ABCMdb

ABCC7 p.Lys335Glu

Predicted by SNAP2:	A: D (66%), C: D (80%), D: D (91%), E: D (75%), F: D (91%), G: D (80%), H: D (80%), I: D (80%), L: D (80%), M: D (80%), N: D (80%), P: D (91%), Q: N (53%), R: N (87%), S: D (66%), T: D (75%), V: D (80%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, 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.

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No. Sentence Comment
395 K335E altered the whole cell anion permeability sequence by converting CFTR from a low to a high IÀ permeability pore. Login to comment
401 In contrast, K335E changed the halide permeability and/or conductance sequences [18] but did not alter the anomalous mole-fraction effects [186]. Login to comment

[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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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. Login to comment
6 However, the Kds for NPPB block were increased to 371 and 1573 mM, for the K335E and R347E mutants, respectively. Login to comment
26 ), and the K335E and R347E mutants obtained from Dr K Kunzelmann (Albert-Ludwigs University, Freiburg, Germany). Login to comment
76 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. Login to comment
77 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. Login to comment
90 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). Login to comment
92 NPPB was less eective in blocking the CFTR currents in oocytes expressing the K335E and R347E channels. Login to comment
93 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. Login to comment
95 Right panel: concentration versus response curve for inhibition of the wild-type and K335E channels by NPPB. Login to comment
98 For the K335E data, each point represents the mean+s.e.mean of three to ®ve experiments. Login to comment
99 The theoretical curve for the K335E data provided a Kd of 371 mM. Login to comment
108 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). Login to comment
109 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). Login to comment
116 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. Login to comment
117 The slopes of the straight lines had values of 0.23 (wild-type), 0.24 (K335E) and 0.30 (R347E). Login to comment
153 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. Login to comment
160 This is supported by our observation that a mutation of K335, a site predicted to reside closer to the external mouth of the channel (Riordan et al., 1989), resulted in a small, but signi®cant change in NPPB potency (IC50=166 mM for wild-type and 371 mM for K335E). Login to comment

[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.

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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. Login to comment
437 Blockade of K335E-CFTR was diminished (KD ‫ס‬ 371 ␮M) while the voltage-dependence was not affected. Login to comment
440 The effect of the K335E mutation is probably representative of a through-space interaction, wherein the negative charge introduced impedes the approach of the negatively charged drug, rather than disruption of an intimate interaction between NPPB and this lysine. Login to comment

[hide] Relationship between anion binding and anion perme... J Physiol. 2001 Feb 15;531(Pt 1):51-66. Linsdell P
Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Physiol. 2001 Feb 15;531(Pt 1):51-66., 2001-02-15 [PMID:11179391]

Abstract [show]
1. Anion binding within the pores of wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in two different mammalian cell lines, was assayed using patch clamp recording. Specifically, experiments measured both the conductance of different anions and the ability of other permeant anions to block Cl- permeation through the pore. 2. Under symmetrical ionic conditions, wild-type CFTR channels showed the conductance sequence Cl- > NO3- > Br- > or = formate > F- > SCN- congruent to ClO4-. 3. High SCN- conductance was not observed, nor was there an anomalous mole fraction effect of SCN- on conductance under the conditions used. Iodide currents could not be measured under symmetrical ionic conditions, but under bi-ionic conditions I- conductance appeared low. 4. Chloride currents through CFTR channels were blocked by low concentrations (10 mM) of SCN-, I- and ClO4-, implying relatively tight binding of these anions within the pore. 5. Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Furthermore, block by SCN-, I- and ClO4- were weakened in both mutants. Both these effects are consistent with altered anion binding within the pore. 6. The effects of mutations on anion permeability and relative anion conductance suggested that, for most anions, increased permeability was associated with increased conductance. This indicates that the CFTR channel pore does not achieve its anion selectivity by selective anion binding within the mutated region. Instead, it is suggested that entry of anions into the region around F337 and T338 facilitates their passage through the pore. In wild-type CFTR channels, anion entry into this crucial pore region is probably dominated by anion hydration energies.

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No. Sentence Comment
34 The hypothesis that anion permeability and anion binding are separable facets of the permeation process in the CFTR Cl¦ channel is supported by the fact that several mutations within the pore have been shown to alter anion binding without strongly affecting anion permeability (e.g. K335E, Anderson et al. 1991; R347D, Tabcharani et al. 1993; G314E, Mansoura et al. 1998). Login to comment
200 Several mutations within the pore of CFTR alter permeant anion binding without strongly affecting anion selectivity (in terms of permeability ratios) (e.g. K335E, Anderson et al. 1991; G314E, Mansoura et al. 1998), suggesting that these mutations affect anion binding sites not intimately involved in the anion selectivity process (Smith et al. 1999; Linsdell et al. 2000). Login to comment

[hide] ATP hydrolysis-coupled gating of CFTR chloride cha... Biochemistry. 2001 May 15;40(19):5579-86. Zou X, Hwang TC
ATP hydrolysis-coupled gating of CFTR chloride channels: structure and function.
Biochemistry. 2001 May 15;40(19):5579-86., 2001-05-15 [PMID:11341822]

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No. Sentence Comment
56 For instance, replacing two positively charged residues with negatively charged residues in the M1 and M2 R-helices of MSD (K95D and K335E) alters the anion selectivity sequence of CFTR (17). Login to comment

[hide] Identification of a region of strong discriminatio... Am J Physiol Lung Cell Mol Physiol. 2001 Oct;281(4):L852-67. McCarty NA, Zhang ZR
Identification of a region of strong discrimination in the pore of CFTR.
Am J Physiol Lung Cell Mol Physiol. 2001 Oct;281(4):L852-67., [PMID:11557589]

Abstract [show]
The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl(-) channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl(-) and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl(-) and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl(-) and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.

