ABCC7 p.Arg347Glu
| ClinVar: |
c.1039C>T
,
p.Arg347Cys
?
, not provided
c.1040G>A , p.Arg347His D , Pathogenic c.1040G>T , p.Arg347Leu D , Pathogenic c.1040G>C , p.Arg347Pro D , Pathogenic |
| CF databases: |
c.1040G>C
,
p.Arg347Pro
D
, CF-causing ; CFTR1: This mutation destroys a Hha I restriciton site and creates an NcoI site and occurred in a family diagnosed as PS. The mutation have been identified and analyzed using the SSCP technique.
c.1040G>A , p.Arg347His D , CF-causing ; CFTR1: The patient is of Italian origin and carries the [delta]F508 mutation on the other chromosome. Initially we thought this was the same mutation as R347 because it destroys the same hhai site; however, R347H does not create the NcoI site. c.1040G>T , p.Arg347Leu (CFTR1) D , A nucleotide change, G->T at position 1172, was detected leading to R347L. The other chromosome carries a [delta]F508. This mutation was found on one chromosome among 150 CF chromosomes screened. c.1039C>T , p.Arg347Cys (CFTR1) ? , This mutation was identified by DGGE and direct sequencing. |
| Predicted by SNAP2: | A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (71%), I: D (95%), K: D (95%), L: D (80%), M: D (95%), N: D (95%), P: D (75%), Q: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%), |
| Predicted by PROVEAN: | A: N, C: D, D: D, E: N, F: D, G: D, 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] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]
Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.
Comments [show]
None has been submitted yet.
No. Sentence Comment
400 R347E had little or no effect on the halide permeability or conductance sequences [18].
X
ABCC7 p.Arg347Glu 16442101:400:0
status: NEW[hide] Cystic fibrosis-associated mutations at arginine 3... J Biol Chem. 1999 Feb 26;274(9):5429-35. Cotten JF, Welsh MJ
Cystic fibrosis-associated mutations at arginine 347 alter the pore architecture of CFTR. Evidence for disruption of a salt bridge.
J Biol Chem. 1999 Feb 26;274(9):5429-35., 1999-02-26 [PMID:10026154]
Abstract [show]
Arginine 347 in the sixth transmembrane domain of cystic fibrosis transmembrane conductance regulator (CFTR) is a site of four cystic fibrosis-associated mutations. To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function. Every Arg-347 mutation examined, except R347K, had a destabilizing effect on the pore, causing the channel to flutter between two conductance states. Chloride flow through the larger conductance state was similar to that of wild-type CFTR, suggesting that the residue at position 347 does not interact directly with permeating anions. We hypothesized that Arg-347 stabilizes the channel through an electrostatic interaction with an anionic residue in another transmembrane domain. To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations. Interestingly, the D924R mutation complemented R347D, yielding a channel that behaved like wild-type CFTR. These data suggest that Arg-347 plays an important structural role in CFTR, at least in part by forming a salt bridge with Asp-924; cystic fibrosis-associated mutations disrupt this interaction.
Comments [show]
None has been submitted yet.
No. Sentence Comment
1 To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function.
X
ABCC7 p.Arg347Glu 10026154:1:122
status: NEW5 To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations.
X
ABCC7 p.Arg347Glu 10026154:5:107
status: NEW24 To better understand the role of Arg-347 in CFTR structure and function, we examined the effect of mutating Arg-347 to cysteine, aspartic acid, glutamic acid, lysine, and leucine on CFTR conductance.
X
ABCC7 p.Arg347Glu 10026154:24:108
status: NEW25 We examined the cytosolic pH (pHc)-dependent behavior of CFTR-R347H and that of the other residue 347 mutants both with (R347C, R347D, R347E, and R347K) and without (R347L) a pHc-titratable residue.
X
ABCC7 p.Arg347Glu 10026154:25:135
status: NEW86 Visual inspection suggested that the lifetimes of OL and OB states were also influenced by the nature of the residue at position 347: R347E and R347H tended to have longer dwell times in the OL and OB states, whereas R347L, R347C, and R347D tended to display shorter dwell times.
X
ABCC7 p.Arg347Glu 10026154:86:134
status: NEW91 Single-channel Conductance of Residue 347 Mutants-To determine whether the residue at position 347 affects single-channel conductance and not merely the conductance state of the channel, we examined the I-V relationship and slope conductance of the mutants with slower pHc-dependent kinetics, R347H and R347E, as well as R347K.
X
ABCC7 p.Arg347Glu 10026154:91:303
status: NEW94 The single-channel conductance at pHc 6.0 of wild-type CFTR, R347K, and the OB state of R347E and R347H were all very similar (in pS): 7.7 Ϯ 0.4, 8.3 Ϯ 0.6, 7.4 Ϯ 0.4, and 6.9 Ϯ 0.2, respectively (n ϭ 3 for each).
X
ABCC7 p.Arg347Glu 10026154:94:88
status: NEW95 The single-channel conductance of the OL states of R347E and R347H at pHc 6.0 were also very similar (in pS): 1.5 Ϯ 0.1 and 1.6 Ϯ 0.1, respectively (n ϭ 3 and 4 for each).
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ABCC7 p.Arg347Glu 10026154:95:51
status: NEW102 The first explanation seems unlikely because the reciprocal lifetime of the OL state which represents the "on" rate for the proton is very slow (e.g. it is 6 ϫ 107 M -1 s-1 for R347E).
X
ABCC7 p.Arg347Glu 10026154:102:183
status: NEW107 Dwell-time Analysis of OL and OB States-We performed a dwell-time analysis of the lifetimes of the OL and OB states of R347E and R347H to enable more quantitative comparisons between them and to better understand their pHc dependence.
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ABCC7 p.Arg347Glu 10026154:107:119
status: NEW113 The observable pK (0 mV) for the equilibrium between OL and OB of R347E and R347H were 6.4 and 6.3, respectively. The faster kinetics of R347D, R347C, and R347L made dwell-time analysis for these mutants less reliable.
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ABCC7 p.Arg347Glu 10026154:113:66
status: NEW119 Single-channel I-V relationships for R347E (OL and OB states), R347H (OL and OB states), R347K, R347D/D924R, and wild-type CFTR at pHc 6.0. n ϭ 2-4 at each data point.
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ABCC7 p.Arg347Glu 10026154:119:37
status: NEW122 As a control for the variance analysis, we examined the R347E mutant on which we had also done dwell-time analysis (Fig. 3A); as expected, Fig. 3B shows that the variance of R347E goes through a maximum between pHc 6.5 and 5.5.
X
ABCC7 p.Arg347Glu 10026154:122:56
status: NEWX
ABCC7 p.Arg347Glu 10026154:122:174
status: NEW124 Fig. 4A shows qualitatively that both conductance states of R347E were voltage-dependent.
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ABCC7 p.Arg347Glu 10026154:124:60
status: NEW125 Fig. 4B shows quantitatively that at pHc 6.0 the OL and OB states for R347E and R347H were both influenced by the transmembrane voltage.
X
ABCC7 p.Arg347Glu 10026154:125:70
status: NEW128 The degree of voltage dependence was similar for both mutants despite the charge differences at residue 347 and yielded a z of 0.25 and 0.21 for R347E and R347H, respectively. The voltage dependence was asymmetrically disposed between the rate of entry into the OB state and the rate of exit from OB (Fig. 4B).
X
ABCC7 p.Arg347Glu 10026154:128:153
status: NEW130 The rate of exit from the OB state (B -1 ) was more voltage-dependent than the rate of exit from the OL state (L -1 ) for both mutants (␦ ϭ 0.8 versus 1 - ␦ ϭ 0.2 for R347E and ␦ ϭ 0.7 versus 1 - ␦ ϭ 0.3 for R347H).
X
ABCC7 p.Arg347Glu 10026154:130:209
status: NEW134 A, dwell-time analysis in the OL and OB conductance states versus pHc for R347E and R347H.
X
ABCC7 p.Arg347Glu 10026154:134:74
status: NEW136 B, open-channel current variance of the R347C, R347D, R347L, and R347E mutants versus pHc.
X
ABCC7 p.Arg347Glu 10026154:136:65
status: NEW142 A, current records from excised, inside-out membrane patch containing R347E channel.
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ABCC7 p.Arg347Glu 10026154:142:70
status: NEW144 B, dwell times (pHc 6.0) in the OL state (closed symbols) or OB state (open symbols) versus voltage for R347E (left, circles) and R347H (right, squares).
X
ABCC7 p.Arg347Glu 10026154:144:104
status: NEW147 arises from charge movement through a voltage field and since R347E and R347H displayed similar voltage dependences and carry different charges at position 347, residue 347 is not likely moving through a transmembrane potential during interchange between OL and OB states.
