ABCMdb

ABCC7 p.Arg334Ala

None has been submitted yet.

Publications

PMID: 11585852 [PubMed] Smith SS et al: "CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction."

No. Sentence Comment

187 MTSES, MTSET, or MTSEA (100 ␮M or 1 mM) were added to the perfusate of oocytes expressing R334A or R334Q CFTR and produced no discernible effect on conductance (unpublished data). Login to comment

PMID: 11927667 [PubMed] Gong X et al: "Molecular determinants of Au(CN)(2)(-) binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl(-) channel pore."

No. Sentence Comment

82 Two other mutants were also transfected into BHK cells; however, even in the presence of PPi, currents carried by R334A and T351A were too small for proper analysis (not shown). Login to comment

PMID: 12679372 [PubMed] Gong X et al: "Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore."

No. Sentence Comment

51 Previously we reported that the mutant R334A could not be expressed in BHK cells (Gong et al. 2002a); in the course of the present study, we confirmed this previous finding, but did find that, in addition to R334C, five other mutants could be studied (Fig. 4). Login to comment

PMID: 12745925 [PubMed] Gupta J et al: "Extent of the selectivity filter conferred by the sixth transmembrane region in the CFTR chloride channel pore."

No. Sentence Comment

124 In most (six of eight) cases, alanine substitution was employed; however, we have previously found that the mutants R334A [15] and T339A [22] fail to express in BHK cells, and for these residues mutants which gave adequate current expression (R334C, T339V) were studied. Login to comment

PMID: 14598388 [PubMed] Liu X et al: "CFTR: what's it like inside the pore?"

No. Sentence Comment

118 Changing the charge at position 334 either by modification of R334C/T338H CFTR with polar thiol reactive reagents or by amino acid substitution (R334A/T338C) shifts the titration curve in a direction that was predicted on the basis of a nearby positive charge being able to stabilize a titratable group (Liu et al., 2001). Login to comment

PMID: 15504721 [PubMed] Ge N et al: "Direct comparison of the functional roles played by different transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

45 Previously we showed that the TM6 mutant R334A did not express but characterized several other Arg-334 mutants (10, 28); in the present study we have used the charge conservative R334K mutant. Login to comment

PMID: 18366345 [PubMed] Caci E et al: "Evidence for direct CFTR inhibition by CFTR(inh)-172 based on Arg347 mutagenesis."

No. Sentence Comment

111 R334A and S341A showed reduced anion transport, although this was significantly greater than cells transfected with the fluorescent protein alone (Figure 1B). Login to comment
127 CFTR form CFTRinh-172 Ki (μM) Hill coefficient I- influx (mM/s) n Wild-type 1.32 + - 0.25 1.03 + - 0.07 0.1336 + - 0.0107 10 S341A 0.57 + - 0.17 1.21 + - 0.37 0.0297 + - 0.0064 4 T338A 3.20 + - 0.86 1.13 + - 0.20 0.1260 + - 0.0225 4 R347A 44.98 + - 4.71** 0.91 + - 0.04 0.1288 + - 0.0154 7 R334A 2.39 + - 0.74 0.93 + - 017 0.0313 + - 0.062 4 A349S 1.23 + - 0.41 1.11 + - 0.25 0.1500 + - 0.011 4 R347D >50 Not determined 0.1160 + - 0.0136 7 R347D/D924R >50 Not determined 0.1008 + - 0.0504 4 R347C >50 Not determined 0.1437 + - 0.0123 4 Mock 0.003 + - 0.001 10 introduced a mutation at position 349 (an alanine residue replaced by a serine residue). Login to comment
143 FRT cells were stably transfected with wild-type, R334A, R347A and R347D CFTR, and transepithelial Cl- currents were measured. Login to comment
157 For comparison, the sensitivity of the R334A mutant was not significantly altered (Ki = 0.50 +- 0.18 μM, n = 6; Figures 3C and 3E), in agreement with the fluorescence assay results. Login to comment
158 Interestingly, the R347D mutant, although insensitive to CFTRinh-172, was fully inhibited by the open-channel blocker GlyH-101 Figure 3 CFTR Cl- current inhibition by CFTRinh-172 (A-D) Representative traces showing recordings of transepithelial Cl- currents measured in FRT cells with stable expression of wild-type (WT), R347A, R334A and R347D-CFTR. Cells were first stimulated with 20 μM forskolin to activate CFTR and then tested with increasing concentrations of CFTRinh-172. Login to comment

PMID: 22160394 [PubMed] Cui G et al: "Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers."

