFP-CFTR with the following mutations: G551D, -G1349D and -2GD. Login to comment 151 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Kinetic parameters for activation (Ks) and inhibition (Ki) of CFTR by PhlxB and genisteina PhlxB Genistein Construct Ks Ki Ks Ki wt-CFTR 3.2 ± 1.6 38 ± 1.4 10 ± 1b 106 ± 1b delF508 3 ± 1.8 33 ± 1 9.8 ± 1b 107 ± 1b G551D 2 ± 1.13 -d 13 ± 1.25b - G1349D - 40 ± 1.01c - 121 ± 1.14c 2GD - - - - a All data (in lM) are n = 4. b Data from Melin et al., 2004. c With activation by Fsk/IBMX. Login to comment 158 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Interestingly our data show that G1349D-CFTR elicited a larger Cl) current than G551D-CFTR, these two glycine residues being located within the NBD1 and NBD2 active sites, respectively (Fig. 1B). Login to comment 161 ABCC7 p.Gly1349Asp Effects of PhlxB on cAMP-stimulated G1349D-CFTR channels. Login to comment 162 ABCC7 p.Gly1349Asp (A) Representative time course of G1349D-CFTR current amplitude in the presence of various agonists. Login to comment 163 ABCC7 p.Gly1349Asp (B) Representative trace of current recorded on G1349D channels with Fsk + IBMX (*) and with Fsk + IBMX + PhlxB (**). Login to comment 164 ABCC7 p.Gly1349Asp (C) I/V relationships for G1349D-CFTR chloride current (mean ± SEM) normalized by cell capacitance. Login to comment 167 ABCC7 p.Gly1349Asp (D) Summary of current amplitudes (mean ± SEM between +40 and )40 mV) recorded on cells transfected with GFP-CFTR- G1349D in various conditions: basal (n = 4), Fsk + IBMX (n = 4), Fsk + IBMX + PhlxB (n = 4), Fsk + IBMX + PhlxB + glibenclamide (Glib, n = 4). Login to comment 171 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp According to these data, we show that the LSGDQ mutated sequence (with G551D) drastically impairs activation of CFTR Cl) channel activity by Fsk/IBMX compared to the LSHDH mutated sequence (with G1349D). Login to comment 174 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Drugs like genistein and PhlxB are associated with direct binding at the NBDs (Randak et al., 1999; Cai & Sheppard, 2002), consistent with our observations that CFTR modulation is altered by NBD mutations G551D and G1349D. Login to comment 185 ABCC7 p.Gly551Asp Effects of PhlxB on cAMP-activated G551D-CFTR channels. Login to comment 186 ABCC7 p.Gly551Asp (A) Representative time course of G551D-CFTR amplitude current in the presence of various agonists. Login to comment 187 ABCC7 p.Gly551Asp (B) Representative trace of current recorded on G551D channels with Fsk + IBMX (*) and with Fsk + IBMX + PhlxB (**). Login to comment 188 ABCC7 p.Gly551Asp (C) I/V relationships for the G551D-CFTR channels (mean ± SEM) normalized by cell capacitance. Login to comment 189 ABCC7 p.Gly551Asp (D) Summary of current amplitudes (mean ± SEM between +40 and )40 mV) recorded on cells transfected with GFP-G551D-CFTR in various conditions: basal (n = 10), Fsk + IBMX (n = 6), Fsk + IBMX + PhlxB (n = 5), Fsk + IBMX + PhlxB + glibenclamide (Glib, n = 3). Login to comment