X
ABCC7 p.Arg347Glu 10026154:147:62
status: NEW153 To identify the Arg-347 interaction partner, we replaced Arg-347 with an anionic residue (R347E or R347D) and introduced an arginine residue in the place of candidate partners in a salt bridge.
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ABCC7 p.Arg347Glu 10026154:153:90
status: NEW154 We studied the conductance properties of the following double mutants: R347D/D924R, R347D/D993R, and R347E/E1104R.
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ABCC7 p.Arg347Glu 10026154:154:101
status: NEW155 The R347D/D993R and R347E/E1104R mutants each had two conductance states with pHc-dependent behavior (Fig. 5).
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ABCC7 p.Arg347Glu 10026154:155:20
status: NEW157 Accordingly, for R347D/D993R and R347E/E1104R the current variance in the open state increased with decreasing pHc (Fig. 5B).
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ABCC7 p.Arg347Glu 10026154:157:33
status: NEW158 Qualitatively, the lifetimes of the OL and OB conductance states in the R347D/D993R and R347E/E1104R were similar to that of the R347D and R347E mutants, respectively. The amplitude of the OL state was larger for both of these double mutants as compared with the single mutants (Figs.
X
ABCC7 p.Arg347Glu 10026154:158:88
status: NEWX
ABCC7 p.Arg347Glu 10026154:158:139
status: NEW160 We also observed an infrequent, additional small conductance state in the R347E/E1104 mutant (see amplitude histogram in Fig. 5A); this is likely due to the E1104R mutation itself.
X
ABCC7 p.Arg347Glu 10026154:160:74
status: NEW171 Additionally, the single-channel slope conductances of R347H and R347E were the same in both OB and OL states.
X
ABCC7 p.Arg347Glu 10026154:171:65
status: NEW179 A, single-channel current tracings from excised, inside-out membrane patches containing R347E/E1104R, R347D/D924R, and R347D/D993R.
X
ABCC7 p.Arg347Glu 10026154:179:88
status: NEW181 B, current variance of R347E/E1104R, R347D/D924R, and R347D/D993R at the indicated pHc was collected as in Fig. 3.
X
ABCC7 p.Arg347Glu 10026154:181:23
status: NEW[hide] Structural and ionic determinants of 5-nitro-2-(3-... Br J Pharmacol. 1999 May;127(2):369-76. Walsh KB, Long KJ, Shen X
Structural and ionic determinants of 5-nitro-2-(3-phenylprophyl-amino)-benzoic acid block of the CFTR chloride channel.
Br J Pharmacol. 1999 May;127(2):369-76., [PMID:10385235]
Abstract [show]
1. The goals of this study were to identify the structural components required for arylaminobenzoate block of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and to determine the involvement of two positively charged amino acid residues, found within the channel, in drug binding. 2. Wild-type and mutant CFTR chloride channels were expressed in Xenopus oocytes and CFTR currents measured using the two microelectrode voltage clamp. Block of the wild-type CFTR current by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) occurred in a voltage-dependent manner with preferential inhibition of the inward currents (Kd = 166 microM at -90 mV). 3. Removal of the phenyl ring from the aliphatic chain of NPPB, with the compound 2-butylamino-5-nitrobenzoic acid, caused only a small change in CFTR inhibition (Kd = 243 microM), while addition of an extra phenyl ring at this position (5-nitro-2-(3,3-diphenylpropylamino)-benzoic acid) increased drug potency (Kd = 58 microM). In contrast, removal of the benzoate ring (2-amino-4-phenylbutyric acid) or the 5-nitro group (2-(3-phenylpropylamino)-benzoic acid) of NPPB severely limited drug block of the wild-type channel. 4. NPPB inhibition of CFTR currents in oocytes expressing the mutants K335E and R347E also occurred in a voltage-dependent manner. However, the Kds for NPPB block were increased to 371 and 1573 microM, for the K335E and R347E mutants, respectively. 5. NPPB block of the inward wild-type CFTR current was reduced in the presence of 10 mM of the permeant anion SCN-. 6. These studies present the first step in the development of high affinity probes to the CFTR channel.
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None has been submitted yet.
No. Sentence Comment
5 4 NPPB inhibition of CFTR currents in oocytes expressing the mutants K335E and R347E also occurred in a voltage-dependent manner.
X
ABCC7 p.Arg347Glu 10385235:5:79
status: NEW6 However, the Kds for NPPB block were increased to 371 and 1573 mM, for the K335E and R347E mutants, respectively.
X
ABCC7 p.Arg347Glu 10385235:6:85
status: NEW26 ), and the K335E and R347E mutants obtained from Dr K Kunzelmann (Albert-Ludwigs University, Freiburg, Germany).
X
ABCC7 p.Arg347Glu 10385235:26:21
status: NEW76 Eect of NPPB on K335E and R347E CFTR mutants The pKa of NPPB is close to 4.5 (Wangemann et al., 1986; Walsh & Wang, 1998), and thus the drug molecules are predominately charged (499%) in the ND-96 solution at pH 7.5.
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ABCC7 p.Arg347Glu 10385235:76:32
status: NEW77 Since the negatively charged drug may interact with positively charged amino acid residues in the pore of the CFTR channel, we examined the eect of NPPB on the mutants K335E and R347E.
X
ABCC7 p.Arg347Glu 10385235:77:184
status: NEW90 The K335E channel displayed a more linear I/V relationship (Figure 5) and the R347E channel a more outward-rectifying I/V relationship (Figure 6) than that measured with the wild-type channel (Figure 3).
X
ABCC7 p.Arg347Glu 10385235:90:78
status: NEW92 NPPB was less eective in blocking the CFTR currents in oocytes expressing the K335E and R347E channels.
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ABCC7 p.Arg347Glu 10385235:92:94
status: NEW93 The Kd for NPPB block of the current was increased from 166 mM for the wild-type to 371 and 1573 mM for the K335E and R347E mutants, respectively (Figures 5 and Figure 5 Eect of NPPB on the K335E CFTR channel. Left panel: I/V relationship for the CFTR current measured in the presence and absence of 100 mM NPPB.
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ABCC7 p.Arg347Glu 10385235:93:118
status: NEW100 Figure 6 Eect of NPPB on the R347E CFTR channel. Left panel: I/V relationship for the CFTR current measured in the presence and absence of 100 mM NPPB.
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ABCC7 p.Arg347Glu 10385235:100:35
status: NEW102 Right panel: concentration versus response curve for inhibition of the wild-type and R347E channels by NPPB.
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ABCC7 p.Arg347Glu 10385235:102:85
status: NEW105 For the R347E data, each point represents the mean+s.e.mean of three to ®ve experiments.
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ABCC7 p.Arg347Glu 10385235:105:8
status: NEW106 The theoretical curve for the R347E data provided a Kd of 1573 mM.
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ABCC7 p.Arg347Glu 10385235:106:30
status: NEW108 Although the sensitivity of the mutant channels to NPPB was reduced, both the K335E and R347E mutants displayed a voltage-dependence to NPPB block that was similar to the wild-type channel (Figure 7).
X
ABCC7 p.Arg347Glu 10385235:108:88
status: NEW109 The slopes of the lines obtained from the relationship between the fractional block (Id/Io) and voltage had values of 0.23 (wild-type), 0.24 (K335E) and 0.30 (R347E).
X
ABCC7 p.Arg347Glu 10385235:109:159
status: NEW116 Relationship between the fractional drug block (Id/I0) and the membrane potential determined for the wild-type (100 mM NPPB), K335E (400 mM NPPB) and R347E (1 mM NPPB) CFTR channels.
X
ABCC7 p.Arg347Glu 10385235:116:150
status: NEW117 The slopes of the straight lines had values of 0.23 (wild-type), 0.24 (K335E) and 0.30 (R347E).
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ABCC7 p.Arg347Glu 10385235:117:88
status: NEW152 This insensitivity is most striking for the R347E mutant, suggesting that this positively charged residue serves as an important determinant in the binding of the negatively charged drug molecules.
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ABCC7 p.Arg347Glu 10385235:152:44
status: NEW153 While the concentration versus response curves for NPPB block of mutants K335E and R347E were shifted to higher concentrations, NPPB produced a voltage-dependent block in the mutants that was almost identical to that of the wild-type channel.
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ABCC7 p.Arg347Glu 10385235:153:83
status: NEW159 Although the anity of the R347E mutant for DPC has not been determined, it is likely that mutations at several sites in the M6 region will eect arylaminobenzoate block.
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ABCC7 p.Arg347Glu 10385235:159:32
status: NEW[hide] Direct comparison of NPPB and DPC as probes of CFT... J Membr Biol. 2000 May 1;175(1):35-52. Zhang ZR, Zeltwanger S, McCarty NA
Direct comparison of NPPB and DPC as probes of CFTR expressed in Xenopus oocytes.
