ABCC7 p.Arg334Asp
| ClinVar: |
c.1000C>T
,
p.Arg334Trp
D
, Pathogenic
c.1001G>T , p.Arg334Leu ? , not provided c.1001G>A , p.Arg334Gln ? , not provided |
| CF databases: |
c.1000C>T
,
p.Arg334Trp
D
, CF-causing ; CFTR1: This mutation has been found in two Spanish CF chromosomes. One of the patients has the [delta]F508 mutation in the other chromosome and the other patient does not. We have not found this mutation on 30 normal chromosomes with the same haplotype, and in 88 CF chromosomes without the [delta]F508, and in 24 with the [delta]F508. The mutation destroys a MapI site and is easily identified by agarose gel electrophoresis after PCR with intron primers.
c.1001G>A , p.Arg334Gln (CFTR1) ? , The above mutation was found by DGGE and direct sequencing in Caucasian patients. c.1001G>T , p.Arg334Leu (CFTR1) D , Missense mutation E334L was detected in a German CBAVD patient who is compound heterozygous for the R334L and I336K mutations. |
| Predicted by SNAP2: | A: D (91%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (91%), I: D (95%), K: D (85%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (91%), S: D (91%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: N, M: N, N: N, P: N, Q: N, S: N, T: N, V: D, W: D, Y: D, |
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[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2012 Mar 20;51(11):2199-212. Epub 2012 Mar 7. Norimatsu Y, Ivetac A, Alexander C, Kirkham J, O'Donnell N, Dawson DC, Sansom MS
Cystic fibrosis transmembrane conductance regulator: a molecular model defines the architecture of the anion conduction path and locates a "bottleneck" in the pore.
Biochemistry. 2012 Mar 20;51(11):2199-212. Epub 2012 Mar 7., [PMID:22352759]
Abstract [show]
We developed molecular models for the cystic fibrosis transmembrane conductance regulator chloride channel based on the prokaryotic ABC transporter, Sav1866. Here we analyze predicted pore geometry and side-chain orientations for TM3, TM6, TM9, and TM12, with particular attention being paid to the location of the rate-limiting barrier for anion conduction. Side-chain orientations assayed by cysteine scanning were found to be from 77 to 90% in accord with model predictions. The predicted geometry of the anion conduction path was defined by a space-filling model of the pore and confirmed by visualizing the distribution of water molecules from a molecular dynamics simulation. The pore shape is that of an asymmetric hourglass, comprising a shallow outward-facing vestibule that tapers rapidly toward a narrow "bottleneck" linking the outer vestibule to a large inner cavity extending toward the cytoplasmic extent of the lipid bilayer. The junction between the outer vestibule and the bottleneck features an outward-facing rim marked by T338 in TM6 and I1131 in TM12, consistent with the observation that cysteines at both of these locations reacted with both channel-permeant and channel-impermeant, thiol-directed reagents. Conversely, cysteines substituted for S341 in TM6 or T1134 in TM12, predicted by the model to lie below the rim of the bottleneck, were found to react exclusively with channel-permeant reagents applied from the extracellular side. The predicted dimensions of the bottleneck are consistent with the demonstrated permeation of Cl(-), pseudohalide anions, water, and urea.
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No. Sentence Comment
367 Similarly, while both extracellular MTSET+ and MTSES- react with a cysteine at position 338, the ratio of the rates of reaction (kMTSET +/kMTSES -) of these oppositely charged reagents was <1.0 for T338C/wt CFTR and >1.0 for T338C/ R334D CFTR as expected if the charge at this position makes a major contribution to the electrostatic potential at the outer rim of the bottleneck.40 Studies of the impact of covalent and noncovalent modifications at position 338 also argue that the electrostatic potential at this site, just on the outward-facing lip of the bottleneck, is critical for anion conduction.
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ABCC7 p.Arg334Asp 22352759:367:232
status: NEW[hide] CFTR: a cysteine at position 338 in TM6 senses a p... Biophys J. 2004 Dec;87(6):3826-41. Epub 2004 Sep 10. Liu X, Zhang ZR, Fuller MD, Billingsley J, McCarty NA, Dawson DC
CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore.
Biophys J. 2004 Dec;87(6):3826-41. Epub 2004 Sep 10., [PMID:15361410]
Abstract [show]
We investigated the accessibility to protons and thiol-directed reagents of a cysteine substituted at position 338 in transmembrane segment 6 (TM6) of CFTR to test the hypothesis that T338 resides in the pore. Xenopus oocytes expressing T338C CFTR exhibited pH-dependent changes in gCl and I-V shape that were specific to the substituted cysteine. The apparent pKa of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. The pKa was shifted toward alkaline values when a nearby positive charge (R334) was substituted with neutral or negatively charged residues, consistent with the predicted influence of the positive charge of R334, and perhaps other residues, on the titration of a cysteine at 338. The relative rates of chemical modification of T338C CFTR by MTSET+ and MTSES- were also altered by the charge at 334. These observations support a model for CFTR that places T338 within the anion conduction path. The apparent pKa of a cysteine substituted at 338 and the relative rates of reaction of charged thiol-directed reagents provide a crude measure of a positive electrostatic potential that may be due to R334 and other residues near this position in the pore.
