ABCC7 p.Lys95Glu
| Predicted by SNAP2: | A: D (75%), C: D (75%), D: D (91%), E: D (85%), F: D (85%), G: D (85%), H: D (53%), I: D (80%), L: D (80%), M: D (75%), N: D (80%), P: D (91%), Q: D (75%), R: N (66%), S: D (63%), T: D (80%), V: D (80%), W: D (91%), Y: D (71%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: D, G: D, H: N, I: D, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[hide] Location of a common inhibitor binding site in the... J Biol Chem. 2005 Mar 11;280(10):8945-50. Epub 2005 Jan 5. Linsdell P
Location of a common inhibitor binding site in the cytoplasmic vestibule of the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Biol Chem. 2005 Mar 11;280(10):8945-50. Epub 2005 Jan 5., 2005-03-11 [PMID:15634668]
Abstract [show]
Chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is inhibited by a broad range of organic anions that enter the channel pore from its cytoplasmic end, physically occluding the Cl- permeation pathway. These open channel blocker molecules are presumed to bind within a relatively wide pore inner vestibule that shows little discrimination between different large anions. The present study uses patch clamp recording to identify a pore-lining lysine residue, Lys-95, that acts to attract large blocker molecules into this inner vestibule. Mutations that remove the fixed positive charge associated with this amino acid residue dramatically weaken the blocking effects of five structurally unrelated open channel blockers (glibenclamide, 4,4'-dinitrostilbene-2,2'-disulfonic acid, lonidamine, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and taurolithocholate-3-sulfate) when applied to the cytoplasmic face of the membrane. Mutagenesis of Lys-95 also induced amino acid side chain charge-dependent rectification of the macroscopic current-voltage relationship, consistent with the fixed positive charge on this residue normally acting to attract Cl- ions from the intracellular solution into the pore. These results identify Lys-95 as playing an important role in attracting permeant anions into the channel pore inner vestibule, probably by an electrostatic mechanism. This same electrostatic attraction mechanism also acts to attract larger anionic molecules into the relatively wide inner vestibule, where these substances bind to block Cl- permeation. Thus, structurally diverse open channel blockers of CFTR appear to share a common molecular mechanism of action that involves interaction with a positively charged amino acid side chain located in the inner vestibule of the pore.
Comments [show]
None has been submitted yet.
No. Sentence Comment
69 In a striking parallel with previous findings involving Arg-334 (13, 16), mutagenesis of Lys-95 had a strongly charge-dependent effect on I-V rectification (Fig. 1); the charge-conservative K95R, like wild type CFTR, showed a practically linear I-V relationship, whereas all of the other mutations, and especially the charge-reversing K95E, caused significant outward rectification.
X
ABCC7 p.Lys95Glu 15634668:69:335
status: NEW91 These results, using a number of different amino acid substitutions of Lys-95, strongly suggest that side chain charge at this position is important in controlling the apparent affinity of glibenclamide block; the apparent Kd at -100 mV was not affected in the charge-conservative K95R but was significantly increased in charge-neutralizing mutants (K95A, K95C, K95Q) and most strongly increased in the charge-reversing K95E mutant.
X
ABCC7 p.Lys95Glu 15634668:91:420
status: NEW[hide] Curcumin opens cystic fibrosis transmembrane condu... J Biol Chem. 2007 Feb 16;282(7):4533-44. Epub 2006 Dec 18. Wang W, Bernard K, Li G, Kirk KL
Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains.
J Biol Chem. 2007 Feb 16;282(7):4533-44. Epub 2006 Dec 18., 2007-02-16 [PMID:17178710]
Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt transport across epithelia. Channel opening normally requires ATP binding to both nucleotide-binding domains (NBDs), probable dimerization of the two NBDs, and phosphorylation of the R domain. How phosphorylation controls channel gating is unknown. Loss-of-function mutations in the CFTR gene cause cystic fibrosis; thus, there is considerable interest in compounds that improve mutant CFTR function. Here we investigated the mechanism by which CFTR is activated by curcumin, a natural compound found in turmeric. Curcumin opened CFTR channels by a novel mechanism that required neither ATP nor the second nucleotide-binding domain (NBD2). Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. The stimulation of NBD2 deletion mutants by curcumin was strongly inhibited by ATP binding to NBD1, which implicates NBD1 as a plausible activation site. Curcumin activation became irreversible during prolonged exposure to this compound following which persistently activated channels gated dynamically in the absence of any agonist. Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization.
