ABCMdb

ABCC7 p.Asp836*

ClinVar:	c.2506G>T , p.Asp836Tyr ? , not provided
CF databases:	c.2506G>T , p.Asp836Tyr (CFTR1) ? , This mutation was found in a French adult patient. The defect on the other chromosome is not yet characterized.

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Publications
[hide] Severed channels probe regulation of gating of cys... J Gen Physiol. 2000 Sep;116(3):477-500. Csanady L, Chan KW, Seto-Young D, Kopsco DC, Nairn AC, Gadsby DC
Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains.
J Gen Physiol. 2000 Sep;116(3):477-500., [PMID:10962022]

Abstract [show]
Opening and closing of a CFTR Cl(-) channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634-836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1-633 and 837-1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.

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337 Interestingly, a half-channel truncated at that same cut site (D836X; Sheppard et al., 1994) also showed a low level of constitutive activity, but was strongly activated by PKA, and had a high apparent affinity for ATP, properties reminiscent of those described here for 835ϩ837 channels. Login to comment

[hide] Mutations at arginine 352 alter the pore architect... J Membr Biol. 2008 Mar;222(2):91-106. Epub 2008 Apr 18. Cui G, Zhang ZR, O'Brien AR, Song B, McCarty NA
Mutations at arginine 352 alter the pore architecture of CFTR.
J Membr Biol. 2008 Mar;222(2):91-106. Epub 2008 Apr 18., [PMID:18421494]

Abstract [show]
Arginine 352 (R352) in the sixth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) previously was reported to form an anion/cation selectivity filter and to provide positive charge in the intracellular vestibule. However, mutations at this site have nonspecific effects, such as inducing susceptibility of endogenous cysteines to chemical modification. We hypothesized that R352 stabilizes channel structure and that charge-destroying mutations at this site disrupt pore architecture, with multiple consequences. We tested the effects of mutations at R352 on conductance, anion selectivity and block by the sulfonylurea drug glipizide, using recordings of wild-type and mutant channels. Charge-altering mutations at R352 destabilized the open state and altered both selectivity and block. In contrast, R352K-CFTR was similar to wild-type. Full conductance state amplitude was similar to that of wild-type CFTR in all mutants except R352E, suggesting that R352 does not itself form an anion coordination site. In an attempt to identify an acidic residue that may interact with R352, we found that permeation properties were similarly affected by charge-reversing mutations at D993. Wild-type-like properties were rescued in R352E/D993R-CFTR, suggesting that R352 and D993 in the wild-type channel may interact to stabilize pore architecture. Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+, while wild-type and R352E/D993R-CFTR were not. These data suggest that R352 plays an important structural role in CFTR, perhaps reflecting its involvement in forming a salt bridge with residue D993.