J Membr Biol. 2000 May 1;175(1):35-52., 2000-05-01 [PMID:10811966]
Abstract [show]
Blockers of CFTR with well-characterized kinetics and mechanism of action will be useful as probes of pore structure. We have studied the mechanism of block of CFTR by the arylaminobenzoates NPPB and DPC. Block of macroscopic currents by NPPB and DPC exhibited similar voltage-dependence, suggestive of an overlapping binding region. Kinetic analysis of single-channel currents in the presence of NPPB indicate drug-induced closed time constants averaging 2.2 msec at -100 mV. The affinity for NPPB calculated from single-channel block, K(D) = 35 microm, exceeds that for other arylaminobenzoates studied thus far. These drugs do not affect the rate of activation of wild-type (WT) channels expressed in oocytes, consistent with a simple mechanism of block by pore occlusion, and appear to have a single binding site in the pore. Block by NPPB and DPC were affected by pore-domain mutations in different ways. In contrast to its effects on block by DPC, mutation T1134F-CFTR decreased the affinity and reduced the voltage-dependence for block by NPPB. We also show that the alteration of macroscopic block by NPPB and DPC upon changes in bath pH is due to both direct effects (i.e., alteration of voltage-dependence) and indirect effects (alteration of cytoplasmic drug loading). These results indicate that both NPPB and DPC block CFTR by entering the pore from the cytoplasmic side and that the structural requirements for binding are not the same, although the binding regions within the pore are similar. The two drugs may be useful as probes for overlapping regions in the pore.
Comments [show]
None has been submitted yet.
No. Sentence Comment
436 Two mutants were studied by Walsh: K335E, predicted to be at the extracellular end of TM6, and R347E, predicted to be at the cytoplasmic end of TM6.
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ABCC7 p.Arg347Glu 10811966:436:95
status: NEW439 Block of R347E-CFTR was also reduced (KD ס 1573 M) and the voltage-dependence was increased significantly.
X
ABCC7 p.Arg347Glu 10811966:439:9
status: NEW443 The results of the R347E mutation studied by Walsh [56] are difficult to interpret because this mutation causes disruption of channel structure due to loss of a salt bridge with an aspartic acid in TM8 [5].
X
ABCC7 p.Arg347Glu 10811966:443:19
status: NEW[hide] Activating cystic fibrosis transmembrane conductan... J Biol Chem. 2005 Jun 24;280(25):23622-30. Epub 2005 Apr 27. Wang W, Li G, Clancy JP, Kirk KL
Activating cystic fibrosis transmembrane conductance regulator channels with pore blocker analogs.
J Biol Chem. 2005 Jun 24;280(25):23622-30. Epub 2005 Apr 27., 2005-06-24 [PMID:15857825]
Abstract [show]
Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is deltaF508-CFTR, which inefficiently traffics to the surfaces of most cells. The deltaF508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and deltaF508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment deltaF508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and deltaF508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of deltaF508-CFTR channels in excised membrane patches by 10-15-fold under conditions in which wild-type channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the deltaF508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface.
Comments [show]
None has been submitted yet.
No. Sentence Comment
68 To determine whether NPPB activates CFTR by binding to the same (or different) site that causes a pore block, we tested its effects on a CFTR pore mutant (R347E) that is resistant to block by NPPB (19).
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ABCC7 p.Arg347Glu 15857825:68:155
status: NEW69 Fig. 1 (E and F) shows that NPPB stimulated the currents mediated by R347E-CFTR at positive potentials to a greater extent compared with wild-type CFTR at moderate levels of phosphorylation (high PKA concentration (110 units/ml), followed by PKI).
X
ABCC7 p.Arg347Glu 15857825:69:69
status: NEW70 Unlike the wild-type channel, the R347E-CFTR currents were stimulated by NPPB even at negative potentials.
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ABCC7 p.Arg347Glu 15857825:70:34
status: NEW71 Thus, NPPB behaves more as a pure agonist for the R347E pore mutant, which implies that this compound stimulates channel opening by binding to a site that is distinct from the pore-blocking site.
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ABCC7 p.Arg347Glu 15857825:71:50
status: NEW138 F, at low doses, NPPB stimulates currents in both directions for moderately phosphorylated R347E-CFTR channels in an HEK-293T patch.
X
ABCC7 p.Arg347Glu 15857825:138:91
status: NEW[hide] Cl- transport by cystic fibrosis transmembrane con... J Physiol. 1998 May 1;508 ( Pt 3):825-36. Briel M, Greger R, Kunzelmann K
Cl- transport by cystic fibrosis transmembrane conductance regulator (CFTR) contributes to the inhibition of epithelial Na+ channels (ENaCs) in Xenopus oocytes co-expressing CFTR and ENaC.
J Physiol. 1998 May 1;508 ( Pt 3):825-36., 1998-05-01 [PMID:9518736]
Abstract [show]
1. Epithelial Na+ channels (ENaCs) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is activated by protein kinase A. Since cAMP-dependent activation of CFTR Cl- conductance is defective in cystic fibrosis (CF), ENaC currents are not inhibited by CFTR. This could explain the enhanced Na+ conductance found in CF. In the present study, we examined possible mechanisms of interaction between CFTR and ENaC co-expressed in Xenopus oocytes. 2. The magnitude of CFTR Cl- currents activated by 3-isobutyl-1-methylxanthine (IBMX) in oocytes co-expressing either wild-type or mutant CFTR and ENaC determined the degree of downregulation of ENaC currents. 3. The ability of CFTR to inhibit ENaC currents was significantly reduced either when extracellular Cl- was replaced by poorly conductive anions, e.g. SCN- or gluconate, or when CFTR was inhibited by diphenylamine-carboxylate (DPC, 1 mmol l-1). 4. Downregulation of ENaC was more pronounced at positive when compared with negative clamp voltages. This suggests that outward currents, i.e. influx of Cl- through activated CFTR most effectively downregulated ENaC. 5. Activation of endogenous Ca2+-activated Cl- currents by 1 micromol l-1 ionomycin did not inhibit ENaC current. This suggests that inhibition of ENaC mediated by Cl- currents may be specific to CFTR. 6. The present findings indicate that downregulation of ENaC by CFTR is correlated to the ability of CFTR to conduct Cl-. The data have implications for how epithelia switch from NaCl absorption to NaCl secretion when CFTR is activated by secretagogues.
Comments [show]
None has been submitted yet.
No. Sentence Comment
35 The following CFTR mutations were generated: CF-associated mutations such as ÄF508, G551D and R117H as well as artificial mutations within MSD1 such as R347E and K335E (Hipper et al. 1995).
X
ABCC7 p.Arg347Glu 9518736:35:157
status: NEW105 Finally, two artificial mutations (R347E and K335E) in the 6th transmembrane spanning domain were initially created in order to examine properties of the putative pore of CFTR (Anderson et al. 1991).
X
ABCC7 p.Arg347Glu 9518736:105:35
status: NEW110 In contrast, other mutations, which still activated whole-cell Cl¦ conductance (R117H, R347E, K335E) downregulated ENaC currents.
X
ABCC7 p.Arg347Glu 9518736:110:92
status: NEW[hide] Cystic fibrosis transmembrane conductance regulato... J Cell Biol. 1998 Nov 2;143(3):645-57. Jiang Q, Mak D, Devidas S, Schwiebert EM, Bragin A, Zhang Y, Skach WR, Guggino WB, Foskett JK, Engelhardt JF
Cystic fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor.
J Cell Biol. 1998 Nov 2;143(3):645-57., 1998-11-02 [PMID:9813087]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl- that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl- conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl- conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl >> Br; R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl- binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl-. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.
Comments [show]
None has been submitted yet.
No. Sentence Comment
6 Despite the lack of a need for Cl-conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl ϾϾ Br; R347P, Cl ϾϾ Br; R347E, Br ϾϾ Cl; R334W, Cl ϭ Br).
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ABCC7 p.Arg347Glu 9813087:6:278
status: NEW80 The cAMP-stimulated Cl- and Br- conductances of the R347E mutant (5.3 Ϯ 2.1 s and 12.15 Ϯ 3.3 s, respectively) were much lower than that of the wild type.
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ABCC7 p.Arg347Glu 9813087:80:52
status: NEW89 For mutant R347, a cDNA segment was cut out from the PTM1-R347E CFTR construct (kindly provided by Dr. M. Welsh, University of Iowa) by restriction enzymes Erev,Br Erev,Cl RT F ------- PBr Br- [ ] PCl Cl- [ ] -------------------------- ln=- MroI and Bst1107I, and was subcloned into the pBQ-CFTR plasmid between the same restriction sites.
