ABCMdb

ABCC7 p.His620Gln

None has been submitted yet.

Publications

PMID: 16442101 [PubMed] Frelet A et al: "Insight in eukaryotic ABC transporter function by mutation analysis."

No. Sentence Comment

296 G622D and R792G have reduced intrinsic chloride channel activities whereas H620Q and A800G resulted in increased intrinsic chloride transport properties [160]. Login to comment

PMID: 11242048 [PubMed] Choi JY et al: "Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis."

No. Sentence Comment

50 Two sets of particularly interesting mutants are G551D and G551S and H620Q and A800G. Login to comment
54 When expressed in oocytes, the H620Q and A800G mutants increased the macroscopic Cl- current about threefold and the channel open probability by 150±180% (ref. 19). Login to comment
57 The H620Q mutant, found in one CF patient with pancreatic insuf®ciency, only stimulated HCO3 transport about 13% as effectively as did CFTR. Login to comment
66 Notably, although a few of these mutants exhibit altered letters to nature NATURE |VOL 410 |1 MARCH 2001 |www.nature.com 95 NO3 - Forskolin 5 µM NO3 - Forskolin 5 µM a G551S b G551D 10mMCl- 200 s NO3 - NO3 - Forskolin 5 µM Forskolin 5 µM f H620Q g A800G h H620Q Forskolin 5 µMCl- free Cl- freeCl- free Cl-free c G551S d G551D Forskolin 5 µM Forskolin 5 µM 0.25pHunits 250 s e A800G 0 0.25 0.50 0.75 1.00 H620Q A800G G551D G551S j WT Forskolin 5 µM 0 0.25 0.50 0.75 1.00 H620Q A800G G551D G551S WT i [Cl- ]change(mMs-1 )HCO3 -transport (∆pH+ min-1 ) Figure 2 cAMP-stimulated Cl- and HCO3 transport by CFTR mutants associated with a severe or a mild form of CF. Login to comment
186 letters to nature 96 NATURE |VOL 410 |1 MARCH 2001 |www.nature.com HCO3 -/Cl- transportratio 0 0.25 0.50 0.75 1.00 WT I148T G178R R297Q G551D H620Q G970R A1067T G1244E S1255P G1349D E193K G551S A800G H949Y R1070Q Pancreatic insufficient Pancreatic sufficientD648V N CI148T G178R E193K R297Q R117H A1067T R1070Q G1244E S1255P G1349D NBD2 RD H949Y G970R CL4CL3CL2CL1 NBD1 G551D G551S H620Q D648V A800G Figure 3 The HCO3:Cl-transport ratio of CFTR mutants associated with CF. Login to comment

PMID: 12940920 [PubMed] Rowntree RK et al: "The phenotypic consequences of CFTR mutations."

No. Sentence Comment

78 Three mutant CFTR proteins, G622D, R792G and E822K, that were transiently expressed in COS cells showed lower chloride channel activities when compared to wild-type CFTR, whereas mutants H620Q and A800G showed increased activities. Login to comment

PMID: 16132229 [PubMed] Eudes R et al: "Nucleotide binding domains of human CFTR: a structural classification of critical residues and disease-causing mutations."

No. Sentence Comment

247 (B) Ribbon representation of the NBD aa1 - aa2 segment of the three experimental MalK structures and of mCFTR NBD1, with the CFTR F508 and MalK F98 being shown in atomic details involves the exposed F508 residue, only a few missense mutations affecting these exposed aromatic residues have been reported: H484R, Y515H, H620P, H620Q and Y1307C (http://www.genet.sickkids.on.ca/cftr). Login to comment

PMID: 17700961 [PubMed] Quinton PM et al: "Too much salt, too little soda: cystic fibrosis."

No. Sentence Comment

177 The activity was CFTR mutation-specific and, for example, mutations such as R117H (the pancreas is spared in compound CF heterozygotes such as R117H/ΔF508)appeared to retain the ability to support exchange even though Cl-conductance was depressed while mutations such as H620Q did not support exchange, but retained a disputed[240] Cl-conductance. Login to comment

PMID: 20435887 [PubMed] Billet A et al: "C terminus of nucleotide binding domain 1 contains critical features for cystic fibrosis transmembrane conductance regulator trafficking and activation."