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No. Sentence Comment
184 To investigate the effect of charge at position 334 on the titration behavior of T338C CFTR, we examined the conductance of oocytes expressing double mutants, T338C/R334A, T338C/R334E, and T338C/R334D CFTR.
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ABCC7 p.Arg334Asp 15361410:184:195
status: NEW185 Shown in Fig. 8 A are representative titration curves for the conductance for T338C CFTR and two of these double mutants (n ¼ 5 each).
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ABCC7 p.Arg334Asp 15361410:185:195
status: NEW187 The substitution of acidic residues, however, did not result in a large additional shift of the apparent pKa to more alkaline values (8.84 6 0.05 for T338C/R334D CFTR, n ¼ 4 and 8.96 6 0.08 for T338C/R334E CFTR, n ¼ 5).
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ABCC7 p.Arg334Asp 15361410:187:156
status: NEW228 in which the arginine at 334 was replaced by aspartic acid (T338C/R334D CFTR).
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ABCC7 p.Arg334Asp 15361410:228:66
status: NEW229 Note that covalent modification of T338C CFTR with either reagent decreased the macroscopic conductance.
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ABCC7 p.Arg334Asp 15361410:229:66
status: NEW234 Introduction of negative charge at 334 (in T338C/R334D) reversed the relative reaction rates so that modification by MTSET1 was more rapid.
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ABCC7 p.Arg334Asp 15361410:234:49
status: NEW237 The time constants for MTSET1 and MTSESÿ modification of T338C/R334D CFTR ([MTS] ¼ 25 mM) averaged 39.8 6 15.8 s (n ¼ 3) and 641 6 27.7 s (n ¼ 3), respectively.
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ABCC7 p.Arg334Asp 15361410:237:68
status: NEW238 Calculations based on a simple kinetic model for the ratio of the rates of reaction of MTSET1 and MTSESÿ , including a correction for the difference in the intrinsic rates of the MTS-thiolate reactions (see Discussion), suggested that charged reagents modifying T338C CFTR sensed an electrostatic potential that was ;54 mV positive with respect to the bath.
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ABCC7 p.Arg334Asp 15361410:238:63
status: NEW247 The effective concentration of MTSET1 in the pipette likely varied because the half-life of MTSET1 is only ;10 min at room temperature according to Stauffer FIGURE 9 Representative time course of modification of T338C CFTR and T338C/R334D CFTR conductance by MTSET1 (n ¼ 3) and MTSESÿ (n ¼ 3).
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ABCC7 p.Arg334Asp 15361410:247:233
status: NEW250 (B) After activation, oocytes expressing T338C/R334D CFTR were first exposed to reducing agents (2-ME) followed by a wash and were then exposed to 25 mM MTSET1 (s) or MTSESÿ (d).
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ABCC7 p.Arg334Asp 15361410:250:47
status: NEW345 The ratio of the rates of modification is given by Eq. 7 (see Supplementary Material), k MTSES k MTSET ¼ k MTSES i k MTSET i exp 2 F RT Cq o : (7) Using the ratio for kMTSES i =kMTSET i of 1/12 measured for 2-ME (Karlin and Akabas, 1998), the relative rates of modification for these two compounds yields a value for Cq o of 54 mV for T338C CFTR and ÿ4 mV for T338C/R334D CFTR, comparable to the values estimated from the shift in cysteine pKa resulting from the replacement of arginine by alanine or aspartic acid.
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ABCC7 p.Arg334Asp 15361410:345:380
status: NEW347 Prediction of the electrostatic effects of R334 If we ignore the possible effects of structural changes in the CFTR protein produced by amino acid substitution, then the change in Cq o calculated from the difference in the pKa of T338C/R334 and T338C/R334A can be taken to be a crude measure of CR334 o ; the component of Cq o due to the native arginine, and we can compare the value derived experimentally with that predicted on the basis of first principles.
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ABCC7 p.Arg334Asp 15361410:347:374
status: NEW188 The substitution of acidic residues, however, did not result in a large additional shift of the apparent pKa to more alkaline values (8.84 6 0.05 for T338C/R334D CFTR, n &#bc; 4 and 8.96 6 0.08 for T338C/R334E CFTR, n &#bc; 5).
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ABCC7 p.Arg334Asp 15361410:188:156
status: NEW235 Introduction of negative charge at 334 (in T338C/R334D) reversed the relative reaction rates so that modification by MTSET1 was more rapid.
X
ABCC7 p.Arg334Asp 15361410:235:49
status: NEW248 The effective concentration of MTSET1 in the pipette likely varied because the half-life of MTSET1 is only ;10 min at room temperature according to Stauffer FIGURE 9 Representative time course of modification of T338C CFTR and T338C/R334D CFTR conductance by MTSET1 (n &#bc; 3) and MTSES (n &#bc; 3).
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ABCC7 p.Arg334Asp 15361410:248:233
status: NEW251 (B) After activation, oocytes expressing T338C/R334D CFTR were first exposed to reducing agents (2-ME) followed by a wash and were then exposed to 25 mM MTSET1 (s) or MTSES (d).
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ABCC7 p.Arg334Asp 15361410:251:47
status: NEW