No. Sentence Comment

57 For simplification, names of mutant channels are often abbreviated (e.g., R334A is R334A-CFTR). Login to comment
119 The major effects of increasing or decreasing sensitivity to Glyb were seen with mutations R334A, K335A, F337A, S341A, I344A, R347A, M348A, V350A, and R352A (Fig. 3 left). Login to comment
125 Mutations R334A and K335A lie in the outer vestibule of the pore of CFTR; surprisingly, the two mutations db ca 2 nA 100 ms 100 ms 2 nA Concentration (μM) 0 200 400 600 800 1000 0.2 0.4 0.6 0.8 1.0 Fractionalblock 100 ms 4 nA Fig. 2 Concentration-dependent block of WT-CFTR by Glyb, Glip and Tolb. Login to comment
133 R334A weakened block while K335A strengthened block by both blockers. Login to comment
135 Also, we reported that R334A and R334C exhibited multiple single-channel conductance levels, including subconductance 1 (s1), subconductance 2 (s2), and full conductance states (f). Login to comment
151 The surprising finding that mutations at six adjacent positions Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** * * * 0.8 0.6 0.4 0.2 0 Fractional block by Glyb50 μM Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** ** ** * * * * * * ** ** Fractional block by Tolb300 μM 0.8 0.6 0.4 0.2 0 Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT * ** ** ** ** ** ** ** ** Fractional block by Glip200 μM 0.8 0.6 0.4 0.2 0 Fig. 3 Alanine-scanning in TM6 to identify the amino acids that interact with the three blockers. Login to comment
158 Among the 20 single amino acid mutants of TM12 that we tested in this paper, none of them exhibited significant change in their single-channel conductance compared to WT-CFTR, while we know that mutations R334A, F337A, S341A, R347A, and R352A in TM6 all exhibited significant change in their single-channel conductance [11, 12, 29, and the present manuscript]; these data strongly suggest that TM6 and TM12 do not equally contribute to the pore of CFTR. Login to comment
162 Although mutations R334A and K335A exhibited opposite effects on steady-state block by Glyb and Glip, neither mutation altered initial block (Fig. 5). Login to comment
163 Effects on time-dependent block by mutations R334A and K335A Fractional block by Glip200 μM V1153A D1152A I1151A S1150A S1149A N1148A V1147A A1146S W1145A Q1144A L1143A T1142A S1141A M1140A I1139A N1138A M1137A A1136S L1135A T1134A WT 0 0.2 0.4 0.6 0.8 * ** ** ** ** ** ** * V1153A D1152A I1151A S1150A S1149A N1148A V1147A A1146S W1145A Q1144A L1143A T1142A S1141A M1140A I1139A N1138A M1137A A1136S L1135A T1134A WT 0 0.2 0.4 0.6 0.8 1.0 * * * * * ** ** ** ** Fractional block by Glyb50 μM Fig. 4 Alanine-scanning in TM12 to identify amino acids that interact with Glyb and Glip. Login to comment
166 Double asterisks indicate significantly different compared to WT-CFTR (p<0.01) Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.3 0.2 0.1 0 * * ** ** 0.4 Initial block by 50 μM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.4 0.3 0.2 0.1 0 ** ** * Initial block by 200 μM Glip Fig. 5 Initial block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in steady-state fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment
168 Double asterisks indicate significantly different compared to WT-CFTR (p<0.01) were similar for Glyb and Glip, although the effect of R334A on Glyb was larger than for Glip and the effect of K335A was larger for Glip than Glyb (Fig. 6). Login to comment
193 Probable orientation of drugs in the pore Glyb and Glip are identical molecules along most of their lengths, differing only in the substituents on the ring at the Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.8 0.6 0.2 0 ** ** ** ** Time-dependent block by 50 μμM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** * ** * Time-dependent block by 200 μM Glip 0.4 0.8 0.6 0.2 00.4 Fig. 6 Time-dependent block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment
221 The effects of mutations R334A and K335A are indirect, likely related to the movement of chloride within the pore, or the stabilization of the outer vestibule. Login to comment

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. Login to comment
185 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). Login to comment
186 Neutralizing the charge at 334 (R334A) resulted in a pKa that was more than one pH unit more basic (8.78 6 0.03, n ¼ 4) than that determined for T338C CFTR. Login to comment
187 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). Login to comment
221 FIGURE 8 The pH-induced changes in the conductances of oocytes expressing T338C/R334A, T338C/R334E, T338H, or T338H/R334C CFTR. Login to comment
222 (A) Sample titration curves of conductances of oocytes expressing T338C CFTR (solid circles), T338C/R334A (open squares), or T338C/ R334E CFTRs (shaded triangles). Login to comment
340 A comparison of the apparent pKa of T338C CFTR with that of the double mutant, T338C/R334A, suggests that the amino acid substitution at position 334 shifted the pKa from ;7.4 to 8.8 or ;1.4 units. Login to comment
342 A comparison of T338C/R334A (pKa ¼ 8.8) with T338C/R334E (pKa ¼ 8.9), would suggest a further change in Cq o of ;ÿ6 mV. Login to comment
347 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. Login to comment
223 (A) Sample titration curves of conductances of oocytes expressing T338C CFTR (solid circles), T338C/R334A (open squares), or T338C/ R334E CFTRs (shaded triangles). Login to comment
344 A comparison of T338C/R334A (pKa &#bc; 8.8) with T338C/R334E (pKa &#bc; 8.9), would suggest a further change in Cq o of ;6 mV. Login to comment
348 The absolute values of Cq o must be interpreted with caution since we do not know to what extent structural differences between these two mutants might have resulted from the amino acid substitution (see below), but both of the measurements used to estimate the electrostatic potential of the vestibule indicated an asymmetry between the impact of basic and acidic residues at 334. 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. Login to comment

PMID: 15130785 [PubMed] Gong X et al: "Maximization of the rate of chloride conduction in the CFTR channel pore by ion-ion interactions."