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ABCC7 p.Arg347Glu 9813087:89:58
status: NEW206 Mutations in the CFTR Channel Pore Alter the Halide Dependence of ATP Release To explore the mechanisms by which changes in the extracellular Cl-concentration affect activation of ATP release, we examined the CFTR mutants R334W, R347P, and R347E.
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ABCC7 p.Arg347Glu 9813087:206:240
status: NEW208 Immunoprecipitation studies using a rabbit antisera raised against a synthetic peptide corresponding to residues 45-65 in the CFTR NH2 terminus demonstrated similar levels of protein expression in wtCFTR, R334W, R347P, and R347E cRNA-injected oocytes (Fig. 6 A).
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ABCC7 p.Arg347Glu 9813087:208:223
status: NEW216 In contrast, the stimulated Cl-conductances were 4and 20-fold lower in the R334W and R347E mutants, respectively (Fig. 6 and Table II).
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ABCC7 p.Arg347Glu 9813087:216:85
status: NEW242 The halide dependence of CFTR-modulated ATP release was similarly analyzed in R334W, R347P, and R347E cRNA-injected oocytes.
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ABCC7 p.Arg347Glu 9813087:242:96
status: NEW247 Most importantly, the halide dependence in the R347E mutant was reversed to Br- ϾϾ Cl- ; JATP in the presence of Cl- (1.5 Ϯ 0.49 pmoles/min) was 7.3-fold lower than that in the presence of Br- (11 Ϯ 4.8 pmoles/min; Fig. 7 and Table II).
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ABCC7 p.Arg347Glu 9813087:247:47
status: NEW248 Interestingly, exposure of R347E-expressing oocytes to extracellular Br- had a lasting affect on JATP, even after extracellular Br- was replaced with Cl- .
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ABCC7 p.Arg347Glu 9813087:248:27
status: NEW279 Furthermore, the R347E had a greater than 20-fold reduced GBr as compared with wtCFTR, yet the magnitude of JATP in the presence of extracellular Br- was sixfold higher for R347E when compared with wtCFTR cRNA-injected oocytes.
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ABCC7 p.Arg347Glu 9813087:279:17
status: NEWX
ABCC7 p.Arg347Glu 9813087:279:173
status: NEW295 Mutants of CFTR that alter charged arginine residues within the channel pore including R334W, R347E, and R347P were used.
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ABCC7 p.Arg347Glu 9813087:295:94
status: NEW298 The Wc I/V relationships for wtCFTR, R347P, R347E, R334W cRNA, and water-injected oocytes are given in B.
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ABCC7 p.Arg347Glu 9813087:298:44
status: NEW303 These results depict average I/V relationships from N experiments for wtCFTR (N ϭ 5), R347E (N ϭ 5), R347P (N ϭ 5), R334W (N ϭ 8), and water (N ϭ 7)-injected oocytes from at least two independent batches of oocytes.
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ABCC7 p.Arg347Glu 9813087:303:92
status: NEW307 We therefore characterized the effects of arginine mutations R334W, R347P, and R347E on CFTR-modulated ATP release.
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ABCC7 p.Arg347Glu 9813087:307:79
status: NEW314 In contrast, the R347E mutation caused pronounced alterations of both electrophysiologic as well as ATP release properties of CFTR.
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ABCC7 p.Arg347Glu 9813087:314:17
status: NEW338 Summary of Electrophysiologic Measurements on CFTR Mutants Wild type R347P R347E R334W Mock ⌬GCl (cAMP) (s) 121 Ϯ 35 125 Ϯ 28 5.3 Ϯ 2.1 32 Ϯ 20 -0.61 Ϯ 0.62 (N ϭ 5) (N ϭ 5) (N ϭ 5) (N ϭ 8) (N ϭ 7) GBr /GCl 0.93 Ϯ 0.01 1.12 Ϯ 0.01 2.36 Ϯ 0.23 1.12 Ϯ 0.03 (N ϭ 5) (N ϭ 5) (N ϭ 5) (N ϭ 8) - PBr /PCl 1.13 Ϯ 0.02 1.02 Ϯ 0.01 1.00 Ϯ 0.04 1.22 Ϯ 0.02 (N ϭ 5) (N ϭ 5) (N ϭ 5) (N ϭ 8) - JATP (Cl) 8.7 Ϯ 3.4 2.0 Ϯ 0.98 1.5 Ϯ 0.49 3.4 Ϯ 1.3 0.021 Ϯ 0.001 (N ϭ 15) (N ϭ 9) (N ϭ 11) (N ϭ 7) (N ϭ 15) JATP (Br) 1.9 Ϯ 0.51 0.26 Ϯ 0.17 11.0 Ϯ 4.8 3.7 Ϯ 1.5 0.013 Ϯ 0.006 (N ϭ 15) (N ϭ 9) (N ϭ 11) (N ϭ 7) (N ϭ 15) JATP (Br)/JATP (Cl) 0.22 0.13 7.3 1.1 - CFTR cRNAs were injected into Xenopus oocytes and evaluated for both electrophysiologic and ATP release characteristics.
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ABCC7 p.Arg347Glu 9813087:338:75
status: NEW354 We thank Dr. Welsh for his mutant CFTR cDNAs R334W, R347E, and R347P, and his thoughtful review of this manuscript.
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ABCC7 p.Arg347Glu 9813087:354:52
status: NEW[hide] Structure and function of the CFTR chloride channe... Physiol Rev. 1999 Jan;79(1 Suppl):S23-45. Sheppard DN, Welsh MJ
Structure and function of the CFTR chloride channel.
Physiol Rev. 1999 Jan;79(1 Suppl):S23-45., [PMID:9922375]
Abstract [show]
Structure and Function of the CFTR Chloride Channel. Physiol. Rev. 79, Suppl.: S23-S45, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ABC transporter family that forms a novel Cl- channel. It is located predominantly in the apical membrane of epithelia where it mediates transepithelial salt and liquid movement. Dysfunction of CFTR causes the genetic disease cystic fibrosis. The CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. Here we review the structure and function of this unique channel, with a focus on how the various domains contribute to channel function. The MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls channel gating. Current knowledge of CFTR structure and function may help us understand better its mechanism of action, its role in electrolyte transport, its dysfunction in cystic fibrosis, and its relationship to other ABC transporters.
Comments [show]
None has been submitted yet.
No. Sentence Comment
104 Thus, depending on the experimental tants, R347E and R1030E, did not alter the anion perme- conditions and whether block of the channel by I0 is con- ability sequence, although PI/PCl values were increased.
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ABCC7 p.Arg347Glu 9922375:104:43
status: NEW[hide] CFTR: mechanism of anion conduction. Physiol Rev. 1999 Jan;79(1 Suppl):S47-75. Dawson DC, Smith SS, Mansoura MK
CFTR: mechanism of anion conduction.
Physiol Rev. 1999 Jan;79(1 Suppl):S47-75., [PMID:9922376]
Abstract [show]
CFTR: Mechanism of Anion Conduction. Physiol. Rev. 79, Suppl.: S47-S75, 1999. - The purpose of this review is to collect together the results of recent investigations of anion conductance by the cystic fibrosis transmembrane conductance regulator along with some of the basic background that is a prerequisite for developing some physical picture of the conduction process. The review begins with an introduction to the concepts of permeability and conductance and the Nernst-Planck and rate theory models that are used to interpret these parameters. Some of the physical forces that impinge on anion conductance are considered in the context of permeability selectivity and anion binding to proteins. Probes of the conduction process are considered, particularly permeant anions that bind tightly within the pore and block anion flow. Finally, structure-function studies are reviewed in the context of some predictions for the origin of pore properties.
Comments [show]
None has been submitted yet.
No. Sentence Comment
432 This behavior is consistent with the notion that iodide can reside in the channel with-(TM1), K335E (TM6), R347E (TM6), and R1030E (TM10).
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ABCC7 p.Arg347Glu 9922376:432:107
status: NEW437 The R347E reducing agent like thiosulfate ion.
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ABCC7 p.Arg347Glu 9922376:437:4
status: NEW557 Ineffect of mutations on anion binding suggests that permeant anions can interact with the channel sufficiently confirmation of these results, Smith and Dawson (unpublished data) found that blockade of CFTR by externalstrongly to impede conduction rates but that the ''tight binding`` is dependent on some element of pore conforma- SCN was abolished in R347E and also in R347Q CFTR, confirming the importance of the positive charge at thistion that is not critical to promote the entry of anions into the pore.
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ABCC7 p.Arg347Glu 9922376:557:353
status: NEW[hide] Mutations to amino acids located in predicted tran... Biochemistry. 1994 Nov 29;33(47):14049-57. Loo TW, Clarke DM
Mutations to amino acids located in predicted transmembrane segment 6 (TM6) modulate the activity and substrate specificity of human P-glycoprotein.