Comments [show]
None has been submitted yet.
No. Sentence Comment
143 D, curcumin also stimulates K95E/⌬1198-CFTR currents, which showstrongoutwardrectification.Inset,I-Vcurvesforcontrol(curve1),afteraddingcurcumin(curve2),afterre-addingcurcuminwithnoATP(curve3),andafter curcuminwashout(curve4).E,curcumintitrationfor⌬1198-CFTRchannelsintheabsenceofbathATP.Inset,meantitrationdatafittoasingleMichaelis-Menten function(EC50 ϭ20.8Ϯ5.4M,nϭ4).F,curcuminalsoactivatesW1282X-CFTRchannels.Alloftherecordsarerepresentativeofatleastfourexperimentsexcept D (n ϭ 2).
X
ABCC7 p.Lys95Glu 17178710:143:28
status: NEW144 ATP-independent CFTR Channel Gating Because the ⌬1198-CFTR construct is new to the CFTR field, we validated that the currents that are mediated by this construct are authentic CFTR currents in two ways: (i) by performing unitary currents recordings, which confirmed that this NBD2 deletion construct exhibits the appropriate single channel conductance (6-8 pS) and linear I-V behavior expected for CFTR channels (Fig. 3) (5, 6) and (ii) by introducing a point mutation (K95E) in this deletion construct that had been shown by Linsdell (30) to induce strong outward rectification of the currents mediated by the full-length channel.
X
ABCC7 p.Lys95Glu 17178710:144:479
status: NEW145 Fig. 2D shows that the currents mediated by K95E/⌬1198-CFTR exhibit strong outward rectification, as expected, and that these rectifying currents are robustly activated by curcumin.
X
ABCC7 p.Lys95Glu 17178710:145:44
status: NEW[hide] Halide permeation in wild-type and mutant cystic f... J Gen Physiol. 1997 Oct;110(4):341-54. Tabcharani JA, Linsdell P, Hanrahan JW
Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels.
J Gen Physiol. 1997 Oct;110(4):341-54., [PMID:9379167]
Abstract [show]
Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22 degrees C or 8.7 pS at 37 degrees C. The current-voltage relationship became linear when patches were excised into symmetrical, -tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22 degrees C to 10.9 pS at 37 degrees C. The conductance at 22 degrees C was approximately 1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl activity was hyperbolic and well fitted by a Michaelis-Menten-type function having a of approximately 38 mM and maximum conductance of 10 pS at 22 degrees C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P/P = 0.003-0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P(1.8) > P(1. 3) > P(1.0) > P(0.17), consistent with a "weak field strength" selectivity site. The same sequence was obtained for external halides, although inward F flow was not observed. Iodide currents were protocol dependent and became blocked after 1-2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I/Cl permeability ratio (P/P< 0.4). The switch to low I permeability was enhanced at potentials that favored Cl entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl and I ions may influence I permeation and be responsible for the wide range of P/P ratios that have been reported for the CFTR channel. The low P/P ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a "weak field strength" anion selectivity sequence.
Comments [show]
None has been submitted yet.
No. Sentence Comment
264 Arg347 May Contribute to a Weak Field Strength Site for Iodide High macroscopic PI/PCl ratios have been reported previously for CFTR channels in which positively charged residues in the membrane spanning regions were mutated to negatively charged residues (K95E, 1.43; K335E, 1.37; R347E, 0.9; R1030E, 0.81; Anderson et al., 1991).
X
ABCC7 p.Lys95Glu 9379167:264:257
status: NEW289 Arg347 May Contribute to a Weak Field Strength Site for Iodide High macroscopic PI/PCl ratios have been reported previously for CFTR channels in which positively charged residues in the membrane spanning regions were mutated to negatively charged residues (K95E, 1.43; K335E, 1.37; R347E, 0.9; R1030E, 0.81; Anderson et al., 1991).
X
ABCC7 p.Lys95Glu 9379167:289:257
status: NEW