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No. Sentence Comment
60 Mutants K335E, K335F, T338A, T339A, S341A, S341T, T1134A, and T1134F were prepared as previously described (33). Login to comment
143 Relative permeabilities for WT and mutant CFTRs for monovalent anions CFTR n NO3 Br SCN I ClO4 Acetate Isethionate Glutamate Gluconate WT 16 1.35Ϯ0.01 1.19Ϯ0.02 2.42Ϯ0.06 0.36Ϯ0.01 0.10Ϯ0.01 0.15Ϯ0.00* 0.24Ϯ0.01 0.24Ϯ0.01 0.18Ϯ0.01 K335A 5 1.35Ϯ0.01 1.36Ϯ0.03 3.10Ϯ0.11† 0.75Ϯ0.02† 0.12Ϯ0.01 0.06Ϯ0.01† 0.07Ϯ0.01† 0.07Ϯ0.01† 0.08Ϯ0.01† K335F 7 1.51Ϯ0.03† 1.36Ϯ0.02† 2.73Ϯ0.14 0.99Ϯ0.03† 0.20Ϯ0.02† 0.13Ϯ0.01 0.18Ϯ0.03 0.30Ϯ0.02 0.20Ϯ0.02 K335E 5 1.24Ϯ0.04 1.17Ϯ0.02 2.60Ϯ0.06 1.10Ϯ0.03† 0.23Ϯ0.01† 0.10Ϯ0.01† 0.11Ϯ0.01† 0.10Ϯ0.01† 0.11Ϯ0.01† T338A 5 1.74Ϯ0.07† 1.59Ϯ0.02† 4.35Ϯ0.24† 2.56Ϯ0.13† 1.84Ϯ0.08† 0.07Ϯ0.01† 0.06Ϯ0.01† 0.08Ϯ0.01† 0.08Ϯ0.01† T338E 3 3.65Ϯ0.19† 1.94Ϯ0.04† 4.29Ϯ0.13† 2.41Ϯ0.24† 1.18Ϯ0.06† 0.16Ϯ0.03 0.37Ϯ0.05† 0.36Ϯ0.01† 0.22Ϯ0.03 T339A 5 1.47Ϯ0.01 1.29Ϯ0.03 2.65Ϯ0.06 0.57Ϯ0.02† 0.24Ϯ0.04 0.10Ϯ0.02 0.19Ϯ0.02 0.18Ϯ0.01 0.15Ϯ0.01 S341A 6 1.91Ϯ0.02† 1.42Ϯ0.01† 3.10Ϯ0.09† 0.59Ϯ0.00*† 0.09Ϯ0.00* 0.11Ϯ0.01† 0.12Ϯ0.00*† 0.11Ϯ0.00*† 0.12Ϯ0.00*† S341E 12 2.01Ϯ0.10† 1.46Ϯ0.05† 2.81Ϯ0.18 0.84Ϯ0.00*† 0.31Ϯ0.03† 0.20Ϯ0.01 0.23Ϯ0.02 0.19Ϯ0.01 0.19Ϯ0.02 S341T 5 1.81Ϯ0.05† 1.39Ϯ0.03 3.15Ϯ0.15† 0.41Ϯ0.01 0.07Ϯ0.00* 0.05Ϯ0.00*† 0.06Ϯ0.00*† 0.03Ϯ0.01† 0.06Ϯ0.01† T1134A 6 1.43Ϯ0.02 1.30Ϯ0.02 2.66Ϯ0.02 0.46Ϯ0.00*† 0.06Ϯ0.00*† 0.08Ϯ0.01† 0.10Ϯ0.01† 0.11Ϯ0.01† 0.10Ϯ0.00*† T1134F 5 1.31Ϯ0.07 1.17Ϯ0.05 2.50Ϯ0.10 0.63Ϯ0.01† 0.08Ϯ0.00* 0.13Ϯ0.01 0.09Ϯ0.01† 0.18Ϯ0.02 0.13Ϯ0.01 T1134E 4 1.68Ϯ0.02† 1.39Ϯ0.05† 2.37Ϯ0.18 0.19Ϯ0.03† 0.20Ϯ0.03 0.06Ϯ0.01† 0.09Ϯ0.01† 0.08Ϯ0.01† 0.10Ϯ0.01† Values are means Ϯ SE with only data from the hyperpolarizing ramp protocol; n, no. of oocytes. Relative permeability, permeability of anion x to that of Cl. Anions are listed in order of increasing ionic radius. Login to comment
167 Selectivity sequences for WT and mutant CFTRs CFTR Selectivity Sequence by Relative Permeability WT SCNϾϾNO3 ϾBrϾClϾϾIϾisethionateϭglutamateϾgluconateϭacetateϾClO4 K335A SCNϾϾBrϭNO3 ϾClϾIϾϾClO4 Ͼgluconateϭisethionateϭglutamateϭacetate K335F SCNϾϾNO3 ϾBrϾClϭIϾϾglutamateϾgluconateϭClO4 ϭisethionateϾacetate K335E SCNϾϾNO3 ϾBrϭIϾClϾϾClO4 Ͼgluconateϭisethionateϭglutamateϭacetate T338A SCNϾϾIϾϾClO4 ϭNO3 ϾBrϾClϾϾgluconateϭisethionateϭglutamateϭacetate T338E SCNϾNO3 ϾIϾBrϾClO4 ϾClϾϾisethionateϭglutamateϾgluconateϭacetate T339A SCNϾϾNO3 ϾBrϾClϾϾIϾϾClO4 ϭisethionateϭglutamateϭgluconateϾacetate S341A SCNϾNO3 ϾBrϾClϾϾIϾϾgluconateϭisethionateϭglutamateϭacetateϭClO4 S341E SCNϾNO3 ϾBrϾClϾIϾϾClO4 Ͼisethionateϭacetateϭglutamateϭgluconate S341T SCNϾϾNO3 ϾBrϾClϾϾIϾϾClO4 ϭisethionateϭgluconateϭacetateϭglutamate T1134A SCNϾϾNO3 ϾBrϾClϾϾIϾϾglutamateϭisethionateϭgluconateϭacetateϭClO4 T1134F SCNϾϾNO3 ϾBrϾClϾϾIϾϾglutamateϾacetateϭgluconateϾisethionateϭClO4 T1134E SCNϾNO3 ϾBrϾClϾϾClO4 ϭIϾgluconateϭisethionateϭglutamateϭacetate L856 A REGION OF STRONG DISCRIMINATION IN THE CFTR PORE AJP-Lung Cell Mol Physiol • VOL 281 • OCTOBER 2001 • www.ajplung.org out propagation to distant sites. Login to comment
191 Relative conductances for WT and mutant CFTRs for monovalent anions CFTR n NO3 Br SCN I ClO4 Acetate Isethionate Glutamate Gluconate WT 16 0.87Ϯ0.01 0.77Ϯ0.01 0.18Ϯ0.01 0.25Ϯ0.01 0.23Ϯ0.01 0.55Ϯ0.01 0.50Ϯ0.01 0.57Ϯ0.02 0.56Ϯ0.02 K335A 5 0.88Ϯ0.04 0.77Ϯ0.02 0.30Ϯ0.02† 0.35Ϯ0.02 0.24Ϯ0.02 0.33Ϯ0.01† 0.32Ϯ0.02† 0.37Ϯ0.02† 0.38Ϯ0.02† K335F 7 1.21Ϯ0.05† 0.87Ϯ0.02† 0.55Ϯ0.02† 0.36Ϯ0.01† 0.19Ϯ0.01 0.34Ϯ0.01† 0.34Ϯ0.01† 0.41Ϯ0.01† 0.37Ϯ0.01† K335E 5 1.16Ϯ0.05† 0.91Ϯ0.02† 0.59Ϯ0.02† 0.51Ϯ0.02† 0.28Ϯ0.01 0.22Ϯ0.01† 0.25Ϯ0.01† 0.22Ϯ0.01† 0.24Ϯ0.01† T338A 5 1.20Ϯ0.13† 1.03Ϯ0.06† 0.98Ϯ0.12† 0.82Ϯ0.02† 0.50Ϯ0.04† 0.18Ϯ0.05† 0.08Ϯ0.01† 0.31Ϯ0.05† 0.29Ϯ0.05† T338E 3 3.66Ϯ0.36† 1.53Ϯ0.09† 1.80Ϯ0.12† 1.39Ϯ0.11† 0.87Ϯ0.03† 0.36Ϯ0.04† 0.56Ϯ0.17 0.44Ϯ0.03† 0.48Ϯ0.03† T339A 5 1.01Ϯ0.02† 0.77Ϯ0.03 0.22Ϯ0.01 0.31Ϯ0.03 0.23Ϯ0.01 0.38Ϯ0.02† 0.48Ϯ0.01 0.48Ϯ0.01 0.52Ϯ0.01 S341A 6 1.67Ϯ0.01† 1.08Ϯ0.01† 0.63Ϯ0.03† 0.26Ϯ0.00* 0.15Ϯ0.01† 0.63Ϯ0.01† 0.54Ϯ0.02 0.63Ϯ0.01 0.63Ϯ0.01 S341E 12 1.74Ϯ0.11† 1.14Ϯ0.02† 1.81Ϯ0.06† 0.48Ϯ0.01† 0.35Ϯ0.02† 0.28Ϯ0.01† 0.69Ϯ0.02† 0.65Ϯ0.01† 0.68Ϯ0.01† S341T 5 0.85Ϯ0.02 0.82Ϯ0.01 0.29Ϯ0.01† 0.22Ϯ0.01 0.13Ϯ0.01† 0.48Ϯ0.01 0.45Ϯ0.02 0.43Ϯ0.02 0.55Ϯ0.01 T1134A 6 0.83Ϯ0.02 0.78Ϯ0.01 0.24Ϯ0.01† 0.21Ϯ0.01 0.09Ϯ0.01† 0.39Ϯ0.01† 0.38Ϯ0.01† 0.39Ϯ0.01† 0.40Ϯ0.01 T1134F 5 0.68Ϯ0.03† 0.69Ϯ0.03† 0.36Ϯ0.01† 0.07Ϯ0.01† 0.16Ϯ0.01 0.48Ϯ0.02 0.30Ϯ0.02† 0.22Ϯ0.01† 0.32Ϯ0.02† T1134E 4 0.99Ϯ0.02† 1.00Ϯ0.02† 0.50Ϯ0.02† 0.20Ϯ0.03 0.26Ϯ0.02 0.32Ϯ0.03† 0.34Ϯ0.01† 0.34Ϯ0.03† 0.34Ϯ0.03† Values are means Ϯ SE with only data from the hyperpolarizing ramp protocol; n, no. of oocytes. Relative conductance, conductance of anion x to that of Cl. Anions are listed in order of increasing ionic radius. Login to comment
213 Vrev Cl in ND96 bath solution for WT and mutant CFTRs CFTR n Vrev Cl WT 16 -21.24Ϯ0.59 K335A 5 -22.12Ϯ0.35 K335F 7 -21.92Ϯ0.90 K335E 5 -22.88Ϯ0.36 T338A 5 -26.97Ϯ0.79* T338E 3 -20.58Ϯ1.07 T339A 5 -22.21Ϯ0.98 S341A 6 -21.21Ϯ0.56 S341E 12 -28.77Ϯ1.36* S341T 5 -26.62Ϯ1.43* T1134A 6 -28.33Ϯ1.23* T1134F 5 -19.74Ϯ0.73 T1134E 4 -27.54Ϯ1.27* Values are means Ϯ SE; n, no. of oocytes. Login to comment
278 K335E CFTR exhibited decreased Px/PCl for large anions and increased Px/PCl for the "sticky" anions I- and ClO4 - (Table 2). Login to comment
280 Gx/GCl values for most anions were altered in K335E CFTR, with decreases in Gx/GCl values for large anions and increases in Gx/GCl values for small anions (Table 4). Login to comment
281 T1134E CFTR exhibited a similar pattern, with changes in Gx/GCl values smaller in magnitude than those for K335E CFTR. Login to comment
357 Mutation K335E led to a slight decrease in GS2O3/GCl, whereas mutations T338E and S341E greatly increased GS2O3/GCl. Login to comment
388 Selectivity between Cl- and the divalent anion S2O3 2CFTR n GS2O3/GCl WT 16 0.39Ϯ0.01 K335A 5 0.37Ϯ0.01 K335F 7 0.39Ϯ0.01 K335E 5 0.34Ϯ0.01* T338A 5 0.38Ϯ0.01 T338E 3 0.70Ϯ0.08* T339A 5 0.39Ϯ0.02 S341A 6 0.27Ϯ0.01* S341E 12 0.54Ϯ0.01* S341T 5 0.38Ϯ0.01 T1134A 6 0.34Ϯ0.02 T1134F 5 0.33Ϯ0.01* T1134E 4 0.44Ϯ0.05 Values are means Ϯ SE; n, no. of oocytes. Login to comment
439 Our results with K335E (outside the region of high discrimination) and S341E (inside the region of high discrimination) show that this interpretation is feasible. Login to comment

[hide] Voltage-sensitive gating induced by a mutation in ... Am J Physiol Lung Cell Mol Physiol. 2002 Jan;282(1):L135-45. Zhang ZR, Zeltwanger S, Smith SS, Dawson DC, McCarty NA
Voltage-sensitive gating induced by a mutation in the fifth transmembrane domain of CFTR.
Am J Physiol Lung Cell Mol Physiol. 2002 Jan;282(1):L135-45., [PMID:11741825]

Abstract [show]
A mutation in the fifth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel (V317E) resulted in whole cell currents that exhibited marked outward rectification on expression in Xenopus oocytes. However, the single-channel unitary current (i)-voltage (V) relationship failed to account for the rectification of whole cell currents. In excised patches containing one to a few channels, the time-averaged single-channel current (I)-V relationship (I = N x P(o) x i, where N is the number of active channels and P(o) is open probability) of V317E CFTR displayed outward rectification, whereas that of wild-type CFTR was linear, indicating that the P(o) of V317E CFTR is voltage dependent. The decrease in P(o) at negative potentials was due to both a decreased burst duration and a decreased opening rate that could not be ameliorated by a 10-fold increase in ATP concentration. This behavior appears to reflect a true voltage dependence of the gating process because the P(o)-V relationship did not depend on the direction of Cl(-) movement. The results are consistent with the introduction, by a point mutation, of a novel voltage-dependent gating mode that may provide a useful tool for probing the portions of the protein that move in response to ATP-dependent gating.