Biochemistry. 1994 Nov 29;33(47):14049-57., [PMID:7947814]
Abstract [show]
Site-directed mutagenesis was used to investigate whether amino acids located in the predicted transmembrane segment, TM6 (residues 330-351), of human P-glycoprotein play essential roles in drug transport. Mutant cDNAs were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to cytotoxic drugs. Four mutations were found to strongly alter the drug resistance profile conferred by P-glycoprotein. Mutation of Val338 to Ala resulted in a mutant P-glycoprotein which conferred enhanced resistance to colchicine and reduced relative resistance to vinblastine. By contrast, mutant Gly341 to Val conferred little resistance to colchicine or doxorubicin, while its ability to confer resistance to vinblastine or actinomycin D was retained. A reduction in the ability of P-glycoprotein to confer resistance to all four drugs was observed for mutant Ala342 to Leu. Mutation of Ser344 to Ala, Thr, Cys, or Tyr resulted in mutant P-glycoproteins which were unable to confer drug resistance. Photolabeling of P-glycoprotein with azidopine in the presence of varying amounts of vinblastine showed that mutation of Ser344 to Tyr required approximately 15-fold more vinblastine to inhibit photolabeling when compared to wild-type enzyme. All of the Ser344 mutants were found to have reduced drug-stimulated ATPase activity relative to wild-type enzyme. These results, together with our previous demonstration that changes to Phe335 affected dissociation of vinblastine, suggest that TM6 may play an important role in drug--protein interaction and coupling of drug binding to ATPase activity.
Comments [show]
None has been submitted yet.
No. Sentence Comment
279 Mutation of Lys335 or Arg347 to Glu in TM6 altered the permeability and/or conductance ratios for halide ions (Anderson et al., 1991).
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ABCC7 p.Arg347Glu 7947814:279:22
status: NEW[hide] Cystic fibrosis: a multiple exocrinopathy caused b... Am J Med. 1998 Jun;104(6):576-90. Schwiebert EM, Benos DJ, Fuller CM
Cystic fibrosis: a multiple exocrinopathy caused by dysfunctions in a multifunctional transport protein.
Am J Med. 1998 Jun;104(6):576-90., [PMID:9674722]
Abstract [show]
Comments [show]
None has been submitted yet.
No. Sentence Comment
243 Other mutations in TMD1 which affect function and cause disease include other arginines in predicted ␣-helix 6, R334W, R347P, and R347E (97,98) and a glycine, G314E, in ␣-helix 5 (99).
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ABCC7 p.Arg347Glu 9674722:243:138
status: NEW[hide] Topological model of membrane domain of the cystic... J Mol Graph Model. 1998 Apr;16(2):72-82, 97-8. Gallet X, Festy F, Ducarme P, Brasseur R, Thomas-Soumarmon A
Topological model of membrane domain of the cystic fibrosis transmembrane conductance regulator.
J Mol Graph Model. 1998 Apr;16(2):72-82, 97-8., [PMID:9879057]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator is a cAMP-regulated chloride channel. We used molecular modelling to predict 3-D models for the CFTR membrane domain. Hydropathy and residue conservation in all CFTRs as well as in other proteins suggested that the membrane domain is a 12-helix bundle. If the domain is enclosing a channel for chloride, it could be made of five helices. We propose two structural models in which both lumenal and cytoplasmic entrances to the chloride pore have a ring of positively charged residues. The inner surface of the channel is covered with neutral polar plus one or two charged residues. Helices that are not directly involved in the chloride channel could organise to form a second channel; a dimeric symmetrical structure is proposed. Analysis raised interest for helix 5: this hydrophobic fragment is conserved in all CFTRs and aligns with segments present in several different ion channels and transporters. The existence of an FFXXFFXXF motif is proposed. Helix 5 could be an important domain of CFTRs. The models agree with available data from pathological mutations but does not account for the membrane insertion of a hydrophilic fragment of NBDI.
Comments [show]
None has been submitted yet.
No. Sentence Comment
232 Mutations associated with mild forms of cystic fibrosis (R117H, R334W, and R347P) implicate three of our inner pore residues in the chloride conductance.50 In other studies, basic amino acids of membrane helices were replaced by acidic residues (K95D, K335E, R347E, and R1030E).
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ABCC7 p.Arg347Glu 9879057:232:259
status: NEW236 Mutations associated with mild forms of cystic fibrosis (R117H, R334W, and R347P) implicate three of our inner pore residues in the chloride conductance.50 In other studies, basic amino acids of membrane helices were replaced by acidic residues (K95D, K335E, R347E, and R1030E).
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ABCC7 p.Arg347Glu 9879057:236:259
status: NEW[hide] Cystic fibrosis transmembrane conductance regulato... Biophys J. 1998 Mar;74(3):1320-32. Mansoura MK, Smith SS, Choi AD, Richards NW, Strong TV, Drumm ML, Collins FS, Dawson DC
Cystic fibrosis transmembrane conductance regulator (CFTR) anion binding as a probe of the pore.
Biophys J. 1998 Mar;74(3):1320-32., [PMID:9512029]
Abstract [show]
We compared the effects of mutations in transmembrane segments (TMs) TM1, TM5, and TM6 on the conduction and activation properties of the cystic fibrosis transmembrane conductance regulator (CFTR) to determine which functional property was most sensitive to mutations and, thereby, to develop a criterion for measuring the importance of a particular residue or TM for anion conduction or activation. Anion substitution studies provided strong evidence for the binding of permeant anions in the pore. Anion binding was highly sensitive to point mutations in TM5 and TM6. Permeability ratios, in contrast, were relatively unaffected by the same mutations, so that anion binding emerged as the conduction property most sensitive to structural changes in CFTR. The relative insensitivity of permeability ratios to CFTR mutations was in accord with the notion that anion-water interactions are important determinants of permeability selectivity. By the criterion of anion binding, TM5 and TM6 were judged to be likely to contribute to the structure of the anion-selective pore, whereas TM1 was judged to be less important. Mutations in TM5 and TM6 also dramatically reduced the sensitivity of CFTR to activation by 3-isobutyl 1-methyl xanthine (IBMX), as expected if these TMs are intimately involved in the physical process that opens and closes the channel.
Comments [show]
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No. Sentence Comment
229 Hipper et al. (1995) reported that the mutations R334E, R334H, K335E, K335H, R347E, and R347H did not alter CFTR conduction properties, but careful inspection of the data presented revealed that the level of CFTR expression was very low so that altered properties of mutant CFTRs might have been easily obscured.
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ABCC7 p.Arg347Glu 9512029:229:77
status: NEW[hide] CFTR Cl- channel and CFTR-associated ATP channel: ... EMBO J. 1998 Feb 16;17(4):898-908. Sugita M, Yue Y, Foskett JK
CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates.
EMBO J. 1998 Feb 16;17(4):898-908., [PMID:9463368]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is regulated by phosphorylation of the R domain and ATP hydrolysis at two nucleotide-binding domains (NBDs). It is controversial whether CFTR conducts ATP or whether CFTR might be closely associated with a separate ATP conductance. To characterize ATP channels associated with CFTR, we analyzed Cl- and ATP single channel-currents in excised inside-out membrane patches from MDCK epithelial cells transiently expressing CFTR. With 100 mM ATP in the pipette and 140 mM Cl- in the bath, ATP channels were associated with CFTR Cl- channels in two-thirds of patches that included CFTR. CFTR Cl- channels and CFTR-associated ATP channels had slope conductances of 7.4 pS and 5.2 pS, respectively, and had distinct reversal potentials and sensitivities to channel blockers. CFTR-associated ATP channels exhibited slow gating kinetics that depended on the presence of protein kinase A and cytoplasmic ATP, similar to CFTR Cl- channels. Gating kinetics of the ATP channels as well as the CFTR Cl- channels were similarly affected by non-hydrolyzable ATP analogues and mutations in the CFTR R domain and NBDs. Our results indicate that phosphorylation- and nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of an associated ATP channel. However, the permeation pathways for Cl- and ATP are distinct and the ATP conduction pathway is not obligatorily associated with the expression of CFTR.
Comments [show]
None has been submitted yet.
No. Sentence Comment
115 We confirmed the reduced single channel conductance of R347E CFTR (1.6 Ϯ 0.1 pS; nϭ3, p Ͻ0.01) (Figure 7C).
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ABCC7 p.Arg347Glu 9463368:115:55
status: NEW117 Effects of DIDS and R347E mutation on CFTR Cl- channels and CFTR-associated ATP channels.