No. Sentence Comment

2 We mutated CFTR amino acids located in the betac5-betac6 hairpin, within the betac5 strand (H620Q), within the beta-turn linking the two beta strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the betac6 strand. Login to comment
3 Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. Login to comment
5 In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Login to comment
33 We have mutated these two glycine residues in aspartic acid (G622D) and arginine (G628R) and considered other mutants in their neighborhood (H620Q, E621G, S623A, and S624A). Login to comment
87 RESULTS Expression of the NBD1 C-terminal CFTR Mutants-We have introduced EGFP-tagged CFTR proteins into HEK293 cells, wt CFTR and six CFTR mutants, i.e. H620Q, E621G, G622D, S623A, S624A, and G628R. Login to comment
90 For the expression of CFTR mutants H620Q, E621G, S623A, and S624A, the profiles of core-glycosylated and mature-glycosylated forms were similar to that of the wt protein. Login to comment
109 Second, with the two mutations introduced on both sides of the beta turn, H620Q and S623A, we recorded an increased (p Ͻ 0.001) Cl- current (supplemental Table 2) compared with wt CFTR. Login to comment
122 Concerning the activation kinetics, only the two CFTR mutants with an increased Cl-transport activity (H620Q and S623A) also present a faster time course of activation (Fig. 5B). Login to comment
124 T50 of the two mutated channels (T50 ϭ 124.45 Ϯ 8.8 s for H620Q, n ϭ 8 and T50 ϭ 114.63 Ϯ 8.8 s for S623A, n ϭ 8) were significantly different compared with wt (T50 ϭ 164.85 Ϯ 10.2 s, n ϭ 8). Login to comment
125 Our results indicated that H620Q and S623A channels could be activated more rapidly than the other CFTR mutant channels studied. Login to comment
130 Interestingly, although the level of current for H620Q and S623A was greater than the response of wt channels (Fig. 3), both mutated CFTRs were activated by MPB-91 to a level similar to that of wt CFTR (Fig. 6) (at 40 mV, current densities were: H620Q, 27.86 Ϯ 4.2 pA/pF, n ϭ 5; S623A, 30.83 Ϯ 3.4 pA/pF, n ϭ 6). Login to comment
163 In particular, the cAMP-activated Cl- current densities elicited by H620Q and S623A channels were increased compared with wt. Login to comment
165 For H620Q mutant, this increase could be explained by the reported increased of open probability (15). Login to comment
169 The H620Q mutation might thus disturb the contacts existing between the beta hairpin and the P-loop of NBD1 and thus might result in a perturbation of the channel gating through an effect on the ATP binding and/or hydrolysis in the noncanonical site. Login to comment
180 In addition, MPB-91 was also able to activate H620Q, E621G, S623A, and S624A mutants. Login to comment
182 Contrary to the activation by Fsk, no difference of kinetic parameters was detected with the two mutants H620Q and S623A. Login to comment

PMID: 20932301 [PubMed] Green DM et al: "Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients."

No. Sentence Comment

74 For Pa, the hazard ratio Table 1 Classification of CFTR alleles Category Mutation Specific mutations Class I Defective Protein Synthesis (nonsense, frameshift, aberrant splicing) 1078delT, 1154 insTC, 1525-2A > G, 1717-1G > A, 1898+1G > A, 2184delA, 2184 insA, 3007delG, 3120+1G > A, 3659delC, 3876delA, 3905insT, 394delTT, 4010del4, 4016insT, 4326delTC, 4374+1G > T, 441delA, 556delA, 621+1G > T, 621-1G > T, 711+1G > T, 875+1G > C, E1104X, E585X, E60X, E822X, G542X, G551D/R553X, Q493X, Q552X, Q814X, R1066C, R1162X, R553X, V520F, W1282X, Y1092X Class II Abnormal Processing and Trafficking A559T, D979A, ΔF508, ΔI507, G480C, G85E, N1303K, S549I, S549N, S549R Class III Defective Channel Regulation/Gating G1244E, G1349D, G551D, G551S, G85E, H199R, I1072T, I48T, L1077P, R560T, S1255P, S549 (R75Q) Class IV Decreased Channel Conductance A800G, D1152H, D1154G, D614G, delM1140, E822K, G314E, G576A, G622D, G85E, H620Q, I1139V, I1234V, L1335P, M1137V, P67L, R117C, R117P, R117H, R334W, R347H, R347P, R347P/ R347H, R792G, S1251N, V232D Class V Reduced Synthesis and/or Trafficking 2789+5G > A, 3120G > A, 3272-26A > G, 3849+10kbC > T, 5T variant, 621+3A > G, 711+3A > G, A445E, A455E, IVS8 poly T, P574H was increased 3 fold for those with 'Minimal` function when compared to those with 'Residual` function. Login to comment

PMID: 9736778 [PubMed] Vankeerberghen A et al: "Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

