Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients

Dean, Michael; White, Marga B.; Amos, Jean; Gerrard, Bernard; Stewart, Claudia; Khaw, Kon Taik; Leppert, M.

doi:10.1016/0092-8674(90)90196-l

Cited by 328 publications

(141 citation statements)

References 37 publications

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“…The Arg-347 residue is targeted by several CF-associated mutations, R347C, R347H, R347L, and R347P (13)(14)(15). 2 Our data suggest that CF-associated as well as other mutations at residue 347 affect CFTR similarly.…”

Section: Discussionmentioning

confidence: 81%

“…At least four CF-associated mutations have been identified at position 347 in M6: R347C, R347H, R347L, and R347P, suggesting that Arg-347 is important for CFTR structure and function (13)(14)(15). 2 Early studies by Sheppard et al (7) showed that mutation of Arg-347 to proline significantly decreased single-channel conductance with little effect on CFTR trafficking to the plasma membrane.…”

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confidence: 99%

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Cystic Fibrosis-associated Mutations at Arginine 347 Alter the Pore Architecture of CFTR

Cotten

Welsh

1999

Journal of Biological Chemistry

107

105

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Arginine 347 in the sixth transmembrane domain of cystic fibrosis transmembrane conductance regulator (CFTR) is a site of four cystic fibrosis-associated mutations. To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function. Every Arg-347 mutation examined, except R347K, had a destabilizing effect on the pore, causing the channel to flutter between two conductance states. Chloride flow through the larger conductance state was similar to that of wildtype CFTR, suggesting that the residue at position 347 does not interact directly with permeating anions. We hypothesized that Arg-347 stabilizes the channel through an electrostatic interaction with an anionic residue in another transmembrane domain. To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations. Interestingly, the D924R mutation complemented R347D, yielding a channel that behaved like wild-type CFTR. These data suggest that Arg-347 plays an important structural role in CFTR, at least in part by forming a salt bridge with Asp-924; cystic fibrosis-associated mutations disrupt this interaction. The cystic fibrosis transmembrane conductance regulator (CFTR)1 contains an anion-selective pore (1-6). Earlier work has shown that amino acid residues in the two membranespanning domains, MSD1 and MSD2, determine the properties of this pore and harbor a number of disease-associated mutations (7-12). Despite the importance of this region to CFTR function, an understanding of the pore and MSD structure is limited.Studies of the effect of cystic fibrosis (CF)-associated mutations have been of value in identifying structurally and functionally important regions of CFTR. At least four CF-associated mutations have been identified at position 347 in M6: R347C, R347H, R347L, and R347P, suggesting that Arg-347 is important for CFTR structure and function (13-15).2 Early studies by Sheppard et al. (7) showed that mutation of Arg-347 to proline significantly decreased single-channel conductance with little effect on CFTR trafficking to the plasma membrane. Other work by Tabcharani et al. (8,16) and Linsdell and Hanrahan (17) emphasized the importance of Arg-347 for anomalous mole-fraction behavior, iodide permeability, and voltagedependent block by DIDS, in addition to single-channel conductance. Interestingly, mutation of Arg-347 to a histidine (R347H) produced a channel that displayed pH-dependent conductance and anomalous mole-fraction behavior (8). These studies suggested that Arg-347 may line the pore and that a positive charge at position 347 is sufficient for wild-type conductance. Because mutation of Arg-347 eliminated anomalous mole-fraction behavior, Arg-347 itself was proposed to be an anion binding site in the CFTR pore (8), and the presumed positive charge introduced upon protonation of His-347 was thought to facilitate interactions with permeating a...