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ABCC7 p.Arg347Glu 9463368:117:20
status: NEW121 (C) Current traces from a MDCK cell expressing R347E in an inside-out patch with 100 mM ATP in the pipette and 140 mM Clin the bath.
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ABCC7 p.Arg347Glu 9463368:121:47
status: NEWX
ABCC7 p.Arg347Glu 9463368:121:55
status: NEW122 R347E mutation had little effect on the slope conductance of the CFTR-associated ATP channels (5.08 Ϯ 0.27 pS; nϭ3) (Figure 7C).
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ABCC7 p.Arg347Glu 9463368:122:0
status: NEW128 We first examined the deletion mutant CFTR"06;R-S660A, Fig. 8.
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ABCC7 p.Arg347Glu 9463368:128:47
status: NEW189 Third, mutation of a residue in the CFTR Cl- channel pore, R347E, had little effect on the conductance of CFTR-associated ATP channels, in contrast to its effect on the CFTR Cl- conductance.
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ABCC7 p.Arg347Glu 9463368:189:59
status: NEW272 Acknowledgements We thank M.Welsh for providing the CFTR∆R-S660A and CFTR S-oct-D mutants, R.Kopito for providing the K1250A and K464A mutants, J.Engelhardt for providing the R347E mutant, U.Patel for her precious technical help and D.Mak for helpful discussions.
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ABCC7 p.Arg347Glu 9463368:272:182
status: NEW123 Effects of DIDS and R347E mutation on CFTR Cl-channels and CFTR-associated ATP channels.
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ABCC7 p.Arg347Glu 9463368:123:20
status: NEW129 R347E mutation had little effect on the slope conductance of the CFTR-associated ATP channels (5.08 afe; 0.27 pS; nafd;3) (Figure 7C).
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ABCC7 p.Arg347Glu 9463368:129:0
status: NEW199 Third, mutation of a residue in the CFTR Cl-channel pore, R347E, had little effect on the conductance of CFTR-associated ATP channels, in contrast to its effect on the CFTR Cl-conductance.
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ABCC7 p.Arg347Glu 9463368:199:58
status: NEW282 Acknowledgements We thank M.Welsh for providing the CFTRƊR-S660A and CFTR S-oct-D mutants, R.Kopito for providing the K1250A and K464A mutants, J.Engelhardt for providing the R347E mutant, U.Patel for her precious technical help and D.Mak for helpful discussions.
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ABCC7 p.Arg347Glu 9463368:282:180
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.
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No. Sentence Comment
214 In this regard, the mutant R347D had normal anion:cation permeability ratios (Linsdell and Hanrahan, unpublished observations) as did R347E (Anderson et al., 1991), but arg352, which has also been proposed to form part of the anion selectivity filter, remains a candidate.
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ABCC7 p.Arg347Glu 9379167:214:134
status: NEW264 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).
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ABCC7 p.Arg347Glu 9379167:264:282
status: NEW265 The PI/PCl ratio obtained for R347D under biionic conditions is intermediate between Iunbl and Ibl in the wild-type channel, and is consistent with the value of 0.9 reported previously for R347E (Anderson et al., 1991).
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ABCC7 p.Arg347Glu 9379167:265:189
status: NEW231 In this regard, the mutant R347D had normal anion:cation permeability ratios (Linsdell and Hanrahan, unpublished observations) as did R347E (Anderson et al., 1991), but arg352, which has also been proposed to form part of the anion selectivity filter, remains a candidate.
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ABCC7 p.Arg347Glu 9379167:231:134
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).
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ABCC7 p.Arg347Glu 9379167:289:282
status: NEW290 The PI/PCl ratio obtained for R347D under biionic conditions is intermediate between Iunbl and Ibl in the wild-type channel, and is consistent with the value of 0.9 reported previously for R347E (Anderson et al., 1991).
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ABCC7 p.Arg347Glu 9379167:290:189
status: NEW[hide] Locating the anion-selectivity filter of the cysti... J Gen Physiol. 1997 Mar;109(3):289-99. Cheung M, Akabas MH
Locating the anion-selectivity filter of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel.
J Gen Physiol. 1997 Mar;109(3):289-99., [PMID:9089437]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel; the site and mechanism of charge selectivity is unknown. We previously reported that cysteines substituted, one at a time, for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353, in and flanking the sixth membrane-spanning segment (M6), reacted with charged, sulfhydryl-specific, methanethiosulfonate (MTS) reagents. We inferred that these residues are on the water-accessible surface of the protein and may line the ion channel. We have now measured the voltage-dependence of the reaction rates of the MTS reagents with the accessible, engineering cysteines. By comparing the reaction rates of negatively and positively charged MTS reagents with these cysteines, we measured the extent of anion selectivity from the extracellular end of the channel to eight of the accessible residues. We show that the major site determining anion vs. cation selectivity is near the cytoplasmic end of the channel; it favors anions by approximately 25-fold and may involve the residues Arg347 and Arg 352. From the voltage dependence of the reaction rates, we calculated the electrical distance to the accessible residues. For the residues from Leu333 to Ser341 the electrical distance is not significantly different than zero; it is significantly different than zero for the residues Thr351 to Gln353. The maximum electrical distance measured was 0.6 suggesting that the channel extends more cytoplasmically and may include residues flanking the cytoplasmic end of the M6 segment. Furthermore, the electrical distance calculations indicate that R352C is closer to the extracellular end of the channel than either of the adjacent residues. We speculate that the cytoplasmic end of the M6 segment may loop back into the channel narrowing the lumen and thereby forming both the major resistance to current flow and the anion-selectivity filter.
Comments [show]
None has been submitted yet.
No. Sentence Comment
20 The mutation R347E, however, had little or no effect on the halide permeability or conductance sequences (Anderson et al., 1991b).
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ABCC7 p.Arg347Glu 9089437:20:13
status: NEW205 Curiously, neither of these mutations nor the mutations R347E and R1030E were reported to alter the Cl- to Naϩ permeability ratio (PCl/PNa), and the latter two mutations had minimal effects on halide permeability or conductance ratios (Anderson et al., 1991b).
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ABCC7 p.Arg347Glu 9089437:205:56
status: NEW[hide] Identification of cystic fibrosis transmembrane co... Biophys J. 1996 Jun;70(6):2688-95. Cheung M, Akabas MH
Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment.
Biophys J. 1996 Jun;70(6):2688-95., [PMID:8744306]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel that is regulated by phosphorylation and ATP binding. Work by others suggested that some residues in the sixth transmembrane segment (M6) might be exposed in the channel and play a role in ion conduction and selectivity. To identify the residues in M6 that are exposed in the channel and the secondary structure of M6, we used the substituted cysteine accessibility method. We mutated to cysteine, one at a time, 24 consecutive residues in and flanking the M6 segment and expressed these mutants in Xenopus oocytes. We determined the accessibility of the engineered cysteines to charged, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) reagents applied extracellularly. The cysteines substituted for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353 reacted with the MTS reagents, and we infer that they are exposed on the water-accessible surface of the protein. From the pattern of the exposed residues we infer that the secondary structure of the M6 segment includes both alpha-helical and extended regions. The diameter of the channel from the extracellular end to the level of Gln353 must be at least 6 A to allow the MTS reagents to reach these residues.
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No. Sentence Comment
190 The multiple ion occupancy effects were eliminated by mutation of Arg347 to Asp or His, and the single-channel conductance was reduced (Tabcharani et al., 1993).
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ABCC7 p.Arg347Glu 8744306:190:24
status: NEW192 Curiously, the mutation R347E was reported to have little effect on the relative halide permeability or conductance sequences of the channel (Anderson et al., 1991b).
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ABCC7 p.Arg347Glu 8744306:192:24
status: NEW[hide] Mutations in the putative pore-forming domain of C... FEBS Lett. 1995 Nov 6;374(3):312-6. Hipper A, Mall M, Greger R, Kunzelmann K
Mutations in the putative pore-forming domain of CFTR do not change anion selectivity of the cAMP activated Cl- conductance.
FEBS Lett. 1995 Nov 6;374(3):312-6., [PMID:7589561]
Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) apparently forms Cl- channels in apical membranes of secretory epithelial cells. A detailed model describes molecular structure and biophysical properties of CFTR and the impact of various mutations as they occur in cystic fibrosis. In the present report mutations were introduced into the putative 6th alpha-helical transmembrane pore forming domain of CFTR. The mutants were subsequently expressed in Xenopus oocytes by injection of the respective cRNAs. Whole cell (wc) conductances could be reversibly activated by IBMX (1 nmol/l) only in oocytes injected with wild-type (wt) or mutant CFTR but not in oocytes injected with water or antisense CFTR. The activated conductance was partially inhibited by (each 100 mumol/l) DIDS (27%) and glibenclamide (77%), but not by 10 mumol/l NPPB. The following mutations were examined: K335E, R347E, R334E, K335H, R347H, R334H. They did not measurably change the wt-CFTR anion permeability (P) and we conductance (G) sequence of: PCl- > PBr- > P1- and GCl- > GBr- > G1-, respectively. Moreover, anomalous mole fraction behavior for the cAMP activated current could not be detected: neither in wt-CFTR nor in R347E-CFTR. Various mutants for which positively charged amino acids were replaced by histidines (K335H, R347H, R334H) did not show pH sensitivity of the IBMX activated wc conductance. We, therefore, cannot confirm previous results. CFTR might have a different molecular structure than previously suggested or it might act as a regulator of ion conductances.