8 Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Login to comment
68 Primers used for mutagenesis Primer Sequence I601F (a1933t) 5'-CTA ACA AAA CTA GGT TTT TGG TCA CTT C-3' L610S (t1961c) 5'-CTA AAA TGG AAC ATT CAA AGA AAG CTG-3' A613T (g1969a) 5'-CAT TTA AAG AAA ACT GAC AAA ATA TTA-3' D614G (a1973g) 5'-CAT TTA AAG AAA GCT GGC AAA ATA TTA A-3' I618T (t1985c) 5'-GAC AAA ATA TTA ACT TTG CAT GAA GG-3' L619S (t1988c) 5'-GAC AAA ATA TTA ATT TCG CAT GAA GGT-3' H620P (a1991c) 5'-CAA AAT ATT AAT TTT GCC TGA AGG TAG C-3' H620Q (t1992g) 5'-AAT ATT AAT TTT GCA GGA AGG TAG CAG-3' G622D (g1997a) 5'-TTG CAT GAA GAT AGC AGC TAT TTT TAT G-3' G628R (g2014c) 5'-GCA GCT ATT TTT ATC GGA CAT TTT C-3' L633P (t2030c) 5'-CAT TTT CAG AAC CCC AAA ATC TAC AGC-3' D648V (a2075t) 5'-CTC ATG GGA TGT GTT TCT TTC GAC C-3' T665S (a2125t) 5'-CAA TCC TAA CTG AGT CCT TAC ACC G-3' F693L (t2209c) 5'-CAG ACT GGA GAG CTT GGG GAA AAA AG-3' R766M (g2429t) 5'-GCA CGA AGG ATG CAG TCT GTC CTG-3' R792G (c2506g) 5'-CAG CAT CCA CAG GAA AAG TGT CAC TG-3' A800G (c2531g) 5'-CTG GCC CCT CAG GGA AAC TTG ACT G-3' I807M (a2553g) 5'-CTG AAC TGG ATA TGT ATT CAA GAA GG-3' E822K (g2596a) 5'-GGC TTG GAA ATA AGT AAA GAA ATT AAC G-3' E826K (g2608a) 5'-GAA GAA ATT AAC AAA GAA GAC TTA AAG-3' Selection primer BstBI 5'-CTC TGG GGT CCG GAA TGA CCG AC-3' Two primers were used for each mutagenesis reaction. Login to comment
77 Mutations detected in patients (I601F, L610S, A613T, D614G, I618T, L619S, H620P, H620Q, D622G, G628R, L633P, T665S, F693L, K698R, V754M, R766M, R792G, A800G, I807M, E822K and E826K) are indicated in bold and underlined, the PKA phosphorylation sites by an arrow and the two acidic domains are boxed. Login to comment
84 Interestingly, two mutations (H620Q and A800G) gave rise to chloride channels with significantly higher chloride transport activities. Login to comment
87 Maturation pattern of RD mutations and their associated phenotype found in patients with the indicated genotype (when the mutation is associated with CF, only the pancreas status is given) Mutation A-form B-form C-form Clinical data Genotype Phenotype Reference I601F + + - I601F/G542X PS M. Schwarz, personal communication L610S + + - Unknown Unknown A613T + + - Unknown Unknown D614G + + - D614G/unknown PI 14 I618T + + - I618T/dF508 PS G.R. Cutting, personal communication L619S + + - L619S/unknown PI B. Tümmler, personal communication H620P + + - H620P/R1158X PS M. Schwarz, personal communication H620Q + + + H620Q/dF508 PI T. Dörk, personal communication G622D + + + G622D/unknown Oligospermia J. Zielenski, personal communication G628R + + - Unknown Unknown L633P + + - L633P/3659delC M. Schwarz, personal communication D648V + + + D648V/3849+10kb C/T PI C. Ferec, personal communication T665S + + + Unknown Unknown F693L + + + F693L/W1282X Healthy C. Ferec; CF Genetic Analysis Consortium R766M + + + R766M/R792G CBAVD D. Glavac, personal communication R792G + + + R766M/R792G CBAVD D. Glavac, personal communication A800G + + + A800G/unknown CBAVD 34 I807M + + + I807M/unknown CBAVD Our observation E822K + + + E822K/unknown PI 35 E826K + + + E826K/unknown Thoracic sarcoidosis C. Bombieri, personal communication +, the protein matures up to that form; -, the protein does not reach the respective maturation step. Login to comment
97 G622D, R792G and E822K gave rise to a CFTR chloride channel with a significantly lower Po than wild-type CFTR; H620Q and A800G CFTR resulted in channels with significantly higher Po. Login to comment
114 When His620 was changed to Gln, noeffectonmaturationwasobserved.WhenHis620waschanged to Pro, an amino acid that dramatically affects the secondary structure of the protein and therefore possibly its correct folding, a non-maturing protein was obtained. Login to comment
123 Mutations that did not affect maturation (H620Q, G622D, D648V, T665S, F693L, R766M, R792G, A800G, I807M, E822K and E826K) were subsequently analysedat theelectrophysiologi- cal level. Login to comment
127 Strikingly, two mutations (H620Q and A800G) showed a significantly increased Po, when compared with wild-type CFTR. Login to comment
130 The 'hyperactive` disease-causing mutations described here, H620Q and A800G, do mature. Login to comment
136 Activation of single channels by expressing wild-type and mutant CFTR (H620Q). Login to comment
137 (A) Single channel current tracings of wild-type and H620Q CFTR before and after addition of the activation cocktail (1 µM IBMX and 0.1 µM forskolin) to the external site of the membrane patch. Login to comment