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No. Sentence Comment
314 Other glutamate substitutions in TM domains 1, 5, 6, and 12 have been investigated [G91E, G314E, and K335E (16); S341E and T1134E (20)], but none of these exhibited voltage-dependent gating (McCarty and Dawson, unpublished observations). Login to comment

[hide] Identification of positive charges situated at the... Pflugers Arch. 2008 Nov;457(2):351-60. Epub 2008 May 1. Zhou JJ, Fatehi M, Linsdell P
Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore.
Pflugers Arch. 2008 Nov;457(2):351-60. Epub 2008 May 1., [PMID:18449561]

Abstract [show]
We have used site-directed mutagenesis and functional analysis to identify positively charged amino acid residues in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that interact with extracellular anions. Mutation of two positively charged arginine residues in the first extracellular loop (ECL) of CFTR, R104, and R117, as well as lysine residue K335 in the sixth transmembrane region, leads to inward rectification of the current-voltage relationship and decreased single channel conductance. These effects are dependent on the charge of the substituted side chain and on the Cl(-) concentration, suggesting that these positive charges normally act to concentrate extracellular Cl(-) ions near the outer mouth of the pore. Side chain charge-dependent effects are mimicked by manipulating charge in situ by mutating these amino acids to cysteine followed by covalent modification with charged cysteine-reactive reagents, confirming the location of these side chains within the pore outer vestibule. State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore. We suggest that ECL1 contributes to the CFTR pore external vestibule and that positively charged amino acid side chains in this region act to attract Cl(-) ions into the pore. In contrast, we find no evidence that fixed positive charges in other ECLs contribute to the permeation properties of the pore.

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60 As shown in Fig. 2, similar inward rectification is observed in the charge-reversing mutants R104E, R117E, and K335E and to a much lesser extent, R1128E, under symmetrical ionic conditions in excised membrane patches. Login to comment
67 In contrast to the linear I-V relationship seen in wild-type CFTR with symmetrical 154 mM Cl- solutions, mutants R104E, R117E, and K335E showed clear inward rectification. Login to comment
73 As shown in Fig. 3b, macroscopic current rectification was indeed sensitive to symmetrical Cl-concentration in R104E, R117E, and K335E, being more pronounced at low Cl-concentration. Login to comment
79 However, current amplitude was significantly reduced in R104E, R117E, and K335E (Fig. 4a, b). Login to comment
91 All mutants depicted (R104Q, R117Q, K335A, R1128Q, R104E, R117E, K335E, R1128E) showed rectification ratios significantly different from wild type (asterisk P<0.05). Login to comment
92 b The degree of rectification in R104E, R117E, and K335E is dependent on the Cl-concentration. Login to comment
116 All mutants depicted (R104Q, R117Q, K335A, R104E, R117E, K335E) significantly different from wild type (asterisk P<0.05). Login to comment
145 The reduction in unitary current for Cl-efflux seen in R104Q, R104E, and K335E (Fig. 4) further suggests that these residues may play an electrostatic role in Fig. 8 Mutation of positively charged residues weakens the apparent inhibitory effect of external Pt(NO2)4 2- ions. Login to comment

[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.

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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). Login to comment
105 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). Login to comment
107 K335E generated Cl¦ conductances very similar to those of CFTR whereas G551D was almost ineffective. Login to comment
110 In contrast, other mutations, which still activated whole-cell Cl¦ conductance (R117H, R347E, K335E) downregulated ENaC currents. Login to comment

[hide] Non-pore lining amino acid side chains influence a... J Physiol. 1998 Oct 1;512 ( Pt 1):1-16. Linsdell P, Zheng SX, Hanrahan JW
Non-pore lining amino acid side chains influence anion selectivity of the human CFTR Cl- channel expressed in mammalian cell lines.
J Physiol. 1998 Oct 1;512 ( Pt 1):1-16., 1998-10-01 [PMID:9729613]

Abstract [show]
1. The effects of individually mutating two adjacent threonine residues in the sixth membrane-spanning region (TM6) of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel on permeation properties were examined using patch clamp recording from mammalian cell lines stably expressing human CFTR. 2. A number of mutations of T338 significantly affected the permeation properties of the channel. Increases and decreases in single channel conductance were observed for different mutants. Anion selectivity was strongly affected, with no two channel variants sharing the same selectivity sequence. Several mutations led to strong inward rectification of the macroscopic current-voltage relationship. The effects of these mutations on permeation properties were correlated with the size of the amino acid side chain substituted, rather than its chemical nature. 3. Most mutations of T339 resulted in a lack of functional channel expression and apparent misprocessing of the protein. One mutant, T339V, was characterized in detail; its permeation properties were significantly altered, although these effects were not as strong as for T338 mutations. 4. These results suggest an important role for T338 in controlling the permeation properties of the CFTR Cl- channel. It is suggested that mutation of this residue alters the interaction between permeating anions and the channel pore via an indirect effect on the orientation of the TM6 helix.

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209 Inward rectification is observed in several other TM6 mutants at the single channel level, e.g. K335E (Tabcharani et al. 1993), T339V (Fig. 11B) and I332K (P. Linsdell, J. A. Tabcharani & J. W. Hanrahan, unpublished observations); however, voltage-dependent gating in T338 mutants cannot be excluded. Login to comment

[hide] ABC transporter-facilitated ATP conductive transpo... Am J Physiol. 1999 Jan;276(1 Pt 1):C1-8. Schwiebert EM
ABC transporter-facilitated ATP conductive transport.
Am J Physiol. 1999 Jan;276(1 Pt 1):C1-8., [PMID:9886914]

Abstract [show]
The concept that the cystic fibrosis (CF) transmembrane conductance regulator, the protein product of the CF gene, can conduct larger multivalent anions such as ATP as well as Cl- is controversial. In this review, I examine briefly past findings that resulted in controversy. It is not the goal of this review to revisit these disparate findings in detail. Rather, I focus intently on more recent studies, current studies in progress, and possible future directions that arose from the controversy and that may reconcile this issue. Important questions and hypotheses are raised as to the physiological roles that ATP-binding cassette (ABC) transporter-facilitated ATP transport and signaling may play in the control of epithelial cell function. Perhaps the identification of key biological paradigms for ABC transporter-mediated extracellular nucleotide signaling may unify and guide the CF research community and other research groups interested in ABC transporters toward understanding why ABC transporters facilitate ATP transport.

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66 Both Cl- and gluconate currents were inhibited by insertion of two well-known Cl- conduction mutations, K335E and R347D, suggesting that CFTR itself is transporting gluconate (26). Login to comment

[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.

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102 This would suggest that like acidic residues (K95D and K335E) altered the whole cell other Cl0 channels, including ligand-gated Cl0 channels in anion permeability sequence by converting CFTR from a neurons (20) and outwardly rectifying Cl0 channels in low I0 permeability pore (Br0 ' Cl0 ú I0 ) to a high I0 epithelia (55), the CFTR pore has a ''weak field strength`` permeability pore (I0 ú Br0 ú Cl0 ) (4). Login to comment
114 However, the mutation K335E did is controversial (51, 59, 72, 93, 99, 100, 111, 129). Login to comment

[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.

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432 This behavior is consistent with the notion that iodide can reside in the channel with-(TM1), K335E (TM6), R347E (TM6), and R1030E (TM10). Login to comment
434 Finkelstein and Cass (55) called attention to the factthe order of 1:10, but two of the substitutions (K95D and K335E) altered the sequence of relative anion permeabilit- that the electrical behavior of planar lipid membranes bathed by iodide-containing solutions may be strongly in-ies by increasing the ratio for iodide, PI/PCl . Login to comment
436 In the two mutant channels, this ratio was increased to Ç1.4, such that the selectivity sequence was their results suggested that the formation of the polyiodides can be effectively eliminated in the presence of aI ú Br ú Cl ú F. These substitutions, particularly K335E, also increased the apparent conductance ratio. Login to comment
441 The PI/PCl ratio was 0.44 for wild-type CFTR and increased F. Cysteine Accessibilityto 1.10 in K335E CFTR and 0.65 in K335D CFTR. Login to comment
449 The ratio PI/PCl was increased in K335E CFTR but commonly employed are metals like Hg, Cd, or Ag or derivatives of methane thiosulfonate (MTS) first intro-was nevertheless substantially less than that predicted by the general pattern (101). Login to comment
581 The K335E construct was identical to wild type with regard to apparent affinity for DPC but did important for the conformational changes that gate the pore.display slight inward rectification. Login to comment

[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.

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279 Mutation of Lys335 or Arg347 to Glu in TM6 altered the permeability and/or conductance ratios for halide ions (Anderson et al., 1991). Login to comment

[hide] Biochemical implications of sequence comparisons o... Arch Biochem Biophys. 2002 May 15;401(2):215-22. Tan AL, Ong SA, Venkatesh B
Biochemical implications of sequence comparisons of the cystic fibrosis transmembrane conductance regulator.
Arch Biochem Biophys. 2002 May 15;401(2):215-22., [PMID:12054472]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is both of medical significance in humans and of interest with regard to osmoregulation in aquatic organisms. CFTR is composed of five domains: two membrane-spanning domains, two nucleotide-binding domains, and a regulatory domain. Notwithstanding the plethora of information concerning the structure and function of CFTR, the biochemistry of many facets of CFTR are not completely understood. In this regard, we have performed a sequence alignment of representative vertebrate CFTR with the aim of generating hypotheses on the functional significance of conserved and variable residues. Postulates on function common to all organisms are: (i) Thr338 in the sixth transmembrane segment could have a function related to that of the pore-lining residue Lys335, and it is possible that Thr338 hydrogen bonds to Lys335, thus indirectly affecting anion permeability; (ii) the fragment (111)PDNKE could be an ion sensor; (iii) motifs in the two nucleotide-binding domains reflect differential ATP binding and hydrolysis; and (iv) an interaction in the R domain involving (765)RRQSVL and the C terminal end of the domain results in an inhibitory conformation. Major adaptations in fishes include variations in the postulated ion sensor (111)PDNKE, and the absence of a proline residue in the R domain with consequent higher chloride efflux.