Comments [show]
None has been submitted yet.
No. Sentence Comment
6 The following mutations were examined: K335E, R347E, R334E, K335H, R347H, R334H.
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ABCC7 p.Arg347Glu 7589561:6:46
status: NEW8 Moreover, anomalous mole fraction behavior for the cAMP activated current could not be detected: neither in wt-CFTR nor in R347E-CFTR.
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ABCC7 p.Arg347Glu 7589561:8:123
status: NEW20 (v) Mutations in the apparent 6th a-helical transmembrane domain of CFTR (K335E, R347E) resulted in changes in the halide selectivity of CFTR [1].
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ABCC7 p.Arg347Glu 7589561:20:81
status: NEW32 Synthesis of mutated CFTR-cDNA was induced by annealing of ampicillin repair oligonucleotide and oligonucleotide primers carrying the respective mutation changing positively charged to negatively charged amino acids (R334E, R347E, K335E) or replacing R and K at these positions by histidines (R334H, R347H, K335H).
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ABCC7 p.Arg347Glu 7589561:32:224
status: NEW80 Next, positively charged amino acids R334, R347, K335 located in the putative 6th pore forming transmembrane a-helical domain of CFTR, were exchanged by histidines (R334H, R347H, K335H) or by the negatively charged glutamate (R334E, R347E, K335E).
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ABCC7 p.Arg347Glu 7589561:80:233
status: NEW81 Wc conductances were activated significantly by IBMX in all 6 mutants but to variable degrees (AG in/.tS): 3.2 + 0.6 (R334E, n = 20), 2.7 + 0.6 (R334H, n = 13), 7.1 + 0.9 (K335E, n-- 20), 2.8 + 0.7 (K335H, n = 10), 3.2 + 0.04 (R347E, n = 32) and 1.8 + 0.3 (R347H, n = 10).
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ABCC7 p.Arg347Glu 7589561:81:227
status: NEW86 SCN conductance in wt and R347E CFTR and pH &sensitivity of histidine mutants Extracellular C1- (101 mmol/1) was partially (7 mmol/1) or almost completely (96 mmol/l) replaced by equal concentrations of SCN- and we currents were measured in IBMX stimulated oocytes.
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ABCC7 p.Arg347Glu 7589561:86:26
status: NEW87 For both wt CFTR and R347E-CFTR we found reduced wc conductance when C1- was replaced by SCN-.
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ABCC7 p.Arg347Glu 7589561:87:21
status: NEW88 Anomalous mole fraction behavior could neither be detected for wt-CFTR nor for R347E mutants (Fig. 4).
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ABCC7 p.Arg347Glu 7589561:88:79
status: NEW92 However, unlike in the previous study in R347H [7] no significant changes of G could be detected when extracellular pH was G wtCFTR R347E 10 - (n=17) 0.8- _T_ T 0~- ~ ~, ~, 0.4- ~ ~ o.2- ~ 0.0-I # 10 0.8 0.60.40.20.0- (n=14) T .__T_ i:I I L J Fig. 4. Summary of the conductance ratios obtained in wt and R347E-CFTR transfected oocytes stimulated by IBMX when 101 mmol/1 extracellular CI- was replaced by (mmol/1) 94 C1- and 7 SCN- (94/7) and 5 C1- and 96 SCN- (5/96), respectively.
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ABCC7 p.Arg347Glu 7589561:92:132
status: NEWX
ABCC7 p.Arg347Glu 7589561:92:304
status: NEW105 In fact, in a previous study lysine and arginine were replaced by negatively charged amino acids in the first and sixth transmembrane domain (K95D, K335E, R347E), respectively [1].
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ABCC7 p.Arg347Glu 7589561:105:155
status: NEW108 In the present study we repeated some of the published (K335E, R347E, R347H) and performed additional mutations (R334E, R334H, K335H) which are all located in the putative sixth transmembrane domain and overexpressed the respective CFTRs in oocytes.
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ABCC7 p.Arg347Glu 7589561:108:63
status: NEW112 Comparable wc measurements were performed in the present study (K335E, R347E compared to R347D in [7]) with SCN- and C1- present in the extracellular bath solution at different concentration ratios.
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ABCC7 p.Arg347Glu 7589561:112:71
status: NEW114 Even more important, SCN- con- ductance was not different for the R347E mutant.
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ABCC7 p.Arg347Glu 7589561:114:66
status: NEW[hide] Analysis of the complete coding region of the CFTR... Hum Genet. 1995 Apr;95(4):397-402. Bonizzato A, Bisceglia L, Marigo C, Nicolis E, Bombieri C, Castellani C, Borgo G, Zelante L, Mastella G, Cabrini G, et al.
Analysis of the complete coding region of the CFTR gene in a cohort of CF patients from north-eastern Italy: identification of 90% of the mutations.
Hum Genet. 1995 Apr;95(4):397-402., [PMID:7535742]
Abstract [show]
A complete coding-region analysis on 225 cystic fibrosis (CF) chromosomes from a cohort that includes all the affected subjects born in two North-Eastern Italian regions over eight years was performed. In a previous study, we identified mutations on 166/225 (73.8%) CF chromosomes after screening for 62 mutations. To characterise the remaining 59 CF chromosomes, we carried out automated direct DNA sequencing (exons 9 and 13), RNA single-strand conformation polymorphism (exons 1-8 and 10-12) and denaturing gradient gel electrophoresis (exons 14a-24) of the 27 exons and flanking regions of the CF transmembrane conductance regulator gene. We identified 22 mutations, four of which are novel, viz. 711 + 5G-->A, R709X, 3132delTG and 2790-2A-->G, and we characterised 90.2% (203/225) of the CF chromosomes. Taking advantage of the homogeneity of the sample, an evaluation of the most important clinical parameters, assessed at the age of 12 years, is presented. We confirm some previously reported genotype-phenotype correlations and we report a new nonsense mutation (R709X) associated with a pancreatic sufficient phenotype.
Comments [show]
None has been submitted yet.
No. Sentence Comment
51 Genotypes were identified in six of them and were AF508/1898+3AG, R 1162X/ R347E AF508/2789 + 5G-~A, AFS08/R709X, 1717-1G--~ A/3849 + 10KbC-~T and R553X/2789 + 5G-~A.
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ABCC7 p.Arg347Glu 7535742:51:75
status: NEW[hide] Amino acid residues lining the chloride channel of... J Biol Chem. 1994 May 27;269(21):14865-8. Akabas MH, Kaufmann C, Cook TA, Archdeacon P
Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 1994 May 27;269(21):14865-8., [PMID:7515047]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator forms a chloride channel that is regulated by phosphorylation and intracellular ATP levels. The structure of the channel-forming domains is undetermined. To identify the residues lining this channel we substituted cysteine, one at a time, for 9 consecutive residues (91-99) in the M1 membrane-spanning segment. The cysteine substitution mutants were expressed in Xenopus oocytes. We determined the accessibility of the engineered cysteine to charged, sulfhydryl-specific methanethiosulfonate reagents added extracellularly. We assume that, among residues in membrane-spanning segments, only those lining the channel will be accessible to react with these hydrophilic reagents and that such a reaction would irreversibly alter conduction through the channel. Only the cysteines substituted for Gly-91, Lys-95, and Gln-98 were accessible to the reagents. We conclude that these residues are in the channel lining. The periodicity of these residues is consistent with an alpha-helical secondary structure.
Comments [show]
None has been submitted yet.
No. Sentence Comment
15 Mutation of Lys-95 to Asp, in M1, and Lys-335 and Arg-347 to Glu, in M6, altered the permeability andlor conductance ratios for halides (6).
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ABCC7 p.Arg347Glu 7515047:15:50
status: NEW[hide] Chloride channels in the apical membrane of normal... Am J Physiol. 1992 Jul;263(1 Pt 1):L1-14. Anderson MP, Sheppard DN, Berger HA, Welsh MJ
Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia.