PMID: 22722932 [PubMed] Hudson RP et al: "Conformational changes relevant to channel activity and folding within the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

1 Results: H620Q mutation associated with increased channel Po, and the corrector/potentiator CFFT-001 both lead to similar conformational shifts in NBD1. Login to comment
5 NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between beta-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. Login to comment
7 Decreases in Tm from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. Login to comment
50 To probe the conformational effects of mutations and binding of small molecule modulators, we have applied a range of biophysical approaches to study H620Q and helix H9 variants of NBD1 and the interaction of the most soluble compound of a series of dual corrector/potentiator compounds provided by the Cystic Fibrosis Foundation, N-cyclohexyl-4-(6-methyl-3-pyridinyl) pyrimidine-2-amine, referred to here as CFFT-001. Login to comment
51 NMR backbone resonance assignments (82%) carried out on a human F508del NBD1 lacking the RI and ending at residue 646 (F508del NBD1 ⌬RI⌬RE) enable us to define conformational changes due to the H620Q mutation. Login to comment
53 Addition of CFFT-001 to F508del NBD1 ⌬RI⌬RE results in NBD1 conformational changes overlapping with those observed for the H620Q mutant protein that has higher open channel probability. Login to comment
54 Differential scanning calorimetry (DSC) data show a reduction in NBD1 thermal melting temperature for H620Q or in the presence of CFFT-001, demonstrating direct binding of the compound to a less thermostable conformation. Login to comment
57 EXPERIMENTAL PROCEDURES Protein Expression and Purification (NMR and DSC Studies)- Human NBD1 (387-646, ⌬405-436) constructs with or without Phe-508 and containing the H620Q mutation or deletion of helix H9(636-646) were expressed as His6-SUMO fusions at 16 °C in BL21(DE3) Codon Plus cells grown in minimal media with [15 N]NH4Cl and/or [13 C]glucose, for NMR studies, or LB for DSC studies, and purified as described previously (31, 32). Login to comment
97 Repeated measurements for the H620Q F508del sample gave identical Tm values to the first decimal place; the general reproducibility of NBD1 Tm values is within Ϯ0.3 °C. Login to comment
116 H620Q Variant Reduces Helicity of H8 and H9 at the C Terminus of F508del NBD1 ⌬RI⌬RE-To address possible conformational changes within NBD1 that may be relevant to channel activity and/or misfolding, we focused on the C-terminal beta-strands (S9 and S10) which have been shown to affect processing, activity and pharmacology of CFTR (56). Login to comment
117 In particular, an H620Q variant (in S9) originally identified in CF patients has an increased Po in single channels (56-58). Login to comment
118 Fig. 2, A and B, presents an overlay of NMR spectra for F508del NBD1 ⌬RI⌬RE and its H620Q variant (F508del H620Q NBD1 ⌬RI⌬RE). Login to comment
119 Arrows indicate a subset of peaks that shift upon H620Q mutation, including Glu-621, Gly-622, Gln-634, Leu-636, Ser-641, Leu-644, and Met-645. Login to comment
134 Chemical shifts in the absence of the mutation demonstrate that these helices are in equilibrium with a coil conformation (supplemental Fig. S4C) (59) and the chemical shift perturbations for the residues of the H8/H9 helices upon replacing His-620 with a Gln reflect a change in the equilibrium further toward the coil conformation. Login to comment
142 H620Q variant reduces helicity of H8 and H9 at the C terminus of F508del NBD1 ⌬RI⌬RE. Login to comment
143 A, overlay of 15 N-1 H correlation spectra at 500 MHz for F508del NBD1 ⌬RI⌬RE (black; background) and the H620Q variant, F508del H620Q NBD1 ⌬RI⌬RE (red; foreground). Login to comment
145 Asterisks show a separate subset of peaks that shift in the H620Q variant but do not shift upon addition of CFFT-001. Login to comment
147 C, same overlay as in B, with a third layer showing the addition of compound to the H620Q variant (green; foreground). Login to comment
149 E, ribbon diagram of WT NBD1 ⌬RI⌬RE (2PZE) with unassigned residues in cyan, and assigned residues that do not shift upon H620Q mutation are shown in light gray. Login to comment
161 Comparison of this set of peaks with a subset of those resulting from the H620Q mutation (compare Fig. 2 with Fig. 4) shows significant similarities, including both the identity of perturbed resonances and the direction of the chemical shift changes. Login to comment
162 These similarities are consistent with a common effect of the H620Q substitution and CFFT-001 on H8 and H9, although additional peak shifts reflecting other conformational changes in the mutant are present as well (Fig. 2A). Login to comment
165 Similar to the H620Q variant, residues on both surfaces of these helices are affected by CFFT-001, as opposed to one surface of the helix as one might expect for a direct drug interaction with the helix. Login to comment
197 Interestingly, titration of the compound into the F508del H620Q NBD1 ⌬RI⌬RE leads to resonances of H9 shifting more toward coil (Fig. 2C, arrows). Login to comment
198 Thus, the compound further pushes the conformational equilibrium shift already present in the H620Q variant, in a qualitatively additive fashion, and the mutation does not inhibit compound binding. Login to comment
227 In all of these binding modes, interaction of CFFT-001 with the surface of strands S3, S9, and S10 is not expected to be significantly perturbed by mutation of H620Q, which is adjacent to but not directly at the proposed binding surface (Fig. 6 and supplemental Fig. S6). Login to comment