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274 However, other authors reported that the amino-terminal portion of CFTR, containing MSD1, NBD1 and the R domain (D836X-CFTR), formed regulated Cl-channels that differed somewhat from WT-CFTR (Sheppard et al. 1994). Login to comment
275 Although the substate behavior of D836X-CFTR was not studied in detail, this mutant was reported to show instability of the open state compared to WT-CFTR; the authors suggested that residues in MSD2 might stabilize the channel complex, perhaps assisting in the arrangement of residues in MSD1 into a functional structure, based on the finding that the number of functional Cl-channels generated from the D836X construct was much lower than expected for WT-CFTR. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 1998 Oct 27;37(43):15222-30. Clancy JP, Hong JS, Bebok Z, King SA, Demolombe S, Bedwell DM, Sorscher EJ
Cystic fibrosis transmembrane conductance regulator (CFTR) nucleotide-binding domain 1 (NBD-1) and CFTR truncated within NBD-1 target to the epithelial plasma membrane and increase anion permeability.
Biochemistry. 1998 Oct 27;37(43):15222-30., 1998-10-27 [PMID:9790686]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the traffic ATPase family that includes multiple proteins characterized by (1) ATP binding, (2) conserved transmembrane (TM) motifs and nucleotide binding domains (NBDs), and (3) molecular transport of small molecules across the cell membrane. While CFTR NBD-1 mediates ATP binding and hydrolysis, the membrane topology and function of this domain in living eukaryotic cells remains uncertain. In these studies, we have expressed wild-type CFTR NBD-1 (amino acids 433-586) or NBD-1 containing the DeltaF508 mutation transiently in COS-7 cells and established that the domain is situated across the plasma membrane by four independent assays; namely, extracellular chymotrypsin digestion, surface protein biotinylation, confocal immunofluorescent microscopy, and functional measurements of cell membrane anion permeability. Functional studies indicate that basal halide permeability is enhanced above control conditions following wild-type or DeltaF508 NBD-1 expression in three different epithelial cell lines. Furthermore, when clinically relevant CFTR proteins truncated within NBD-1 (R553X or G542X) are expressed, surface localization and enhanced halide permeability are again established. Together, these findings suggest that isolated CFTR NBD-1 (with or without the DeltaF508 mutation) is capable of targeting the epithelial cell membrane and enhancing cellular halide permeability. Furthermore, CFTR truncated at position 553 or 542 and possessing the majority of NBD-1 demonstrates surface localization and also confers increased halide permeability. These findings indicate that targeting to the plasma membrane and assumption of a transmembrane configuration are innate properties of the CFTR NBD-1. The results also support the notion that components of the halide-selective pore of CFTR reside within NBD-1.

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No. Sentence Comment
227 On the other hand, Sheppard et al. have demonstrated that a truncated CFTR molecule containing TM-1, NBD-1, and the R domain (D836X) remains capable of forming anion-selective channels with phosphorylation-dependent regulation (49). 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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131 The amino-terminal portion of CFTR (D836X, which contains MSD1, NBD1 andfraction behavior of CFTR was lost (128). Login to comment
136 Based on biochemical and functional data, we speculated that D836X generates a Cl0 channel byveats noted above regarding the contribution of residues to the pore versus less specific alterations of structure forming a homomultimer (116). Login to comment
138 To identify residues that line the CFTR pore and make below), then in a D836X multimer MSD1 sequences may substitute for MSD2 sequences to form the pore. Login to comment
504 First, those sites were phosphor- interference between R domains in a D836X homomultimer.ylated in vivo after stimulation with cAMP agonists. Login to comment
526 In addition, deletion studies of portions of the R domain suggest thatout that PKA cannot interact with CFTRDR-S660A, even though it does interact with CFTRDR. there are two halves to the R domain with the first conserved in other ABC transporters and the second uniqueLike CFTRDR, the amino-terminal portion of CFTR (D836X, which contains MSD1, NBD1, and the R domain) to CFTR (102, 103). Login to comment
528 The activation of D836X Cl0 channels by PKA indicates that E. A Model of R Domain Functionthe MSD1-NBD1 motif must contain many of the sequences with which the R domain interacts to control CFTR. Login to comment
529 The PKA-independent activity of D836X Cl0 chan- The R domain was originally proposed to regulate CFTR by keeping the channel closed at rest. Login to comment
533 Alternatively, the PKA-independent activity of D836X could be due to As described above, phosphorylation by PKA, deletion of FIG. 11 Effect of a recombinant R domain (R1, residues 645-834) on CFTRDR/S660A channel in presence of PKA. Login to comment

[hide] CFTR protein analysis of splice site mutation 2789... J Cyst Fibros. 2008 Mar;7(2):165-7. Epub 2007 Aug 16. van Barneveld A, Stanke F, Claass A, Ballmann M, Tummler B
CFTR protein analysis of splice site mutation 2789+5 G-A.
J Cyst Fibros. 2008 Mar;7(2):165-7. Epub 2007 Aug 16., [PMID:17707141]

Abstract [show]
Ex vivo biochemical analysis of rectal biopsies of a carrier of the mild 2789+5 G-A CFTR frameshift splice site mutation revealed mutant truncated CFTR of expected size and an imbalance of more core-glycosylated and less mature full-length CFTR. This first immunoblot analysis of a non-F508del CFTR mutant protein derived from human tissue demonstrates that splice site mutations should not only be investigated at the mRNA, but also at the protein level to properly interpret the associations between genotype, molecular pathology and disease.

