ABCC7 p.Arg334Asp
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PMID: 22352759
[PubMed]
Norimatsu Y et al: "Cystic fibrosis transmembrane conductance regulator: a molecular model defines the architecture of the anion conduction path and locates a "bottleneck" in the pore."
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
PMID: 15361410
[PubMed]
Liu X et al: "CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore."
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.
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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