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54 The mutation K335E effected a modification of the relative halide permeability from BrÀ > ClÀ > IÀ > FÀ to IÀ > BrÀ > ClÀ > FÀ [28]. Login to comment
58 In salmon II, the Ser residue, whose charge is intermediate between positive and negative (referring to the mutation K335E), could render the permeability to be of the order BrÀ > IÀ > ClÀ > FÀ , i.e., relatively less permeable to chloride than other forms of CFTR. Login to comment
65 If the nonhalide anions are disregarded, the trend is strikingly analogous to what was observed for K335E, suggesting that Thr338 may exert an influence on Lys335, or that the roles of these two residues with regard to pore function are related. Login to comment

[hide] Characterization of mutations located in exon 18 o... FEBS Lett. 1998 Oct 16;437(1-2):1-4. Vankeerberghen A, Wei L, Teng H, Jaspers M, Cassiman JJ, Nilius B, Cuppens H
Characterization of mutations located in exon 18 of the CFTR gene.
FEBS Lett. 1998 Oct 16;437(1-2):1-4., [PMID:9804160]

Abstract [show]
In order to get a better insight into the function of amino acid residues located in the second transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, all exon 18 mutations found in cystic fibrosis (CF) patients were characterized at the protein and at the electrophysiological level. Of the different mutations present in transmembrane helix 12 (M1137V, M1137R, I11139V and deltaM1140), and the intracytoplasmic loop connecting TM12 and NBD2 (D1152H and D1154G), only M1137R interfered with the proper maturation of the protein. Permeability studies performed after injection of the different wild-type and mutant cRNAs in Xenopus laevis oocytes indicated that the mutations did not alter the permeability sequence of the CFTR channels. The whole cell cAMP activated chloride currents, however, were significantly reduced for M1137V, I1139V, D1152H and D1154G and close to zero for deltaM1140, indicating that these mutations interfere with the proper gating of the chloride channels.

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15 The anion selectivity 'lter itself seems to be formed by the transmembrane helices [11], since mutagenesis of lysine 95 to aspartate and of lysine 335 to glutamate changed the anion selectivity of the channel. Login to comment

[hide] Adenosine triphosphate-dependent asymmetry of anio... J Gen Physiol. 1998 Apr;111(4):601-14. Linsdell P, Hanrahan JW
Adenosine triphosphate-dependent asymmetry of anion permeation in the cystic fibrosis transmembrane conductance regulator chloride channel.
J Gen Physiol. 1998 Apr;111(4):601-14., [PMID:9524141]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) forms a tightly regulated channel that mediates the passive diffusion of Cl- ions. Here we show, using macroscopic current recording from excised membrane patches, that CFTR also shows significant, but highly asymmetrical, permeability to a broad range of large organic anions. Thus, all large organic anions tested were permeant when present in the intracellular solution under biionic conditions (PX/PCl = 0.048-0.25), whereas most were not measurably permeant when present in the extracellular solution. This asymmetry was not observed for smaller anions. ATPase inhibitors that "lock" CFTR channels in the open state (pyrophosphate, 5'-adenylylimidodiphosphate) disrupted the asymmetry of large anion permeation by allowing their influx from the extracellular solution, which suggests that ATP hydrolysis is required to maintain asymmetric permeability. The ability of CFTR to allow efflux of large organic anions represents a novel function of CFTR. Loss of this function may contribute to the pleiotropic symptoms seen in cystic fibrosis.

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18 m e t h o d s Experiments were carried out on baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells stably expressing either wild-type or mutant (K335E or R347D) CFTR (Tabcharani et al., 1991, 1993; Linsdell and Hanrahan, 1996a). Login to comment
131 To determine whether gluconate currents were carried directly via CFTR, we examined gluconate efflux mediated by two low conductance CFTR pore mutants, R347D and K335E (Tabcharani et al., 1993). Login to comment
133 Both R347D (Fig. 7 A) and K335E (Fig. 7 B) had similar permeabilities to gluconate in the intracellular solution under biionic conditions to that of wild-type CFTR (PGluconate/PCl ϭ 0.069 Ϯ 0.010, n ϭ 9, for R347D and 0.064 Ϯ 0.008, n ϭ 7, for K335E), suggesting that relative permeability to large organic anions from the intracellular solution is not disrupted in either of these mutants. Login to comment
148 3.9 fA (n ϭ 3) for R347D and 18.1 Ϯ 3.4 fA (n ϭ 5) for K335E, in both cases significantly smaller than wild type under these conditions (P Ͻ 0.05, two-tailed t test). Login to comment
160 Both R347D (A) and K335E (B) mediate macroscopic gluconate efflux with a similar apparent gluconate permeability to wild type (see Figs. 1 A, 2 B, 3, B and C, 5 D, and 8, A and B). Login to comment
161 (C and D) Relationship between mean gluconate current (I) and current variance (␴2) at -50 mV under symmetrical ionic conditions for R347D (C) and K335E (D), calculated as described in Fig. 6 A. Login to comment
210 However, anion export in BHK cell patches was due to CFTR itself and not the result of modification of an anion transporter endogenous to these cells, since the apparent unitary gluconate current amplitude was significantly reduced in two CFTR mutants with reduced Cl- conductance, R347D and K335E (Fig. 7). Login to comment
249 We thank Shu-Xian Zheng and Jie Liao for technical assistance and Dr. J.M. Rommens (Hospital for Sick Children, Toronto, Ontario, Canada) for providing R347D and K335E cDNA. Login to comment
151 To determine whether gluconate currents were carried directly via CFTR, we examined gluconate efflux mediated by two low conductance CFTR pore mutants, R347D and K335E (Tabcharani et al., 1993). Login to comment
153 Both R347D (Fig. 7 A) and K335E (Fig. 7 B) had similar permeabilities to gluconate in the intracellular solution under biionic conditions to that of wild-type CFTR (PGluconate/PCl 5 0.069 6 0.010, n 5 9, for R347D and 0.064 6 0.008, n 5 7, for K335E), suggesting that relative permeability to large organic anions from the intracellular solution is not disrupted in either of these mutants. Login to comment
168 3.9 fA (n 5 3) for R347D and 18.1 6 3.4 fA (n 5 5) for K335E, in both cases significantly smaller than wild type under these conditions (P , 0.05, two-tailed t test). Login to comment
180 Both R347D (A) and K335E (B) mediate macroscopic gluconate efflux with a similar apparent gluconate permeability to wild type (see Figs. 1 A, 2 B, 3, B and C, 5 D, and 8, A and B). Login to comment
181 (C and D) Relationship between mean gluconate current (I) and current variance (s2) at 250 mV under symmetrical ionic conditions for R347D (C) and K335E (D), calculated as described in Fig. 6 A. Login to comment
230 However, anion export in BHK cell patches was due to CFTR itself and not the result of modification of an anion transporter endogenous to these cells, since the apparent unitary gluconate current amplitude was significantly reduced in two CFTR mutants with reduced Cl2 conductance, R347D and K335E (Fig. 7). Login to comment
269 We thank Shu-Xian Zheng and Jie Liao for technical assistance and Dr. J.M. Rommens (Hospital for Sick Children, Toronto, Ontario, Canada) for providing R347D and K335E cDNA. Login to comment

[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.

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189 This is in agreement with experiments by Sheppard et al., that report that mutation R334W modifies the conductance.50 On the other hand, mutation K335E affects channel selectivity.51 We propose that K335 could be implicated in a salt bridge with E873 (H7) in model I and with E92 (H1) in model II, since those acidic residues are located at the same level in the membrane. Login to 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). Login to comment
193 This is in agreement with experiments by Sheppard et al., that report that mutation R334W modifies the conductance.50 On the other hand, mutation K335E affects channel selectivity.51 We propose that K335 could be implicated in a salt bridge with E873 (H7) in model I and with E92 (H1) in model II, since those acidic residues are located at the same level in the membrane. Login to comment
236 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). Login to comment

[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.