230 The data in Fig. 7A illustrate that both the H620Q variant and the deletion of H9 reduce the thermal stability of NBD1. Login to comment
231 The midpoint of thermal denaturation, Tm, is reduced by ϳ1-2 degrees in each, comparing F508del NBD1 ⌬RI⌬RE (Tm in 2 mM ATP ϭ 49.4 °C and 5 mM ATP ϭ 51.0 °C) to either F508del H620Q NBD1 ⌬RI⌬RE (bottom curve, Tm in 2 mM ATP ϭ 47.1 °C) or F508del NBD1 ⌬RI⌬H9 (middle curve, Tm in 5 mM ATP ϭ 50.4 °C). Login to comment
244 Overall, the DSC data are in agreement with the effects of deletion of H9, an H620Q variant, and addition of CFFT-001 on NBD1 inferred from NMR data for these constructs. Login to comment
250 A, DSC traces for WT and F508del NBD1 ⌬RI⌬RE (upper curves) in the absence (solid lines) and presence (dashed lines) of CFFT-001; buffer includes 2 mM ATP. DSC traces for F508del NBD1 ⌬RI⌬H9 (middle curves) in the absence (solid line) and presence (dashed line) of CFFT-001; buffer includes 5 mM ATP. DSC traces for F508del H620Q NBD1 ⌬RI⌬RE (lower curve); buffer includes 2 mM ATP. Login to comment
264 NMR has enabled us to probe these effects at a residue-specific level and demonstrate that the H620Q substitution associated with higher channel open probability and a dual corrector/potentiator compound give rise to similar conformational changes within NBD1. Login to comment
266 The significance of the DSC results for the H620Q and H9 deletion can be better appreciated in the context of our current understanding of the NBD1 thermal unfolding pathway derived from a comprehensive analysis of previous DSC data on WT and F508del NBD1 (32, 64). Login to comment
268 The H620Q and ⌬H9 mutations also may reduce the thermodynamic stability of NBD1, or accelerate the rate of aggregation, or both. Login to comment
283 The H620Q variant, originally identified in CF patients demonstrating pancreatic insufficiency, has previously been shown to increase the Po of single CFTR channels, indicating an effect on the gating properties of CFTR (56-58). Login to comment
284 Both the H620Q variant and the CFFT-001 compound cause a shift of helices H8 and H9 from a preexisting helix-coil conformational equilibrium toward the coil state. These results suggest that perturbations affecting this conformational equilibrium coincide with conditions that promote channel opening and/or impede channel closing. Login to comment
285 Because the H620Q variant displays additional peak shifts relative to those observed for compound binding, there are certainly other consequences of the mutation. Login to comment
287 Although H620Q does not affect maturation/processing, an H620P mutation does, highlighting the importance of this position and the nature of the residue in it. Login to comment
294 Thus, we hypothesize that conformational changes, as elicited by H620Q or CFFT-001, result in a shift in an underlying conformational equilibrium of regulatory interactions that are normally involved in gating. Login to comment
296 This should promote NBD dimerization and lead to an enhanced open probability, increased channel activity for H620Q mutant channels and the potentiating effect of CFFT-001. Login to comment
297 It is possible that H620Q and CFFT-001 act to force an "unnatural" gating; however, the likelihood of this is low, considering that the portion of NBD1 affected appears to be a regulatory hot spot based on our sequence analysis and the observed effects of mutations here (56). Login to comment
305 Addition of compound or the H620Q mutation shifts this equilibrium by reducing H9 helicity and contacts with the NBD1, subsequently leading to release of the RE/R region from the dimerization interface, relieving the inhibition and facilitating NBD1/NBD2 heterodimerization and channel opening. Login to comment
316 The large number of substitutions in NBD1 that can suppress the F508del mutation supports such a general allosteric view of NBD1 with the structural changes we observe in the H620Q variant and upon CFFT-001 binding being one part of the NBD1 conformational equilibria. Login to comment
320 Because the H620Q mutation and CFFT-001 are both associated with an increase in the open probability of CFTR channels and because H8/H9 lead directly into the RE/R region within the context of full-length CFTR, we have hypothesized that this conformational shift at H8/H9 releases the R region from the NBD1/ NBD2 dimerization interface, allowing heterodimers to form and thereby enhancing channel open probability and possibly processing. Login to comment
48 To probe the conformational effects of mutations and binding of small molecule modulators, we have applied a range of biophysical approaches to study H620Q and helix H9 variants of NBD1 and the interaction of the most soluble compound of a series of dual corrector/potentiator compounds provided by the Cystic Fibrosis Foundation, N-cyclohexyl-4-(6-methyl-3-pyridinyl) pyrimidine-2-amine, referred to here as CFFT-001. Login to comment
49 NMR backbone resonance assignments (82%) carried out on a human F508del NBD1 lacking the RI and ending at residue 646 (F508del NBD1 èc;RIèc;RE) enable us to define conformational changes due to the H620Q mutation. Login to comment
52 Differential scanning calorimetry (DSC) data show a reduction in NBD1 thermal melting temperature for H620Q or in the presence of CFFT-001, demonstrating direct binding of the compound to a less thermostable conformation. Login to comment