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No. Sentence Comment
63 The recombinant N-terminal portion of CFTR (D836X) forms proper chloride channels in vitro [13], albeit the cooperation between the two non-equivalent NBDs in the binding and hydrolysis of nucleotides cannot take place [9]. Login to comment

[hide] Conformation, independent of charge, in the R doma... Biophys J. 2000 Mar;78(3):1293-305. Xie J, Zhao J, Davis PB, Ma J
Conformation, independent of charge, in the R domain affects cystic fibrosis transmembrane conductance regulator channel openings.
Biophys J. 2000 Mar;78(3):1293-305., [PMID:10692317]

Abstract [show]
The R domain of cystic fibrosis transmembrane conductance regulator (CFTR), when phosphorylated, undergoes conformational change, and the chloride channel opens. We investigated the contribution of R domain conformation, apart from the changes induced by phosphorylation, to channel opening, by testing the effect of the peptidyl-prolyl isomerase, cyclophilin A, on the CFTR channel. When it was applied after the channel had been opened by PKA phosphorylation, cyclophilin A increased the open probability of wild-type CFTR (from P(o) = 0.197 +/- 0.010 to P(o) = 0.436 +/- 0. 029) by increasing the number of channel openings, not open time. Three highly conserved proline residues in the R domain, at positions 740, 750, and 759, were considered as candidate targets for cyclophilin A. Mutations of these prolines to alanines (P3A mutant) resulted in a channel unresponsive to cyclophilin A but with pore properties similar to the wild type, under strict control of PKA and ATP, but with significantly increased open probability (P(o) = 0.577 +/- 0.090) compared to wild-type CFTR, again due to an increase in the number of channel openings and not open time. Mutation of each of the proline residues separately and in pairs demonstrated that all three proline mutations are required for maximal P(o). When P3A was expressed in 293 HEK cells and tested by SPQ assay, chloride efflux was significantly increased compared to cells transfected with wild-type CFTR. Thus, treatments favoring the trans-peptidyl conformation about conserved proline residues in the R domain of CFTR affect openings of CFTR, above and beyond the effect of PKA phosphorylation.

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No. Sentence Comment
272 Although other CFTR mutants have chloride transport in excess of WT CFTR, they are either not processed efficiently (e.g., P574H or H949Y) (Sheppard et al., 1996a, b; Seibert et al., 1996) or open without PKA stimulation (e.g., CFTR-D836X), and thus are not subject to physiologic regulation (Sheppard et al., 1994). Login to comment
274 Although other CFTR mutants have chloride transport in excess of WT CFTR, they are either not processed efficiently (e.g., P574H or H949Y) (Sheppard et al., 1996a, b; Seibert et al., 1996) or open without PKA stimulation (e.g., CFTR-D836X), and thus are not subject to physiologic regulation (Sheppard et al., 1994). Login to comment