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62 Expression levels Wild-type and 11 mutant CFTR constructs were used in this study: G91A, G91E, G91R, G314A, G314D, G314E, G314Q, K335R, K335A, K335D, and K335E. Login to comment
103 Only one mutation, K335E, significantly altered the sequence of permeability ratios derived from shifts in reversal potential. Login to comment
113 The K335D and K335E constructs also exhibited increased conductance ratios for I, a result previously reported by Anderson et al. (1991) for K335E CFTR. Login to comment
137 Permeability Ratios Wild type 4-9 3.42 Ϯ 0.28 1.42 Ϯ 0.04 1.22 Ϯ 0.02 0.39 Ϯ 0.01 0.44 Ϯ 0.03 G91A 3-6 3.24 Ϯ 0.26 1.53 Ϯ 0.04 1.27 Ϯ 0.02 0.37 Ϯ 0.04 0.40 Ϯ 0.04 G91E 3-7 3.50 Ϯ 0.54 1.59 Ϯ 0.04 1.27 Ϯ 0.01 0.35 Ϯ 0.01 0.51 Ϯ 0.04 G91R 3-4 5.26 ؎ 0.46* 1.60 Ϯ 0.03 1.40 ؎ 0.01* 0.32 Ϯ 0.04 0.64 ؎ 0.04* G314A 3-4 2.87 Ϯ 0.17 1.45 Ϯ 0.03 1.19 Ϯ 0.02 0.31 Ϯ 0.03 0.33 Ϯ 0.03 G314D 4 3.42 Ϯ 0.34 1.44 Ϯ 0.05 1.25 Ϯ 0.04 0.33 Ϯ 0.03 0.51 Ϯ 0.05 G314E 3-4 3.72 Ϯ 0.56 1.65 ؎ 0.09* 1.35 ؎ 0.03* 0.49 Ϯ 0.04 0.53 Ϯ 0.04 G314Q 3-4 3.89 Ϯ 0.37 1.62 Ϯ 0.11 1.27 Ϯ 0.04 0.36 Ϯ 0.03 0.62 Ϯ 0.05 K335R 3-5 3.44 Ϯ 0.29 1.35 Ϯ 0.04 1.22 Ϯ 0.03 0.40 Ϯ 0.05 0.41 Ϯ 0.07 K335A 5-6 5.34 ؎ 0.58* 1.48 Ϯ 0.06 1.28 Ϯ 0.04 0.37 Ϯ 0.03 0.60 Ϯ 0.06 K335D 4-6 3.02 Ϯ 0.19 1.50 Ϯ 0.03 1.10 ؎ 0.02* 0.54 ؎ 0.04* 0.65 ؎ 0.06* K335E 5-8 3.64 Ϯ 0.21 1.48 Ϯ 0.06 1.29 Ϯ 0.03 0.46 Ϯ 0.04 1.10 ؎ 0.04* B. Conductance Ratios Wild type 4-9 0.14 Ϯ 0.02 0.75 Ϯ 0.02 0.64 Ϯ 0.02 0.52 Ϯ 0.03 0.18 Ϯ 0.03 G91A 3-6 0.14 Ϯ 0.01 0.77 Ϯ 0.02 0.61 Ϯ 0.02 0.47 Ϯ 0.02 0.19 Ϯ 0.02 G91E 3-7 0.15 Ϯ 0.03 0.73 Ϯ 0.02 0.60 Ϯ 0.01 0.50 Ϯ 0.04 0.30 Ϯ 0.02 G91R 3-4 0.14 Ϯ 0.00 0.84 Ϯ 0.01 0.63 Ϯ 0.01 0.32 ؎ 0.01* 0.14 Ϯ 0.01 G314A 3-4 0.30 Ϯ 0.09 0.89 ؎ 0.01* 0.66 Ϯ 0.01 0.48 Ϯ 0.09 0.24 Ϯ 0.01 G314D 4 0.28 Ϯ 0.05 0.82 Ϯ 0.01 0.70 Ϯ 0.02 0.49 Ϯ 0.06 0.27 Ϯ 0.03 G314E 3-4 0.62 ؎ 0.07* 1.18 ؎ 0.04* 0.84 ؎ 0.05* 0.42 Ϯ 0.05 0.29 Ϯ 0.09 G314Q 3-4 0.63 ؎ 0.02* 1.01 ؎ 0.04* 0.82 ؎ 0.03* 0.50 Ϯ 0.02 0.42 ؎ 0.02* K335R 3-5 0.14 Ϯ 0.01 0.76 Ϯ 0.03 0.61 Ϯ 0.02 0.59 Ϯ 0.06 0.16 Ϯ 0.03 K335A 6 0.20 Ϯ 0.03 0.77 Ϯ 0.02 0.61 Ϯ 0.02 0.45 Ϯ 0.03 0.21 Ϯ 0.02 K335D 4-6 0.65 ؎ 0.04* 1.25 ؎ 0.02* 0.89 ؎ 0.02* 0.61 Ϯ 0.06 0.58 ؎ 0.06* K335E 5-8 0.50 ؎ 0.06* 1.19 ؎ 0.03* 0.89 ؎ 0.02* 0.53 Ϯ 0.03 0.48 ؎ 0.03* (A) The apparent permeability ratios (PS/PCl) for each substitute anion were calculated from the shift in reversal potential using the Goldman-Hodgkin-Katz relation (noted in Materials and Methods). Login to comment
144 Similar negative results were obtained for the K335R and K335A constructs, but in the K335D and K335E mutants maximal attenuation by [SCN]o was reduced by nearly 50%, although the apparent binding affinity was unaffected. Login to comment
145 The result is consistent with the notion that the affinity of anion binding was not diminished in the K335D and K335E constructs, but the impact of binding on conduction was reduced. Login to comment
146 Sensitivity to [SCN]o was identical to wtCFTR in a construct bearing a mutation in NBF2, K1250A (data not shown), which exhibits severely altered activation in the form of a highly stabilized active state (Wilkinson et al., 1996). Login to comment
158 At the K335 locus, substitution by either glutamate (K335E) or aspartate (K335D) significantly reduced the mean value of RR by 25% and 38%, respectively. Login to comment
167 In contrast, values for the K1/2(IBMX) for both K335D and K335E were significantly greater than that for wtCFTR, an indication of diminished sensitivity to activating conditions. Login to comment
169 TABLE 4 Quantitative analyses of the macroscopic I-V shape changes Mutant ⌬ Net charge n RR g(ϩ30)/g(-30) RR/RRWT Wild type 5 1.220 Ϯ 0.06 1.00 G91A 0 4 1.293 Ϯ 0.06 1.06 G91E -1 5 1.512 ؎ 0.10* 1.24 G91R 1 4 8.041 ؎ 0.87* 6.59 G314A 0 4 1.201 Ϯ 0.09 0.98 G314D -1 4 1.362 Ϯ 0.08 1.12 G314E -1 7 1.405 Ϯ 0.08 1.15 G314Q 0 5 1.376 Ϯ 0.10 1.13 K335R 0 4 1.209 Ϯ 0.06 0.99 K335A -1 4 1.295 Ϯ 0.07 1.06 K335D -2 5 0.762 ؎ 0.02* 0.62 K335E -2 4 0.919 ؎ 0.02* 0.75 The slope conductance was measured at ϩ30 mV and -30 mV with respect to the reversal potential. Login to comment
173 TABLE 5 Concentration-dependent activation of wtCFTR, G91, G314, and K335 variants by IBMX in the presence of 10 ␮M forskolin Mutant n K1/2(IBMX) (mM) Wild type 15 0.35 Ϯ 0.04 G91A 5 0.42 Ϯ 0.06 G91E 8 0.51 ؎ 0.06* G91R 5 0.49 Ϯ 0.09 G314A 10 1.21 ؎ 0.11* G314D 3 1.35 ؎ 0.16* G314E 8 6.39 ؎ 1.35* G314Q 4 14.26 ؎ 6.64* K335R 4 0.46 Ϯ 0.04 K335A 2 0.35 Ϯ 0.15 K335D 7 0.87 ؎ 0.13* K335E 3 0.95 ؎ 0.07* The steady-state slope conductance was measured at -60 mV as increasing concentrations of IBMX (0.02-5.0 mM) were added to the perfusate in the continued presence of 10 mM forskolin. Login to comment
198 The results presented here are consistent with the notion that the binding of anions within the CFTR pore is a sensitive indicator of changes in pore structure whereas permeability ratios appear to be rather insensitive to similar TABLE 6 Qualitative summary of the functional consequences of mutations at G91, G314, and K335 Property G91 (TM1) K335 (TM6) G314 (TM5) G91A G91E G91R K335R K335A K335D K335E G314A G314D G314E G314Q I-V shape - - ϩϩϩ - - ϩϩ ϩ - - - - Psub/PCl - - - - - - ϩϩ - - - - gsub/gCl - - - - - ϩϩϩ ϩϩϩ ϩϩ - ϩϩϩ ϩϩϩ SCN- binding - - - - - ϩϩϩ ϩϩϩ ϩϩ - ϩϩϩϩ ϩϩϩϩ Activation - - - - - ϩϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩϩϩ ϩϩϩϩ Results are expressed as follows: -, function of the CFTR construct with the indicated substitution was indistinguishable from wild type; ϩ to ϩϩϩϩ, semiquantitative indication of the magnitude of the change in the function compared with wild type. Login to 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. Login to comment
245 The fact that the apparent affinity of SCN binding was not affected by the addition of a negative charge but the maximal effect was reduced suggests that SCN binding in K335D or K335E CFTR is similar to that seen with wtCFTR but that the bound anion is not as effective in obstructing the conduction path. Login to comment
253 We found, as did Anderson et al. (1991), that the sequence for Cl and I was reversed in K335E CFTR. Login to comment
254 In the present study, however, the sequence for the mutant was not that predicted solely by hydration energies, suggesting that I interacts in a unique way with both wtCFTR and K335E. Login to comment
104 Only one mutation, K335E, significantly altered the sequence of permeability ratios derived from shifts in reversal potential. Login to comment
114 The K335D and K335E constructs also exhibited increased conductance ratios for I, a result previously reported by Anderson et al. (1991) for K335E CFTR. Login to comment
138 Permeability Ratios Wild type 4-9 3.42 afe; 0.28 1.42 afe; 0.04 1.22 afe; 0.02 0.39 afe; 0.01 0.44 afe; 0.03 G91A 3-6 3.24 afe; 0.26 1.53 afe; 0.04 1.27 afe; 0.02 0.37 afe; 0.04 0.40 afe; 0.04 G91E 3-7 3.50 afe; 0.54 1.59 afe; 0.04 1.27 afe; 0.01 0.35 afe; 0.01 0.51 afe; 0.04 G91R 3-4 5.26 d1e; 0.46* 1.60 afe; 0.03 1.40 d1e; 0.01* 0.32 afe; 0.04 0.64 d1e; 0.04* G314A 3-4 2.87 afe; 0.17 1.45 afe; 0.03 1.19 afe; 0.02 0.31 afe; 0.03 0.33 afe; 0.03 G314D 4 3.42 afe; 0.34 1.44 afe; 0.05 1.25 afe; 0.04 0.33 afe; 0.03 0.51 afe; 0.05 G314E 3-4 3.72 afe; 0.56 1.65 d1e; 0.09* 1.35 d1e; 0.03* 0.49 afe; 0.04 0.53 afe; 0.04 G314Q 3-4 3.89 afe; 0.37 1.62 afe; 0.11 1.27 afe; 0.04 0.36 afe; 0.03 0.62 afe; 0.05 K335R 3-5 3.44 afe; 0.29 1.35 afe; 0.04 1.22 afe; 0.03 0.40 afe; 0.05 0.41 afe; 0.07 K335A 5-6 5.34 d1e; 0.58* 1.48 afe; 0.06 1.28 afe; 0.04 0.37 afe; 0.03 0.60 afe; 0.06 K335D 4-6 3.02 afe; 0.19 1.50 afe; 0.03 1.10 d1e; 0.02* 0.54 d1e; 0.04* 0.65 d1e; 0.06* K335E 5-8 3.64 afe; 0.21 1.48 afe; 0.06 1.29 afe; 0.03 0.46 afe; 0.04 1.10 d1e; 0.04* B. Conductance Ratios Wild type 4-9 0.14 afe; 0.02 0.75 afe; 0.02 0.64 afe; 0.02 0.52 afe; 0.03 0.18 afe; 0.03 G91A 3-6 0.14 afe; 0.01 0.77 afe; 0.02 0.61 afe; 0.02 0.47 afe; 0.02 0.19 afe; 0.02 G91E 3-7 0.15 afe; 0.03 0.73 afe; 0.02 0.60 afe; 0.01 0.50 afe; 0.04 0.30 afe; 0.02 G91R 3-4 0.14 afe; 0.00 0.84 afe; 0.01 0.63 afe; 0.01 0.32 d1e; 0.01* 0.14 afe; 0.01 G314A 3-4 0.30 afe; 0.09 0.89 d1e; 0.01* 0.66 afe; 0.01 0.48 afe; 0.09 0.24 afe; 0.01 G314D 4 0.28 afe; 0.05 0.82 afe; 0.01 0.70 afe; 0.02 0.49 afe; 0.06 0.27 afe; 0.03 G314E 3-4 0.62 d1e; 0.07* 1.18 d1e; 0.04* 0.84 d1e; 0.05* 0.42 afe; 0.05 0.29 afe; 0.09 G314Q 3-4 0.63 d1e; 0.02* 1.01 d1e; 0.04* 0.82 d1e; 0.03* 0.50 afe; 0.02 0.42 d1e; 0.02* K335R 3-5 0.14 afe; 0.01 0.76 afe; 0.03 0.61 afe; 0.02 0.59 afe; 0.06 0.16 afe; 0.03 K335A 6 0.20 afe; 0.03 0.77 afe; 0.02 0.61 afe; 0.02 0.45 afe; 0.03 0.21 afe; 0.02 K335D 4-6 0.65 d1e; 0.04* 1.25 d1e; 0.02* 0.89 d1e; 0.02* 0.61 afe; 0.06 0.58 d1e; 0.06* K335E 5-8 0.50 d1e; 0.06* 1.19 d1e; 0.03* 0.89 d1e; 0.02* 0.53 afe; 0.03 0.48 d1e; 0.03* (A) The apparent permeability ratios (PS/PCl) for each substitute anion were calculated from the shift in reversal potential using the Goldman-Hodgkin-Katz relation (noted in Materials and Methods). Login to comment