Am J Physiol. 1992 Jul;263(1 Pt 1):L1-14., [PMID:1322048]
Abstract [show]
Cl- channels located in the apical membrane of secretory epithelia play a key role in epithelial fluid and electrolyte transport. Dysfunction of one of these channels, cystic fibrosis transmembrane conductance regulator (CFTR), causes the genetic disease cystic fibrosis (CF). We review here the properties and regulation of the different types of Cl- channels that have been reported in airway and intestinal epithelia. We begin by describing the properties of the CFTR Cl- channel and then use those properties as a point of reference. We focused particularly on the evidence that localizes specific types of Cl- channel to the apical membrane. With that background, we assess the biological function of various Cl- channels in airway and intestinal epithelia.
Comments [show]
None has been submitted yet.
No. Sentence Comment
69 Relative anion permeability of CAMP-regulated channels in apical membrane and in cells expressing wild-type and mutant CFTR PXlPCl- Gx/Gcl- Br- ClI- Br ClI- CAMP 3T3 fibroblasts CFTR 1.11 1.00 0.59 1.26 1.00 0.29 HeLa cells CFTR 1.24 1.00 0.57 1.02 1.00 0.39 K95D 1.25 1.00 1.43 1.39 1.00 0.75 K335E 1.06 1.00 1.37 1.71 1.00 1.43 R347E 1.24 1.00 0.90 1.46 1.00 0.47 Rl030E 1.46 1.00 0.81 1.50 1.00 0.28 Human airway epithelia Apical ND 1.00 0.41 ND 1.00 0.35 T84 epithelia Apical 1.21 1.00 0.56 0.92 1.00 0.47 over cations.
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ABCC7 p.Arg347Glu 1322048:69:330
status: NEW[hide] Cystic fibrosis transmembrane conductance regulato... Neuron. 1992 May;8(5):821-9. Welsh MJ, Anderson MP, Rich DP, Berger HA, Denning GM, Ostedgaard LS, Sheppard DN, Cheng SH, Gregory RJ, Smith AE
Cystic fibrosis transmembrane conductance regulator: a chloride channel with novel regulation.
Neuron. 1992 May;8(5):821-9., [PMID:1375035]
Abstract [show]
Comments [show]
None has been submitted yet.
No. Sentence Comment
92 Mutation of 2 other basic residues, R347E and R1030E, did not change the selectivity sequence.
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ABCC7 p.Arg347Glu 1375035:92:36
status: NEW[hide] Acute inhibition of the cystic fibrosis transmembr... Am J Physiol Cell Physiol. 2013 Oct 15;305(8):C817-28. doi: 10.1152/ajpcell.00052.2013. Epub 2013 Jun 19. Cai Z, Li H, Chen JH, Sheppard DN
Acute inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel by thyroid hormones involves multiple mechanisms.
Am J Physiol Cell Physiol. 2013 Oct 15;305(8):C817-28. doi: 10.1152/ajpcell.00052.2013. Epub 2013 Jun 19., [PMID:23784545]
Abstract [show]
The chemical structures of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) resemble those of small-molecules that inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. We therefore tested the acute effects of T3, T4 and reverse T3 (rT3) on recombinant wild-type human CFTR using the patch-clamp technique. When added directly to the intracellular solution bathing excised membrane patches, T3, T4, and rT3 (all tested at 50 muM) inhibited CFTR in several ways: they strongly reduced CFTR open probability by impeding channel opening; they moderately decreased single-channel current amplitude, and they promoted transitions to subconductance states. To investigate the mechanism of CFTR inhibition, we studied T3. T3 (50 muM) had multiple effects on CFTR gating kinetics, suggestive of both allosteric inhibition and open-channel blockade. Channel inhibition by T3 was weakly voltage dependent and stronger than the allosteric inhibitor genistein, but weaker than the open-channel blocker glibenclamide. Raising the intracellular ATP concentration abrogated T3 inhibition of CFTR gating, but not the reduction in single-channel current amplitude nor the transitions to subconductance states. The decrease in single-channel current amplitude was relieved by membrane depolarization, but not the transitions to subconductance states. We conclude that T3 has complex effects on CFTR consistent with both allosteric inhibition and open-channel blockade. Our results suggest that there are multiple allosteric mechanisms of CFTR inhibition, including interference with ATP-dependent channel gating and obstruction of conformational changes that gate the CFTR pore. CFTR inhibition by thyroid hormones has implications for the development of innovative small-molecule CFTR inhibitors.
Comments [show]
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No. Sentence Comment
251 The occurrence of subconductance states is more frequent in some site-directed mutations in the MSDs [e.g., R334C-CFTR (70), R347E-CFTR (11), and R352E-CFTR (13)], while wild-type murine CFTR resides for prolonged periods in a minuscule subconductance state and only transitions infrequently to the full open-state (37).
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ABCC7 p.Arg347Glu 23784545:251:125
status: NEW[hide] Sulfonylurea receptors regulate the channel pore i... Biochem J. 2014 Dec 15;464(3):343-54. doi: 10.1042/BJ20140273. Lodwick D, Rainbow RD, Rubaiy HN, Al Johi M, Vuister GW, Norman RI
Sulfonylurea receptors regulate the channel pore in ATP-sensitive potassium channels via an intersubunit salt bridge.
Biochem J. 2014 Dec 15;464(3):343-54. doi: 10.1042/BJ20140273., [PMID:25236767]
Abstract [show]
ATP-sensitive potassium channels play key roles in many tissues by coupling metabolic status to membrane potential. In contrast with other potassium channels, the pore-forming Kir6 subunits must co-assemble in hetero-octameric complexes with ATP-binding cassette (ABC) family sulfonylurea receptor (SUR) subunits to facilitate cell surface expression. Binding of nucleotides and drugs to SUR regulates channel gating but how these responses are communicated within the complex has remained elusive to date. We have now identified an electrostatic interaction, forming part of a functional interface between the cytoplasmic nucleotide-binding domain-2 of SUR2 subunits and the distal C-terminus of Kir6 polypeptides that determines channel response to nucleotide, potassium channel opener and antagonist. Mutation of participating residues disrupted physical interaction and regulation of expressed channels, properties that were restored in paired charge-swap mutants. Equivalent interactions were identified in Kir6.1- and Kir6.2-containing channels suggesting a conserved mechanism of allosteric regulation.
Comments [show]
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No. Sentence Comment
192 Similar to Kir6.2-Lys338 , single charge reversal, Kir6.1-R347E, expressed with WT SUR2A, resulted in channels with decreased EC50 for pinacidil (0.71 + - 1.2 bc;M compared with WT, 43.9 + - 1.3 bc;M, Figures 9a and 9b).
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ABCC7 p.Arg347Glu 25236767:192:58
status: NEW193 Reinstatement of the salt bridge by charge reversals in both subunits (Kir6.1-R347E-SUR2A-E1318R) moved the EC50 back towards WT (23.5 + - 1.3 bc;M, Figure 9b).
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ABCC7 p.Arg347Glu 25236767:193:78
status: NEW194 Similarly, when glibenclamide sensitivity was considered, like Kir6.2-Lys338 , single charge reversal of Kir6.1-R347E resulted in an increase in IC50 (241.6 + - 1.1 nM compared with WT, 6.2 + - 1.1 nM), which was restored towards that of WT channels when single charge reversals in both subunits of residues involved in this salt bridge (Kir6.1-R347E-SUR2A-E1318R) were made (13.8 + - 1.1 nM) (Figure 9c).
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ABCC7 p.Arg347Glu 25236767:194:112
status: NEWX
ABCC7 p.Arg347Glu 25236767:194:345
status: NEW197 Disruption of native Figure 9 Kir6.1-R347E-SUR2A-E1318R charge swap also restores WT channel pharmacology (a) Example recording of WT Kir6.1-SUR2A channel activation by 1, 10 and 100 bc;M pinacidil and inhibition with 10 bc;M glibenclamide.
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ABCC7 p.Arg347Glu 25236767:197:39
status: NEW199 Kir6.1-R347E and SUR2A WT formed channels which had a marked increase in sensitivity to pinacidil which was reversed by co-expression with the putative charge-swap partner SUR2A-E1318R (EC50 = 0.7 + - 0.1 bc;M compared with 44.9 + - 8.1 bc;M for WT channels, P <0.001, and 23.5 + - 13.4 bc;M with Kir6.1-R347E and SUR2A-E1318R, P > 0.05, ANOVA with Bonferroni`s post-hoc test, n6 cells for each data point).
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ABCC7 p.Arg347Glu 25236767:199:7
status: NEWX
ABCC7 p.Arg347Glu 25236767:199:313
status: NEW201 Kir6.1-R347E co-expression with SUR2A WT showed a leftward shift in concentration-inhibition curve with an IC50 changing from 5.7 + - 0.1 nM to 480 + - 27 nM (P <0.0001).
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ABCC7 p.Arg347Glu 25236767:201:7
status: NEW