[hide] Expression and intracellular processing of chimeri... Biochim Biophys Acta. 2000 Jan 3;1500(1):59-69. Pollet JF, Van Geffel J, Van Stevens E, Van Geffel R, Beauwens R, Bollen A, Jacobs P
Expression and intracellular processing of chimeric and mutant CFTR molecules.
Biochim Biophys Acta. 2000 Jan 3;1500(1):59-69., [PMID:10564718]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride channel comprising two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs) and a unique regulatory (R) domain. The most frequent cystic fibrosis (CF) mutation, a deletion of Phe508 in NBD1, results in the retention of the DeltaF508 CFTR in the endoplasmic reticulum, as do many other natural or constructed mutations located within the first NBD. In order to further define the role of NBD1 in CFTR folding and to determine whether the higher frequency of mutations in NBD1 with respect to NBD2 results from its position in the molecule or is related to its primary sequence, we constructed and expressed chimeric CFTRs wherein NBD domains were either exchanged or deleted. Synthesis, maturation and activity of the chimeras were assessed by Western blotting and iodide efflux assay after transient or stable expression in COS-1 or CHO cells respectively. The data showed that deletion of NBD1 prevented transport of CFTR to the cytoplasmic membrane whereas deletion of NBD2 did not impair this process but resulted in an inactive chloride channel. On the other hand, substituting or inverting NBDs in the CFTR molecule impaired its processing. In addition, while the NBD1 R555K mutation is known to partially correct the processing of CFTR DeltaF508 and to increase activity of both wild-type and DeltaF508 individual channels, it showed no positive effect when introduced into the double NBD1 chimera. Taken together, these observations suggest that the proper folding process of CFTR results from complex interactions between NBDs and their surrounding domains (MSDs and/or R domain).

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27 Nevertheless, the CFTR truncation mutant (D836X), which contains the 'rst MSD, NBD1, and the R domain, is capable of forming regulated Cl3 channels when expressed in HeLa cells [22] and the protein was shown to cosediment with mature CFTR on a sucrose gradient, suggesting that this 'rst half of the CFTR forms homodimers. 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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229 Nevertheless, one paper demonstrates that mutant D836X encodes a truncated protein (up to amino acid D836, i.e., including MSD1, NBD1, and the R domain but not MSD2 and NBD2). Login to comment
233 Nevertheless, one paper demonstrates that mutant D836X encodes a truncated protein (up to amino acid D836, i.e., including MSD1, NBD1, and the R domain but not MSD2 and NBD2). Login to comment

[hide] CFTR: domains, structure, and function. J Bioenerg Biomembr. 1997 Oct;29(5):443-51. Devidas S, Guggino WB
CFTR: domains, structure, and function.
J Bioenerg Biomembr. 1997 Oct;29(5):443-51., [PMID:9511929]

Abstract [show]
Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) (Collins, 1992). Over 500 naturally occurring mutations have been identified in CF gene which are located in all of the domains of the protein (Kerem et al., 1990; Mercier et al., 1993; Ghanem et al., 1994; Fanen et al., 1992; Ferec et al., 1992; Cutting et al., 1990). Early studies by several investigators characterized CFTR as a chloride channel (Anderson et al.; 1991b,c; Bear et al., 1991). The complex secondary structure of the protein suggested that CFTR might possess other functions in addition to being a chloride channel. Studies have established that the CFTR functions not only as a chloride channel but is indeed a regulator of sodium channels (Stutts et al., 1995), outwardly rectifying chloride channels (ORCC) (Gray et al., 1989; Garber et al., 1992; Egan et al., 1992; Hwang et al., 1989; Schwiebert et al., 1995) and also the transport of ATP (Schwiebert et al., 1995; Reisin et al., 1994). This mini-review deals with the studies which elucidate the functions of the various domains of CFTR, namely the transmembrane domains, TMD1 and TMD2, the two cytoplasmic nucleotide binding domains, NBD1 and NBD2, and the regulatory, R, domain.

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144 (1994) who created an artificial mutant CFTR missing TMD2 and NBD2 but containingthe R domain (D836X). 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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No. Sentence Comment
254 5) We have shown that a CFTR construct containing only MSD1, NBD1, and the R domain (D836X) could form a Cl- channel with relatively normal conductive and permeability properties, albeit the efficiency of production was greatly reduced and regulation was altered (29). Login to comment
278 This speculation is also consistent with our previous studies of D836X (29). Login to comment
273 5) We have shown that a CFTR construct containing only MSD1, NBD1, and the R domain (D836X) could form a Cl2 channel with relatively normal conductive and permeability properties, albeit the efficiency of production was greatly reduced and regulation was altered (29). Login to comment
297 This speculation is also consistent with our previous studies of D836X (29). Login to comment