[hide] Transmembrane domain of cystic fibrosis transmembr... Biochemistry. 1998 Jan 20;37(3):844-53. Wigley WC, Vijayakumar S, Jones JD, Slaughter C, Thomas PJ
Transmembrane domain of cystic fibrosis transmembrane conductance regulator: design, characterization, and secondary structure of synthetic peptides m1-m6.
Biochemistry. 1998 Jan 20;37(3):844-53., [PMID:9454574]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) give rise to cystic fibrosis (CF), the most common genetic disease in the Caucasian population. CFTR is organized into five putative domains, including two that are predicted to be transmembrane and consist of six membrane-spanning segments each. CFTR mediates regulated anion transport across the apical membrane of epithelial cells. The pore through which CFTR transports its solutes is thought to be formed by some combination of the amino-terminal membrane-spanning segments. Although these sequences are predicted to be alpha-helical in secondary structure, to date, no direct structural evidence has been presented testing this hypothesis. Here, we present the biophysical characterization of six peptides (m1-m6) representing the predicted amino-terminal membrane-spanning domain of CFTR. The peptides can be incorporated into liposomes and are soluble in SDS micelles and trifluoroethanol (TFE). FTIR and CD spectroscopy indicate all six peptides adopt a stable, predominantly alpha-helical secondary structure in these environments. In contrast, peptide m6 undergoes a shift from alpha-helix to beta-sheet when dissolved in 20% methanol. Additionally, the peptides show an increase in beta-sheet in TFE, a known inducer of alpha-helices, relative to that seen in the nativelike environments. These results have implications for the folding of this complex membrane protein and suggest that the possible functional role of m6 is manifested through a shift in secondary structure.

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287 For example, mutational data demonstrated that the substitution of glutamic acid for lysine 335 alters channel permeability of CFTR (4), and mutation of arginine 347 to histidine gives rise to pH-dependent ion selectivity of the channel (66). Login to comment

[hide] Probing the structural and functional domains of t... J Bioenerg Biomembr. 1997 Oct;29(5):453-63. Akabas MH, Cheung M, Guinamard R
Probing the structural and functional domains of the CFTR chloride channel.
J Bioenerg Biomembr. 1997 Oct;29(5):453-63., [PMID:9511930]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) forms an anion-selective channel involved in epithelial chloride transport. Recent studies have provided new insights into the structural determinants of the channel's functional properties, such as anion selectivity, single-channel conductance, and gating. Using the scanning-cysteine-accessibility method we identified 7 residues in the M1 membrane-spanning segment and 11 residues in and flanking the M6 segment that are exposed on the water-accessible surface of the protein; many of these residues may line the ion-conducting pathway. The pattern of the accessible residues suggests that these segments have a largely alpha-helical secondary structure with one face exposed in the channel lumen. Our results suggest that the residues at the cytoplasmic end of the M6 segment loop back into the channel, narrowing the lumen, and thereby forming both the major resistance to ion movement and the charge-selectivity filter.

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140 The mutations K95D and K335E altered the halide permeability sequence, leading to the suggestion that these residues might be involved in anion binding (Anderson et al., 1991b). Login to comment
141 The mutation K335E, however,did not alter the SCN- - induced anomalous mole fraction effects (Tabcharani et al., 1993), and our data imply that Lys335 is on the opposite side of the helix from the channel-liningface (Fig. 2B) (Cheung and Akabas, 1996), making it unlikely that this residue is part of an anion binding site. Login to comment
142 More likely,the K335E mutationinduces aconfor- mational change in residues that form ananion-binding site, possibly at quite a distance from this residue. Login to comment

[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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264 Arg347 May Contribute to a Weak Field Strength Site for Iodide High macroscopic PI/PCl ratios have been reported previously for CFTR channels in which positively charged residues in the membrane spanning regions were mutated to negatively charged residues (K95E, 1.43; K335E, 1.37; R347E, 0.9; R1030E, 0.81; Anderson et al., 1991). Login to comment
277 Halide selectivity in CFTR cannot be attributed exclusively to the region around arg347; however, because high PI/PCl ratios have been reported previously for other pore mutants (K95D and K335E; Anderson et al., 1991), and because R347D retains some preference for Br- over Cl- and selects strongly against F- (our unpublished observations). Login to comment
278 PI/PCl was altered by a mutation that abolished the anomalous mole fraction effect (AMFE, R347D); however, these properties are not strictly correlated because K335E also had high PI/PCl in a previous study (Anderson et al., 1991) and yet displays an AMFE in SCN--Cl- mixtures (Tabcharani et al., 1993). Login to comment
289 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). Login to comment
305 Halide selectivity in CFTR cannot be attributed exclusively to the region around arg347; however, because high PI/PCl ratios have been reported previously for other pore mutants (K95D and K335E; Anderson et al., 1991), and because R347D retains some preference for Br2 over Cl2 and selects strongly against F2 (our unpublished observations). Login to comment
306 PI/PCl was altered by a mutation that abolished the anomalous mole fraction effect (AMFE, R347D); however, these properties are not strictly correlated because K335E also had high PI/PCl in a previous study (Anderson et al., 1991) and yet displays an AMFE in SCN2-Cl2 mixtures (Tabcharani et al., 1993). Login to comment

[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.

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21 In contrast, the mutation K335E changed the halide permeability and/or conductance sequences (Anderson et al., 1991b) but did not alter the anomalous mole-fraction effects (Tabcharani et al., 1993). Login to comment
204 Based on the effects of the mutations K95D and K335E on halide selectivity sequences, Anderson et al. (1991b) concluded that Lys95 and Lys335 were determinants of halide selectivity. Login to comment
206 Furthermore, the K335E mutation had no effect on anomalous mole-fraction effects suggesting that Lys335 is not part of an anion binding site in the channel (Tabcharani et al., 1993). Login to comment

[hide] Rectification of cystic fibrosis transmembrane con... Biophys J. 1996 Nov;71(5):2458-66. Zhao J, Zerhusen B, Xie J, Drumm ML, Davis PB, Ma J
Rectification of cystic fibrosis transmembrane conductance regulator chloride channel mediated by extracellular divalent cations.
Biophys J. 1996 Nov;71(5):2458-66., [PMID:8913585]

Abstract [show]
We report here distinct rectification of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel reconstituted in lipid bilayer membranes. Under the symmetrical ionic condition of 200 mM KCl (with 1 mM MgCl2 in cis intracellular and 0 MgCl2 in trans extracellular solutions, pH in both solutions buffered at 7.4 with 10 mM HEPES), the inward currents (intracellular-->extracellular chloride movement) through a single CFTR channel were approximately 20% larger than the outward currents. This inward rectification of the CFTR channel was mediated by extracellular divalent cations, as the linear current-voltage relationship of the channel could be restored through the addition of millimolar concentrations of MgCl2 or CaCl2 to the trans solution. The dose responses for [Mg]zero and [Ca]zero had half-dissociation constants of 152 +/- 72 microM and 172 +/- 40 microM, respectively. Changing the pH buffer from HEPES to N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid did not alter rectification of the CFTR channel. The nonlinear conductance property of the CFTR channel seemed to be due to negative surface charges on the CFTR protein, because in pure neutral phospholipid bilayers, clear rectification of the channel was also observed when the extracellular solution did not contain divalent cations. The CFTR protein contains clusters of negatively charged amino acids on several extracellular loops joining the transmembrane segments, which could constitute the putative binding sites for Ca and Mg.