55 EXPERIMENTAL PROCEDURES Protein Expression and Purification (NMR and DSC Studies)- Human NBD1 (387-646, èc;405-436) constructs with or without Phe-508 and containing the H620Q mutation or deletion of helix H9(636-646) were expressed as His6-SUMO fusions at 16 &#b0;C in BL21(DE3) Codon Plus cells grown in minimal media with [15 N]NH4Cl and/or [13 C]glucose, for NMR studies, or LB for DSC studies, and purified as described previously (31, 32). Login to comment
141 H620Q variant reduces helicity of H8 and H9 at the C terminus of F508del NBD1 èc;RIèc;RE. Login to comment
144 Asterisks show a separate subset of peaks that shift in the H620Q variant but do not shift upon addition of CFFT-001. Login to comment
146 C, same overlay as in B, with a third layer showing the addition of compound to the H620Q variant (green; foreground). Login to comment
148 E, ribbon diagram of WT NBD1 èc;RIèc;RE (2PZE) with unassigned residues in cyan, and assigned residues that do not shift upon H620Q mutation are shown in light gray. Login to comment
160 Comparison of this set of peaks with a subset of those resulting from the H620Q mutation (compare Fig. 2 with Fig. 4) shows significant similarities, including both the identity of perturbed resonances and the direction of the chemical shift changes. Login to comment
164 Similar to the H620Q variant, residues on both surfaces of these helices are affected by CFFT-001, as opposed to one surface of the helix as one might expect for a direct drug interaction with the helix. Login to comment
196 Interestingly, titration of the compound into the F508del H620Q NBD1 èc;RIèc;RE leads to resonances of H9 shifting more toward coil (Fig. 2C, arrows). Login to comment
225 In all of these binding modes, interaction of CFFT-001 with the surface of strands S3, S9, and S10 is not expected to be significantly perturbed by mutation of H620Q, which is adjacent to but not directly at the proposed binding surface (Fig. 6 and supplemental Fig. S6). Login to comment
228 The data in Fig. 7A illustrate that both the H620Q variant and the deletion of H9 reduce the thermal stability of NBD1. Login to comment
229 The midpoint of thermal denaturation, Tm, is reduced by b03;1-2 degrees in each, comparing F508del NBD1 èc;RIèc;RE (Tm in 2 mM ATP afd; 49.4 &#b0;C and 5 mM ATP afd; 51.0 &#b0;C) to either F508del H620Q NBD1 èc;RIèc;RE (bottom curve, Tm in 2 mM ATP afd; 47.1 &#b0;C) or F508del NBD1 èc;RIèc;H9 (middle curve, Tm in 5 mM ATP afd; 50.4 &#b0;C). Login to comment
242 Overall, the DSC data are in agreement with the effects of deletion of H9, an H620Q variant, and addition of CFFT-001 on NBD1 inferred from NMR data for these constructs. Login to comment
248 A, DSC traces for WT and F508del NBD1 èc;RIèc;RE (upper curves) in the absence (solid lines) and presence (dashed lines) of CFFT-001; buffer includes 2 mM ATP. DSC traces for F508del NBD1 èc;RIèc;H9 (middle curves) in the absence (solid line) and presence (dashed line) of CFFT-001; buffer includes 5 mM ATP. DSC traces for F508del H620Q NBD1 èc;RIèc;RE (lower curve); buffer includes 2 mM ATP. Login to comment
262 NMR has enabled us to probe these effects at a residue-specific level and demonstrate that the H620Q substitution associated with higher channel open probability and a dual corrector/potentiator compound give rise to similar conformational changes within NBD1. Login to comment
281 The H620Q variant, originally identified in CF patients demonstrating pancreatic insufficiency, has previously been shown to increase the Po of single CFTR channels, indicating an effect on the gating properties of CFTR (56-58). Login to comment
282 Both the H620Q variant and the CFFT-001 compound cause a shift of helices H8 and H9 from a preexisting helix-coil conformational equilibrium toward the coil state. These results suggest that perturbations affecting this conformational equilibrium coincide with conditions that promote channel opening and/or impede channel closing. Login to comment
292 Thus, we hypothesize that conformational changes, as elicited by H620Q or CFFT-001, result in a shift in an underlying conformational equilibrium of regulatory interactions that are normally involved in gating. Login to comment
295 It is possible that H620Q and CFFT-001 act to force an "unnatural" gating; however, the likelihood of this is low, considering that the portion of NBD1 affected appears to be a regulatory hot spot based on our sequence analysis and the observed effects of mutations here (56). Login to comment
303 Addition of compound or the H620Q mutation shifts this equilibrium by reducing H9 helicity and contacts with the NBD1, subsequently leading to release of the RE/R region from the dimerization interface, relieving the inhibition and facilitating NBD1/NBD2 heterodimerization and channel opening. Login to comment
314 The large number of substitutions in NBD1 that can suppress the F508del mutation supports such a general allosteric view of NBD1 with the structural changes we observe in the H620Q variant and upon CFFT-001 binding being one part of the NBD1 conformational equilibria. Login to comment
318 Because the H620Q mutation and CFFT-001 are both associated with an increase in the open probability of CFTR channels and because H8/H9 lead directly into the RE/R region within the context of full-length CFTR, we have hypothesized that this conformational shift at H8/H9 releases the R region from the NBD1/ NBD2 dimerization interface, allowing heterodimers to form and thereby enhancing channel open probability and possibly processing. Login to comment