[hide] Both the wild type and a functional isoform of CFT... Am J Physiol. 1996 Jun;270(6 Pt 2):F1038-48. Morales MM, Carroll TP, Morita T, Schwiebert EM, Devuyst O, Wilson PD, Lopes AG, Stanton BA, Dietz HC, Cutting GR, Guggino WB
Both the wild type and a functional isoform of CFTR are expressed in kidney.
Am J Physiol. 1996 Jun;270(6 Pt 2):F1038-48., [PMID:8764323]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.

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323 These data are in agreement with those recently published by Sheppard et al. (28) who created an artificial mutant CFTR missing TMD2 and NBD2 but containing the R domain (D836X). Login to comment

[hide] Complex cystic fibrosis allele R334W-R1158X result... Hum Mutat. 1996;8(2):134-9. Duarte A, Amaral M, Barreto C, Pacheco P, Lavinha J
Complex cystic fibrosis allele R334W-R1158X results in reduced levels of correctly processed mRNA in a pancreatic sufficient patient.
Hum Mutat. 1996;8(2):134-9., [PMID:8844211]

Abstract [show]
CFTR alleles containing two mutations have been very rarely found in cystic fibrosis (CF) patients. They provide an opportunity to study the effect of two in cis-interacting gene defects on gene expression. Here, we describe a three-generation CF family with a complex CFTR allele that has not been previously described, containing the missense mutation R334W in exon 7 and the nonsense mutation R1158X in exon 19. Lymphocyte RNA analysis showed that (1) the mRNA corresponding to the complex allele is present although at markedly reduced levels; and (2) the nonsense mutation does not lead to detectable skipping of exon 19. The clinical picture of the patients with the genotype R334W-R1158X/delta F508 is characterized by pancreatic sufficiency and an atypical course of the disease.

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No. Sentence Comment
80 The fact that a truncated CFTR protein may have conductive properties was shown by the Iowa group (Sheppard et al., 1994) for the CETR protein resulting from the D836X mutation (exon 14a), which contains only its N-terminal part (the membrane-spanning domains 1-6, the first ATP-binding fold, and the regulatoryregion). Login to comment

[hide] The amino-terminal portion of CFTR forms a regulat... Cell. 1994 Mar 25;76(6):1091-8. Sheppard DN, Ostedgaard LS, Rich DP, Welsh MJ
The amino-terminal portion of CFTR forms a regulated Cl- channel.
Cell. 1994 Mar 25;76(6):1091-8., [PMID:7511062]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel consists of two motifs (each containing a membrane-spanning domain [MSD] and a nucleotide-binding domain [NBD]) linked by an R domain. We tested the hypothesis that one MSD-NBD motif could form a Cl- channel. The amino-terminal portion of CFTR (D836X, which contains MSD1, NBD1, and the R domain) formed Cl- channels with conductive properties identical to those of CFTR. However, channel regulation differed. Although phosphorylation increased activity, channels opened without phosphorylation. MgATP stimulated D836X more potently than CFTR and may interact at more than one site. These data and migration of D836X on sucrose density gradients suggest that D836X may function as a multimer. Thus, the amino-terminal portion of CFTR contains all of the structures required to build a regulated Cl- channel.