No. Sentence Comment

38 Two other mutants, R334A and R334T, did not express any detectable Cl current in BHK cells. Login to comment

PMID: 25024266 [PubMed] Cui G et al: "Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR."

No. Sentence Comment

154 ECL1 mutations shift the reversal potential in macroscopic currents To further verify that these ECL1 amino acids do not strongly or directly affect ion conduction and permeation, we compared the reversal potentials (Vrev) of D110R-, K114D-, E116R-, and R117A-CFTR with WT-CFTR and with the R334A mutant, which has been shown to have a profound effect on Vrev compared with WT-CFTR, consistent with the role of R334 in providing charge in the outer mouth of the open channel. Login to comment
156 E116R- and R117A-CFTR exhibited significantly right-shifted reversal potentials compared with WT-CFTR, but the effects were not as large as for R334A-CFTR. Login to comment
159 As previously reported, R334A-CFTR exhibited outward rectification but with a significantly reduced rectification ratio: 1.33 &#b1; 0.14 (n = 9, P < 0.01). Login to comment
224 Tab l e 1 Reversal potentials of WT-CFTR and mutants in ND96 bath solution CFTR n Vrev mV WT 14 &#e032;27.75 &#b1; 0.78 R334A 6 &#e032;12.15 &#b1; 1.64a R117A 6 &#e032;22.51 &#b1; 0.85a E116R 5 &#e032;21.45 &#b1; 1.14a K114D 5 &#e032;24.68 &#b1; 3.22 D110R 5 &#e032;27.64 &#b1; 3.29 R104E 5 &#e032;21.15 &#b1; 1.08a R899C 4 &#e032;25.30 &#b1; 3.94 D891C 6 &#e032;25.81 &#b1; 2.44 K892E 5 &#e032;23.70 &#b1; 3.62 E1124R 5 &#e032;18.32 &#b1; 0.43a E1126R 5 &#e032;20.67 &#b1; 3.16b R117E/E1126R 6 &#e032;23.06 &#b1; 1.37b R104E/E116R 6 &#e032;27.17 &#b1; 1.08 Values are mean &#b1; SEM. Login to comment

PMID: 26209275 [PubMed] Cui G et al: "Murine and human CFTR exhibit different sensitivities to CFTR potentiators."

No. Sentence Comment

131 WT 0.0 0.1 0.2 0.3 Fractional inhibition by 2.5 &#b5;M GlyH-101 # R334C R334A T338A R352A # # # 0.4 0.5 0.4 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO R334C- hCFTR A B C D ND96 ISO ISO+ GlyH ND96 ISO T338A-hCFTR 1 &#b5;A 50 s 1.0 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO WT-hCFTR Fig. 5. Login to comment
155 We have previously reported with the TEVC technique 1.0 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO WT-mCFTR Fractional inhibition by GlyH 0.0 0.1 0.3 0.4 0.2 0.5 20 40 60 80 100 Concentration (&#b5;M) Kd = 32.39 &#b5;M Fractional activation by GlyH 0.0 0.1 0.3 0.4 0.2 20 40 60 80 100 Concentration (&#b5;M) Kd = 103.56 &#b5;M 0.5 A B C D E Fractional activation by 25 &#b5;M GlyH-101 0.0 0.3 0.1 0.2 F Fractional inhibition by 25 &#b5;M GlyH-101 0.0 0.6 0.2 0.4 # * # 0.5 &#b5;A 50 s R334A-mCFTR ND96 ISO ISO+ GlyH ND96 ISO Fig. 6. Login to comment
162 D: representative current trace of R334A-mCFTR recorded at VM afd; afa;60 mV. Login to comment
163 Extracellular 25 òe;M GlyH-101 failed to block but only potentiated R334A-mCFTR. Summary data for fractional inhibition (E) and fractional potentiation (F) of WT-, V100L-, R147H-, I201V-, R334A-, and T338A-mCFTR with 25 òe;M GlyH-101. Login to comment
180 GlyH-101 at 2.5 òe;M blocked WT-hCFTR 28% at VM afd; afa;60 mV in the continuing presence of 10 òe;M ISO (used to activate CFTR via the beta2-adrenergic receptor; see MATERIALS AND METHODS), but the blocking effect was completely lost with both the R334A and R334C mutations (Fig. 5). Login to comment
200 A representative current trace of R334A-mCFTR is shown in Fig. 6D. GlyH-101 completely lost its blocking effect and only exhibited the potentiation function at R334A-mCFTR. Summary data for inhibition are shown in Fig. 6E. GlyH-101 displayed significantly strengthened block of V100L-mCFTR and distinctly weakened block of I201V- mCFTR. Login to comment
311 In fact, we can separate the inhibitory and stimulatory effects of GlyH-101 on mCFTR because the R334A mutation completely abolished the inhibitory effect of GlyH-101 on mCFTR without affecting potentiation. Login to comment