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Section: Discussionmentioning

confidence: 81%

mentioning

confidence: 99%

Cystic Fibrosis-associated Mutations at Arginine 347 Alter the Pore Architecture of CFTR

Cotten

Welsh

1999

Journal of Biological Chemistry

107

105

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“…Similarly, some mutations in the CF gene alter the conduction of the Cl Ϫ channel CFTR, without affecting its targeting (35). Of note, most of these peculiar CF mutations are located within the membrane-spanning domains of CFTR (36,37), and both mutations that were identified here are located within the central hydrophobic domain of NCC: S555L lies at the end of TM10, whereas A588V is within TM12. It has been suggested that the central domain of NCC determines ion translocation and thiazide-binding specificity (38,39), which could provide a basis for the pathogenic role of these missense mutations.…”

Section: Discussionmentioning

confidence: 97%

Transcriptional and Functional Analyses of SLC12A3 Mutations

Riveira-Muñoz¹,

Chang²,

Godefroid³

et al. 2007

Journal of the American Society of Nephrology

142

125

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Gitelman syndrome (GS) is a recessive salt-losing tubulopathy that is caused by mutations in the SLC12A3 gene that encodes the sodium-chloride co-transporter (NCC). GS is characterized by significant inter-and intrafamilial phenotype variability, with early onset and/or severe clinical manifestations in some patients. No correlations between the disease variability and the position/nature of SLC12A3 mutations have been investigated thus far. In this study, extensive mutational analyses of SLC12A3 were performed in 27 patients with GS, including genomic DNA sequencing, multiplex ligation-dependent probe amplification, cDNA analysis, and quantification of allele-specific transcripts, in parallel with functional analyses in Xenopus laevis oocytes and detailed phenotyping. Twenty-six SLC12A3 mutations were identified in 25 patients with GS, including eight novel (detection rate 80%). Transcript analysis demonstrated that splicing mutations of SLC12A3 lead to frameshifted mRNA subject to degradation by nonsense-mediated decay. Heterologous expression documented a novel class of NCC mutants with defective intrinsic transport activity. A subgroup of patients presented with early onset, growth retardation, and/or detrimental manifestations, confirming the potential severity of GS. The mutations that were associated with a severe presentation were the combination at least for one allele of a missplicing resulting in a truncated transcript that was downregulated by nonsense-mediated decay or a nonfunctional, cell surface-absent mutant. The most recurrent mutation on the second allele was a newly described NCC mutant that affected the functional properties of the co-transporter. These data suggest that the nature/position of SLC12A3 mutation, combined with male gender, is a determinant factor in the severity of GS and provide new insights in the underlying pathogenic mechanisms of the disease.

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“…The recent identification of the gene (2)(3)(4) and mutations (4)(5)(6)(7)(8)(9) responsible for CF has provided insight into the basic defect in this disease. The protein product of this gene has an estimated molecular mass of 170 kDa with transmembrane and nucleotidebinding-fold domains.…”

mentioning

confidence: 99%

cAMP-inducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator.

Rommens

Dho

Bear

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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A cAMP-inducible chloride permeability has been detected in mouse fibroblast (L cell) lines upon stable integration of a full-length cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR). As indicated by a Cl-indicator dye, the Cl permeability of the plasma membrane increases by 10-to 30-fold within 2 min after treatment of the cells with forskolin, an activator of adenylyl cyclase. The properties of the conductance are similar to those described in secretory epithelial cells; the whole-cell currentvoltage relationship is linear and there is no evidence of voltage-dependent inactivation or activation. In contrast, this cAMP-dependent Cl-flux is undetectable in the untransfected cells or cells harboring defective cDNA constructs, including one with a phenylalanine deletion at amino acid position 508 (AF5OS), the most common mutation causing cystic fibrosis.These observations are consistent with the hypothesis that the CFTR is a cAMP-dependent Cl-channel. The availability of a heterologous (nonepithelial) cell type expressing the CFTR offers an excellent system to understand the basi mechanims underlying this CFTR-assoclated ion permeability and to study the structure and function of the CFIR.

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Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients

Cited by 328 publications

References 37 publications

Cystic Fibrosis-associated Mutations at Arginine 347 Alter the Pore Architecture of CFTR

Cystic Fibrosis-associated Mutations at Arginine 347 Alter the Pore Architecture of CFTR

Transcriptional and Functional Analyses of SLC12A3 Mutations

cAMP-inducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator.

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