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190 The studies of Tabcharani et al. (1993) showed that TM6 is also involved in the conduction process of the CFTR channel, as point mutations of K335E and R347D altered the multi-ion pore behavior of the CFTR channel. Login to comment
191 The studies of Tabcharani et al. (1993) showed that TM6 is also involved in the conduction process of the CFTR channel, as point mutations of K335E and R347D altered the multi-ion pore behavior of the CFTR channel. Login to comment

[hide] Function of Xenopus cystic fibrosis transmembrane ... J Biol Chem. 1996 Oct 11;271(41):25184-91. Price MP, Ishihara H, Sheppard DN, Welsh MJ
Function of Xenopus cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels and use of human-Xenopus chimeras to investigate the pore properties of CFTR.
J Biol Chem. 1996 Oct 11;271(41):25184-91., [PMID:8810276]

Abstract [show]
To explore the relationship between structure and function in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, we studied Xenopus CFTR. We found that the anion permeability sequence of cAMP-activated Cl- currents in the apical membrane of Xenopus A6 epithelia differed from that of cAMP-activated Cl- currents in human epithelia expressing CFTR. To understand the molecular basis for this difference and to learn whether CFTR from another species would have properties similar to human CFTR, we assembled a full-length Xenopus CFTR cDNA from A6 cells. Expression of Xenopus CFTR in HeLa cells generated cAMP-activated whole-cell currents and cAMP-dependent protein kinase-activated single channels that resembled those of human CFTR with the exception that the anion permeability sequence was different (Br- = I- > Cl- in Xenopus CFTR and Br- = Cl- > I- in human). In addition, the single-channel conductance of Xenopus CFTR was increased. To investigate protein regions that account for these differences, we constructed chimeric proteins by replacing either the first or second membrane-spanning domain of human CFTR with the equivalent region of Xenopus CFTR (hX1-6 and hX7-12, respectively) and examined their function in HeLa cells. We found that the anion permeability sequence (Br- = I- > Cl-) and single-channel conductance of hX1-6 resembled that of Xenopus CFTR expressed in HeLa cells, whereas hX7-12 had properties like those of human CFTR. However, the gating of hX1-6 showed a flickery behavior. The altered gating of hX1-6 was attributed to residues in the first extracellular loop of Xenopus CFTR because mutation of residues in that region to the corresponding residues of human CFTR produced gating behavior similar to that of human CFTR. These data suggest that sequence differences in the first membrane-spanning domains are responsible for the differences in the permeation properties of human and Xenopus CFTR and that the first extracellular loop influences channel gating.

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260 In MSD1, two mutations (P99L and K335E) that alter both anion permeability and conductance have been identified (15, 30); these are conserved in human and Xenopus CFTR. Login to comment
279 In MSD1, two mutations (P99L and K335E) that alter both anion permeability and conductance have been identified (15, 30); these are conserved in human and Xenopus CFTR. Login to comment

[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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25 A mutation, K335E, in M6 altered the permeability and conductance ratios for halides (Anderson et al., 1991b). Login to comment
193 The mutation K335E, a water-accessible residue, altered the relative halide permeability and conductance sequences (Anderson et al., 199lb). Login to comment
191 The mutation K335E, a water-accessible residue, altered the relative halide permeability and conductance sequences (Anderson et al., 199lb). Login to comment

[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.

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6 The following mutations were examined: K335E, R347E, R334E, K335H, R347H, R334H. Login to comment
20 (v) Mutations in the apparent 6th a-helical transmembrane domain of CFTR (K335E, R347E) resulted in changes in the halide selectivity of CFTR [1]. Login to comment
32 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). Login to comment
76 I R334EIIR334HI K335E...... I K335H I R347EII R347H l (n=16) n=10) (n=10) (n=24) (n=9) "° ,11 ml I'lnt;"i' ii Illll 111 0.8 X T °., I ~ 0.4 0.2 I o.o ~ ~ ~ 6!~ 6 ~ 8 ~ ,I I ~ ...... ] J I I L ...... ,j I I t 1 I * *J t........ ~,_J L * * I * *I _ J I .......... I I , * * * , (n=18) (n=lO) (n=22) (n=7) 1.o - T T (n=8) (n=14) T / T T T o.eT T T o 1 "~ 0.4-O 0.2- oo_ L__J , i I i t - - I 1 I I I ~ t J L ' t * J I__~ * I [ * * I l * * j l. Login to comment
80 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). Login to comment
81 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). Login to comment
105 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]. Login to comment
108 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. Login to comment
112 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. Login to comment

[hide] The CFTR chloride channel of mammalian heart. Annu Rev Physiol. 1995;57:387-416. Gadsby DC, Nagel G, Hwang TC
The CFTR chloride channel of mammalian heart.
Annu Rev Physiol. 1995;57:387-416., [PMID:7539989]

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18 For example, expression in 3T3 cells of two CFTR mutants, K95D and K335E, in which negatively charged amino acids replaced positively charged lysines in the first (Ml) and sixth (M6) putative transmembrane a-helices (Figure I) yielded Cl- channels with altered anion permeability sequences, 1- > B..- > Cl- instead of the normal Br > CI- > 1- (3). Login to comment

[hide] Novel pore-lining residues in CFTR that govern per... Neuron. 1994 Sep;13(3):623-34. McDonough S, Davidson N, Lester HA, McCarty NA
Novel pore-lining residues in CFTR that govern permeation and open-channel block.
Neuron. 1994 Sep;13(3):623-34., [PMID:7522483]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is both a member of the ATP-binding cassette superfamily and a Cl(-)-selective ion channel. We investigated the permeation pathway of human CFTR with measurements on conduction and open-channel blockade by diphenylamine-2-carboxylic acid (DPC). We used site-directed mutagenesis and oocyte expression to locate residues in transmembrane domain (TM) 6 and TM 12 that contact DPC and control rectification and single-channel conductances. Thus, TM 12 and the previously investigated TM 6 line the CFTR pore. In each TM, residues in contact with DPC are separated by two turns of an alpha helix. The contributions of TM 6 and TM 12 to DPC block and Cl- permeation, however, are not equivalent. The resulting structural model for the conduction pathway may guide future studies of permeation in other Cl- channels and ATP-binding cassette transporters.

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71 (F) K335E mutation, indicating inward rectification and unchanged DPC binding affinity. Login to comment
78 Affinity and Voltage Dependence for Block of CFTR Variants by DPC Construct TM Ko( - 100) (PM) 0 I-V Relation n Properties Wild type Wild type low [Cl-], (10 mM) K335E 6 K335F 6 T338A 6 T339A 6 S341A 6 S341T 6 S1118A 11 T1134A 12 T1134F 12 S1141A 12 Triple 6,12 276 f 14 181 f 13" 303 -t 14 351 * 15' 220 * 14 284 * 47 1251 f 116a 530 f 80" 243 * 37 230 * 20 74 * 3" 220 * 13 325 * 26b 0.41 f 0.01 0.32 f 0.02" 0.42 f 0.01 0.42 f 0.02 0.36 f 0.02" 0.44 * 0.12 0.49 * 0.03" 0.35 f 0.09 0.40 f 0.02 0.35 * 0.02" 0.41 f 0.01 0.42 f 0.03 0.21 * O.Ol",b Linear, E,,, = -8 f 1 mV Ere\ = +48+2mV Inward rectification Linear Linear Linear Strong inward rectification Inward rectification Linear Linear Linear Linear Strong inward rectification Affinity for DPC was determined empirically at -100 mV, from whole-cell currents measured in the presence of 200 uM DPC (see Experimental Procedures). Login to comment
92 In contrast, K335E displayed slight inward rectification of unblocked currents, but this mutant had the same DPC affinity as wild-type CFTR (Figure 3F). Login to comment
156 Also in TM 6, mutation K335F slightly reduces DPC block, and mutation K335E gives inward rectification (see also Tabcharani et al., 1993). Login to comment
173 Previous studies of CFTR have found that mutations of positively charged residues in TM 1 and TM 6 (including K335E) give small changes in ionic selectivity (Anderson et al., 1991b) and that naturally occurring mutations of positively charged residues just extracellular to TM 2, or within TM 6 (R347P and R334W), reduce single-channel conductance and alter kinetics (Sheppard et al., 1993). Login to comment

[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.

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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). Login to comment

[hide] Cystic fibrosis gene update. J R Soc Med. 1994;87 Suppl 21:2-4. Cuthbert A
Cystic fibrosis gene update.
J R Soc Med. 1994;87 Suppl 21:2-4., [PMID:7515441]

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14 (c) Directed mutagenesis (ie conversion oflysine 95 to aspartate and lysine 335 to glutamate) altered the ion selectivity of CFTR chloride channels from Br>Cl->I->Fto I->Br->Cl->F-6. Login to comment

[hide] Detection of 98.5% of the mutations in 200 Belgian... Genomics. 1993 Dec;18(3):693-7. Cuppens H, Marynen P, De Boeck C, Cassiman JJ
Detection of 98.5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene.
Genomics. 1993 Dec;18(3):693-7., [PMID:7508414]

Abstract [show]
We have previously shown that about 85% of the mutations in 194 Belgian cystic fibrosis alleles could be detected by a reverse dot-blot assay. In the present study, 50 Belgian chromosomes were analyzed for mutations in the cystic fibrosis transmembrane conductance regulator gene by means of direct solid phase automatic sequencing of PCR products of individual exons. Twenty-six disease mutations and 14 polymorphisms were found. Twelve of these mutations and 3 polymorphisms were not described before. With the exception of one mutant allele carrying two mutations, these mutations were the only mutations found in the complete coding region and their exon/intron boundaries. The total sensitivity of mutant CF alleles that could be identified was 98.5%. Given the heterogeneity of these mutations, most of them very rare, CFTR mutation screening still remains rather complex in our population, and population screening, whether desirable or not, does not appear to be technically feasible with the methods currently available.