PMID: 19019741 [PubMed] Tang L et al: "Mechanism of direct bicarbonate transport by the CFTR anion channel."

No. Sentence Comment

101 Fig. 5 shows macroscopic currents carried by three of these mutants, two associated with loss of cellular HCO3 - transport and PI disease (G178R, H620Q) and one with retained HCO3 - transport and PS disease (G551 S) [10]. Login to comment
103 It can be seen that each of these mutants is capable of mediating HCO3 - efflux; in fact, reversal potential measurements indicate PHCO3/PCl values of 0.289±0.038 (n=3) for G178R, 0.264±0.035 (n=3) for G551S, and 0.237±0.033 (n=3) for H620Q, none of which were significantly different from the value of 0.250±0.037 (n=4) estimated from wild type under the same conditions (see Fig. 1B). Login to comment
138 Bicarbonate permeability of CF mutant forms of CFTR Example leak-subtracted macroscopic I-V relationships for G178R, G551S, and H620Q-CFTR under the same ionic conditions used in Fig. 1B. Login to comment
146 Our direct measurements of CFTR HCO3 - currents also showed no change in HCO3 - permeability in three CF-associated CFTR mutants (G178R, G551S, H620Q; Fig. 5) that were previously associated with different effects on cellular HCO3 - transport relative to Cl- transport [10]. Login to comment
147 This result suggests that loss of direct HCO3 - permeation through the channel is not responsible for the selective loss of HCO3 - transport relative to Cl- transport previously associated with the severe, PI mutations G178R and H620Q. Login to comment

PMID: 9849891 [PubMed] Wei L et al: "Phosphorylation site independent single R-domain mutations affect CFTR channel activity."