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17 To test whether the amino-terminal portion of CFfR could form a regulated Cl- channel, we constructed the CFTR mutant D836X. Login to comment
20 We used two antibodies: M13-1 against the R domain, which is present in D636X, and M24-1 against the carboxyl terminus, which is absent in D836X. Login to comment
30 Under basal conditions, whole-cell currents from cells expressing D836X had a relatively linear current-voltage (I-V) relationship and a reversal potential consistent with Cl--selectivity, whereas basal whole-cell currents from cells expressing wild-type CFTR were much less Clse- lective (Table 1). Login to comment
32 The anion permeability and conductance sequences of D836X whole-cell currents were similar to those of wild-type CFTR (Br > Cl- > I-; Table 1). Login to comment
42 After excision of membrane patches from cells expressing D836X, we observed no channel activity. Login to comment
45 Phosphorylation with PKA (75 nM) significantly increased the P, of D836X channels. Login to comment
48 These data suggest that some aspects of the relationship between the R domain and the rest of the channel may be altered in D836X, while others may remain intact. Login to comment
51 The single-channel slope conductance of D836X (8.03 * 0.23 pS; n = 8) was not different from that of wild-type CFTR (8.29 -c 0.15 pS; n = 4). Login to comment
54 Consistent with this interpretation is the finding that CAMP-activated whole-cell currents were observed in 10 of 59 cells expressing D836X (17%) compared with 10 of 14 cells expressing wild-type CFTR (71%). Login to comment
58 Because D836X lacks NBDZ, we speculated that the interaction with intracellular nucleotides might be altered. Login to comment
63 As we previously reported for wild-type CFTR (Anderson and Welsh, 1992), an Eadie-Hofstee plot of the D836X data generated a curved line(Figure 3C). Login to comment
67 Figures 4A and 48 show that intracellular ADP (1 mM) produced equivalent reductions in the activity of both D836X and wild-type channels. Login to comment
68 D636X Sediments as a Multlmer on Sucrose Gradient Centrlfugatlon Based on these results and considerations discussed below, we asked whether D836X might exist as a multimer. Login to comment
71 1094 A ATP D836X ...... PKA + ATP Is CFTR ATP _..... PKA + ATP _._... B 0.6 0.4 PO 0.2 0 0.6 0.4 PO 0.2 0 I CFTR \TP PKA + ATP ATP PKA + ATP Figure 2. Login to comment
81 These sedimentation patterns A D836X C ~PAL 1s 0.4 PO 0.2 0 :;::::` 0 pl&~P] (2) 4.0 c k 5 2.0 n" 0 -_i 0 0.40.2 PO Figure 3. Login to comment
95 However, our previous data suggest that full-length CFTR does not associate to form a multimer ;h&mino-Terminal Half of CFTR Forms a Cl- Channel A D836X ATP CFTR B ---- - IS 1s Figure 4. Login to comment
96 Intracellular ADP Inhibits D&36X Cl- Channels (A) Comparison of the effect of ADP on the activity of three D836X Cl- channels and a single wild-type Cl- channel following PKAdependent phosphorylation. Login to comment
98 ATP (0.88 mM MgATP) and ADP (1 mM) were added to the intracellular solution as indicated. Voltage was -86 mV (D836X) and -83 mV (CFTR), respectively. Login to comment
101 Voltage was -86 f 1 mV (D836X) and -85 * 2 mV (CFTR). Login to comment
124 The Membrane-Spanning Domains The conductive properties of Cl- channels formed by D636X were remarkably similar to those of wild-type CFTR: the channels had the same anion-to-cation permeability, anion selectivity sequence, single-channel conduc- 1096 A kDa 210 170 116 94 76 67 i B kDa 210 170 116 94 76 57 D836X 6% MO% Total % Sucrow Lysate CFTR % Sucrose ~0% Total Lysate 246 kDa 9/oSucrose - 20% Figure 5. Login to comment
141 We found that although large amountsof protein were produced in cellsexpressing D836X, the number of functional Cl- channels generated was much less than that observed with wild-type CFfR. Login to comment
142 Thus, D836X was not as efficient as wild type at generating functional channels. Login to comment
149 Could a CF-associated mutant that made protein with properties similar to those of D836X produce sufficient residual activity to confer a milder clinical phenotype? Login to comment
150 Given the limited expression of functional D836X Cl- channels despite relatively high-level protein expression, such a possibility seems unlikely. Login to comment