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80 It has been shown that mutagenesis of the neighboring amino acid 335 from a basic amino acid to an acidic amino acid (K335E) results in a CFTR protein with altered anion permeability sequence and conductivity sequence properties of the different halides (2). Login to comment
79 It has been shown that mutagenesis of the neighboring amino acid 335 from a basic amino acid to an acidic amino acid (K335E) results in a CFTR protein with altered anion permeability sequence and conductivity sequence properties of the different halides (2). Login to comment

[hide] Effect of ATP-sensitive K+ channel regulators on c... J Gen Physiol. 1992 Oct;100(4):573-91. Sheppard DN, Welsh MJ
Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.
J Gen Physiol. 1992 Oct;100(4):573-91., [PMID:1281220]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cAMP-dependent phosphorylation and by intracellular ATP. Intracellular ATP also regulates a class of K+ channels that have a distinct pharmacology: they are inhibited by sulfonylureas and activated by a novel class of drugs called K+ channel openers. In search of modulators of CFTR Cl- channels, we examined the effect of sulfonylureas and K+ channel openers on CFTR Cl- currents in cells expressing recombinant CFTR. The sulfonylureas, tolbutamide and glibenclamide, inhibited whole-cell CFTR Cl- currents at half-maximal concentrations of approximately 150 and 20 microM, respectively. Inhibition by both agents showed little voltage dependence and developed slowly; > 90% inhibition occurred 3 min after adding 1 mM tolbutamide or 100 microM glibenclamide. The effect of tolbutamide was reversible, while that of glibenclamide was not. In contrast to their activating effect on K+ channels, the K+ channel openers, diazoxide, BRL 38227, and minoxidil sulfate inhibited CFTR Cl- currents. Half-maximal inhibition was observed at approximately 250 microM diazoxide, 50 microM BRL 38227, and 40 microM minoxidil sulfate. The rank order of potency for inhibition of CFTR Cl- currents was: glibenclamide < BRL 38227 approximately equal to minoxidil sulfate > tolbutamide > diazoxide. Site-directed mutations of CFTR in the first membrane-spanning domain and second nucleotide-binding domain did not affect glibenclamide inhibition of CFTR Cl- currents. However, when part of the R domain was deleted, glibenclamide inhibition showed significant voltage dependence. These agents, especially glibenclamide, which was the most potent, may be of value in identifying CFTR Cl- channels. They or related analogues might also prove to be of value in treating diseases such as diarrhea, which may involve increased activity of the CFTR Cl- channel.

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48 We also transiently expressed another mutant (CFI'R-K335E, in which lysine 335 in the first transmembrane domain was changed to glutamic acid) in HeLa cells using the vaccinia virus-T7 hybrid expression system (Rich et al., 1990; Elroy-Stein, Fuerst, and Moss, 1989). Login to comment
161 We therefore examined the effect of glibenclamide, the most potent inhibitor we had identified, on CI- currents generated by several CFFR mutants. We studied 100 • 1992 CFTR containing mutations that affect each of the three types of domains of CFTR: CFFP~R where part of the R domain has been deleted (amino acids 708-835); CVFR-K335E, which contains a mutation in the sixth putative membrane-spanning sequence; and CFTR-K1250M, which contains a mutation in the second NBD. Login to comment
171 CFFR-K335E forms CI- channels that are similar to wild-type channels, except that the anion selectivity is altered such that I- > Br- > C1- and currents show some outward rectification (Anderson et al., 1991b and Fig. 7, D and F). Login to comment
172 Glibenclamide inhibited CFTR-K335E CI- currents, and the effect was similar to that observed with wild-type CFTR (Fig. 7, E and F). Login to comment
176 +SO mV 0 m V ~ -go mV CFTFI~ A~ ......~-1 baseline D CFTR-K335E I ii..i.Iii cAMP .... 13, CFTR-K1250M 1cAMP baseline + gllbenclamlde 250 pA L 50 ms C 1500 1000 5OO -500 o1~0 -100 -75 -50 -25 0 V (mV) ---o-- baseline ---4-- gllbenclamlde E) I cAMP + gllbenclamlde / tp----e F 1000 750 5OO -100 -75 -50 -25 0 V (mV) H ) 1000 PAL__ 50 ms I i cAMP + gllbenclamlde --o-- cAMP --e-- gllbanclamlde / _~_ _ 4OOO 3OOO ~q]O0 I000 0 I000 2OOO 30OO mOO -100 -75 -50 -25 0 V (mV) 25 50 25 50 25 50 FIGURE 7. Login to comment
178 Traces are from C127 cells stably expressing CFTP-~R (A-C), HeLa cells transiently expressing CFFR-K335E (D-F), and NIH 3T3 cells stably expressing CFIR-K1250M (G-I). Login to comment
180 CFFRAR currents were recorded in the absence of cAMP, but for CFFR-K335E and CFTR-K1250M currents cAMP agonists were present. Login to comment
185 Data for steady-state current values measured at +50 and -90 mV are shown for wild-type CF-FR (A), CFTRAR (B), CFFR-K335E (C), and CFTR-K1250M (D). Login to comment
186 The dose-response curves for wild-type CFFR, CFTR-K335E, and CFTR-K1250M were similar. Login to comment
187 Although the effect was not marked, in each case inhibition was slightly more potent at -90 mV than at +50 inV. A 100 8O O 60 c g 20 B 100 8O o 6o eo o m 20 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992 C CFTR 100 ' --o-- 50mV 80 e 6o C g,m 2O 0 20 40 60 80 1C10 0 gllbenclamlde (,aM) D CFTRAR 100 80 2 60 c i 2O 1 ~ ~ 866o o glil~nclamlde (gM) CFTR-K335E 0 20 40 ~ 80 100 glibenclamlda (,ttM) CFTR-K1250M "il o 2i~ ,o 6i) 8o lOO glibenclamlde (i.tM) FIGURE 8. Login to comment
191 (A) Wild-type CFTR; (B) CFTR~R; (C) CFTR-K335E; (D) CFFR-K1250M. Login to comment
196 Half-maximal inhibition by glibenclamide was similar in all cases, occurring at ~ 20 TABLE I1 Effect of Glibenclamide on CFTR Mutants 4-50 mV -90 mV Mutant Experiments Ki n Ki n my/ u2v/ CFTR 21.8 ± 5.2 0.8 - 0.1 19.0 - 5.6* 0.7 ± 0.1 7 CFTR-K335E 25.9 ± 4.1 0.9 ± 0.I 20.4 +--3.7* 0.9 ± 0.1 6 CFTR-K1250M 16.8 _ 4.2 0.8 ± 0.1 31.2 ± 2.5 1.0 - 0.1 5 Ki and n values were calculated as described in Table I for currents measured at +50 and -90 mV for the number of experiments listed. Login to comment
199 Half-maximal inhibition by glibenclamide, measured at -90 mV, showed a small but statistically significant increase in potency compared with that at +50 mV for wild-type CFTR and CFTR-K335E, but not CFFR-K1250M. Login to comment
248 Inhibition of CFTR-K335E and CFTR-K1250M CI- currents by glibenclamide resembled that of wild-type CFFR. Login to comment
249 This suggests that residues K335E and K1250M do not form a critical part of the glibenclamide interaction site. Login to comment

[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.

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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. Login to comment

[hide] The biochemical defect in cystic fibrosis. J R Soc Med. 1992;85 Suppl 19:2-5. Cuthbert AW
The biochemical defect in cystic fibrosis.
J R Soc Med. 1992;85 Suppl 19:2-5., [PMID:1375960]

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70 Changing lysine 95 to aspartic acid and lysine 335 to glutamic acid alters the selectivity of the chloride conductance fiom chloride>iodide to iodide>chloride When the R-domain was deleted the CFTR chloride conductance was increased in the unstimulated state (ie absence ofcAMP) suggesting that the R-domain is involved in thegatingofthe ion channeL A similar mechanism has beenproposedfor the gating ofpotassium channels. Login to comment

[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]

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87 Yet two mutations, K95D and K335E, each altered anion selectivity. Login to comment
91 The two mutations were not, however, equivalent: unlike the changes in permeability, only K335E changed the conductivity sequence from BrZ Cl- > Ito Br- > I- > Cl-. Login to comment

[hide] Beta-adrenergic receptors couple to CFTR chloride ... Biochim Biophys Acta. 2003 Dec 30;1618(2):140-52. Larsen EH, Amstrup J, Willumsen NJ
Beta-adrenergic receptors couple to CFTR chloride channels of intercalated mitochondria-rich cells in the heterocellular toad skin epithelium.
Biochim Biophys Acta. 2003 Dec 30;1618(2):140-52., [PMID:14729151]

Abstract [show]
In the heterocellular toad skin epithelium the beta-adrenergic receptor agonist isoproterenol activates cyclic AMP-dependent Cl(-) channels that are not located in the principal cells. With four experimental approaches, in the present study, we tested the hypothesis that the signalling pathway targets cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channels of mitochondria-rich cells. (i) Serosal application of isoproterenol (log(10)EC(50)=-7.1+/-0.2; Hill coefficient=1.1+/-0.2), as well as noradrenaline, activated an anion pathway with an apical selectivity sequence, G(Cl)>G(Br)> or =G(NO(3))>G(I), comparable to the published selectivity sequence of cloned human CFTR expressed in Xenopus oocytes. (ii) Known modulators of human CFTR, glibenclamide (200 micromol/l) and genistein (50 micromol/l), depressed and activated, respectively, the receptor-stimulated G(Cl). Genistein did not modify the anion selectivity. (iii) Transcellular voltage clamp studies of single isolated mitochondria-rich cells revealed functional beta-adrenergic receptors on the basolateral membrane. With approximately 60,000 mitochondria-rich cells per cm(2), the saturating activation of 11.9+/-1.6 nS/cell accounted for the measured isoproterenol-activated transepithelial conductance of 600-900 microS/cm(2). In forskolin-stimulated cells, glibenclamide (200 micromol/l) reversibly inhibited the transcellular conductance by 9.6+/-1.6 nS/cell. (iv) With primers constructed from cloned Xenopus CFTR and PCR amplification of reverse-transcribed mRNA from toad skin, full-length Bufo CFTR cDNA was generated. The derived protein of 1466 residues shows 86% homology with xCFTR and 89% homology with hCFTR. All major functional sequences, that is, the R- and the NBF1- and NBF2-domains are well-conserved as are the predicted transmembrane segments proposed to form the pore of the channel protein. These new results taken together with our previously identified small-conductance CFTR-like Cl(-) channel in the apical membrane of the mitochondria-rich cells lead to the conclusion that the toad's CFTR gene codes for a functional Cl(-) channel in the apical plasma membrane of this minority cell type.

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245 Mutation of the basic lysine at this position of hCFTR expressed in HeLa cells to the acidic residue, glutamic acid, resulted in a sequence selectivity shift from GClfGBr>GI>GF (wild-type) to GBrfGI>GCI>GF (K335E) [38]. Login to comment