No. Sentence Comment

1 All mutations were found in cystic fibrosis (CF) patients (H620Q, E822K and E826K). Login to comment
2 The macroscopic CFTR chloride conductance induced by phosphorylation was significantly enhanced in Xenopus oocytes injected with mRNA of H620Q but reduced in the E822K and E826K mutants compared to wild type CFTR. Login to comment
4 Cell attached single channel studies in COS cells revealed that both open channel probability and/or the number of functional channels were either higher (H620Q) or lower (E822K and E826K) than in wild type CFTR. Login to comment
25 Three di¡erent mutations, t1992g (= H620Q), g2596a (= E822K) and g2608a (= E826K), were introduced using the Transformer Site-Directed Mutagenesis kit (Clontech). Login to comment
76 COS cells were transfected with wt CFTR, E822K CFTR (top), H620Q CFTR (bottom) and E826K CFTR (bottom) and selected for 2 weeks with G418. Login to comment
88 In this study, the maturation pattern of three mutant R-domain proteins (H620Q-CFTR, E822K-CFTR and E826K-CFTR) has been characterized. Login to comment
92 Whole cells currents of mutants in Xenopus oocytes Whole cell membrane currents were recorded from Xenopus oocytes injected with RNA transcribed from either wild type or mutant (H620Q, E822K, E826K) constructs. Login to comment
101 Oocytes expressing the mutant R-domain protein (H620Q), in which a predominantly positively charged histidine was substituted by a less charged glutamine (at pH 7.2), showed a much larger conductance activated by application of phos-cock. Login to comment
104 The conductance activated by phos-cock in H620Q expressed cells was 27.84 þ 5.7 WS (n = 6). Login to comment
134 We found that the open probability of the E822K and E826K mutants was signi'cantly lower than that of wild type CFTR, whereas that of the H620Q mutant was strongly enhanced compared to wild type (not shown). Login to comment
136 The average number of activated channels is 2.6 þ 0.306 (n = 10) for wild-type CFTR, 3.51 þ 0.428 (n = 6) for H620Q, 1.0 þ 0.000 (n = 4) for E822K and 1.66 þ 0.211 (n = 6) for E826K. Login to comment
145 A comparison of the three R-domain mutants leads to a remarkable conclusion: both mutations, E822K and E826K, in which negatively charged glutamic acids were replaced by positively charged lysine had a signi'cantly reduced CFTR channel activity, whereas the H620Q mutation, in which positively charged histidine was replaced by the more neutral amino acid glutamine, had a much higher channel activity. Login to comment
74 COS cells were transfected with wt CFTR, E822K CFTR (top), H620Q CFTR (bottom) and E826K CFTR (bottom) and selected for 2 weeks with G418. Login to comment
86 In this study, the maturation pattern of three mutant R-domain proteins (H620Q-CFTR, E822K-CFTR and E826K-CFTR) has been characterized. Login to comment
90 Whole cells currents of mutants in Xenopus oocytes Whole cell membrane currents were recorded from Xenopus oocytes injected with RNA transcribed from either wild type or mutant (H620Q, E822K, E826K) constructs. Login to comment
99 Oocytes expressing the mutant R-domain protein (H620Q), in which a predominantly positively charged histidine was substituted by a less charged glutamine (at pH 7.2), showed a much larger conductance activated by application of phos-cock. Login to comment
102 The conductance activated by phos-cock in H620Q expressed cells was 27.84 &#fe; 5.7 WS (n = 6). Login to comment
132 We found that the open probability of the E822K and E826K mutants was signi'cantly lower than that of wild type CFTR, whereas that of the H620Q mutant was strongly enhanced compared to wild type (not shown). Login to comment
143 A comparison of the three R-domain mutants leads to a remarkable conclusion: both mutations, E822K and E826K, in which negatively charged glutamic acids were replaced by positively charged lysine had a signi'cantly reduced CFTR channel activity, whereas the H620Q mutation, in which positively charged histidine was replaced by the more neutral amino acid glutamine, had a much higher channel activity. Login to comment

PMID: 11448786 [PubMed] Wine JJ et al: "Cystic fibrosis: the 'bicarbonate before chloride' hypothesis."

No. Sentence Comment

52 Ion transport (% WT) 42 41 69 75 >100 >100 98 + 103 100 + + 120 Pancreatic sufficient Pancreatic insufficient Bicarbonate Chloride - intermediate Chloride - high Unknown WT D648V R117H R1070Q H949Y G551S H620Q I148T A1067T G178R G970R S1255P G1244E G551D G1349D 0 0.5 1 1.5 2 2.5 Current Biology ࢞F508 Dispatch R absence of the vas deferens [16]. Login to comment
76 Critical information was provided by Thilo D&#f6;rk (H620Q); R. Moss (R117H & G551D homozygotes); David Kessler, Theresa Grebe and Elizabeth Perkett (D648V); Monica Brooks and contributors to the Cystic Fibrosis Foundation Registry (G178R and G1244E); Aleksey Savov and Luba Kalaydjieva (R1070Q); and Christiane De Boeck and Harry Cuppens (G970R). Login to comment

PMID: 24727426 [PubMed] Wang Y et al: "Understanding how cystic fibrosis mutations disrupt CFTR function: from single molecules to animal models."

No. Sentence Comment

2023 Interestingly, the authors demonstrated that G178R and H620Q, two CF-PI mutations that disrupt cellular HCO3 - transport, were without effect on CFTR-mediated HCO3 - transport, arguing that CF mutations influence HCO3 --transport indirectly. Login to comment

PMID: 24855632 [PubMed] Kirby EF et al: "Enhancing the Potency of F508del Correction: A Multi-Layer Combinational Approach to Drug Discovery for Cystic Fibrosis."

No. Sentence Comment

84 H620Q substitution at the C-terminal region of NBD1 is known to increase the channel open probability of CFTR [34]. Login to comment
86 NMR studies of human F508del NBD1 either carrying the H620Q mutation or treated with CFFT-001 revealed a similar disruption of the interactions between the (b2;-strands S3, S9, and S10 and the C-terminal helices H8 and H9, suggesting a similar conformational shift [35]. Login to comment
87 Differential scanning calorimetry showed a reduced Tm of NBD1 Kirby et al. Page 5 in the presence of H620Q or CFFT-001, suggesting that CFFT-001 binds to a less stable conformation of NBD1 [35]. Login to comment