ABCMdb

ABCC7 p.Met265Val

ClinVar:	c.794T>G , p.Met265Arg ? , not provided
CF databases:	c.794T>G , p.Met265Arg (CFTR1) ? , M265R was detected by SSCP and identified by direct DNA sequencing. This mutation was detected in an obligate carrier of CF, whose daughter died at 1 day old with meconium ileus. The child is inferred to have been a compound heterozygote of [delta]F508 and M265R since her father is a carrier of [delta]F508. M265R was found only once in 50 non-[delta]F508 chromosome screened.
Predicted by SNAP2:	A: D (75%), C: D (71%), D: D (91%), E: D (91%), F: D (66%), G: D (91%), H: D (85%), I: N (82%), K: D (91%), L: D (53%), N: D (85%), P: D (91%), Q: D (85%), R: D (91%), S: D (71%), T: D (80%), V: N (78%), W: D (91%), Y: D (80%),
Predicted by PROVEAN:	A: N, C: N, D: D, E: D, F: N, G: D, H: D, I: N, K: D, L: N, N: D, P: D, Q: D, R: D, S: N, T: N, V: N, W: D, Y: N,

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Publications
[hide] Cystic fibrosis transmembrane conductance regulato... J Cell Biol. 1998 Nov 2;143(3):645-57. Jiang Q, Mak D, Devidas S, Schwiebert EM, Bragin A, Zhang Y, Skach WR, Guggino WB, Foskett JK, Engelhardt JF
Cystic fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor.
J Cell Biol. 1998 Nov 2;143(3):645-57., 1998-11-02 [PMID:9813087]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl- that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl- conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl- conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl >> Br; R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl- binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl-. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.

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No. Sentence Comment
93 The NH2-terminal truncation mutant ⌬259-M265V CFTR was constructed as previously described (Piazza Carroll et al., 1995). Login to comment
157 Dissociation of Cl-Conductance and ATP Efflux Functions Associated with CFTR Because the results indicated that CFTR was necessary but not sufficient for ATP permeability, we attempted to identify the important domains in CFTR by comparing wild-type CFTR with two deletion mutants, TMD1 and ⌬259-M265V. Login to comment
158 The TMD1 mutant encompassing the NH2-terminal portion of CFTR encoded the first 369 amino acids (first six transmembrane helices), while in the ⌬259-M265V mutant the first 259 amino acids were deleted and the methionine 265 was mutated to a valine. Login to comment
161 In contrast, expression of the ⌬259-M265V mutant was associated with near wild-type rates of ATP efflux in the absence of detectable Cl-conductances (Fig. 3). Login to comment
195 Comparison of CFTR-modulated ATP release between wild-type, TMD1, and ⌬259-M265V CFTR cRNA-injected Xenopus oocytes. Login to comment
200 An equal number (N) of wt CFTR, TMD1, and ⌬259-M265V cRNA-injected oocytes were assessed for ATP efflux within each of two batches of oocytes. Login to comment
274 However, the CFTR mutant ⌬259-M265V, which failed to conduct Cl- , also conferred near wild-type rates of ATP release. Login to comment
277 First, the CFTR mutant ⌬259-M265V demonstrated near wild-type rates of CFTR-modulated ATP release despite its inability to conduct Cl- . Login to comment

[hide] CFTR is a conductance regulator as well as a chlor... Physiol Rev. 1999 Jan;79(1 Suppl):S145-66. Schwiebert EM, Benos DJ, Egan ME, Stutts MJ, Guggino WB
CFTR is a conductance regulator as well as a chloride channel.
Physiol Rev. 1999 Jan;79(1 Suppl):S145-66., [PMID:9922379]

Abstract [show]
CFTR Is a Conductance Regulator as well as a Chloride Channel. Physiol. Rev. 79, Suppl.: S145-S166, 1999. - Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter gene family. Although CFTR has the structure of a transporter that transports substrates across the membrane in a nonconductive manner, CFTR also has the intrinsic ability to conduct Cl- at much higher rates, a function unique to CFTR among this family of ABC transporters. Because Cl- transport was shown to be lost in cystic fibrosis (CF) epithelia long before the cloning of the CF gene and CFTR, CFTR Cl- channel function was considered to be paramount. Another equally valid perspective of CFTR, however, derives from its membership in a family of transporters that transports a multitude of different substances from chemotherapeutic drugs, to amino acids, to glutathione conjugates, to small peptides in a nonconductive manner. Moreover, at least two members of this ABC transporter family (mdr-1, SUR) can regulate other ion channels in the membrane. More simply, ABC transporters can regulate somehow the function of other cellular proteins or cellular functions. This review focuses on a plethora of studies showing that CFTR also regulates other ion channel proteins. It is the hope of the authors that the reader will take with him or her the message that CFTR is a conductance regulator as well as a Cl- channel.

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92 In sharp contrast, a similar truncation to amino acid 259 Taken together, these preliminary results utilizing truncations of the CFTR cDNA from the NH2 and COOHbut with methionine-265 altered to a valine (M265V) failed to function as a cAMP-stimulated Cl0 channel in termini suggest that the domains critical for Cl0 channel function and ORCC regulatory coupling are distinct. Login to comment
94 However, despite this lack of Cl0 channel activity, M265V conferred cAMP regulation function could be eliminated with the other intact, and vice versa. Login to comment
101 Similar to the results with M265V, insertion of ''dual`` NBD1 in bilayers (7) and mammalian cells (34) (see also below). Login to comment
219 Wild-type and D259- M265V-CFTR promote cAMP-stimulated ATP release from oocytes, whereas TMD-1 CFTR fails to promote cAMP-stimulated ATP release. Login to comment

[hide] Chloride channel and chloride conductance regulato... Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2674-9. Schwiebert EM, Morales MM, Devidas S, Egan ME, Guggino WB
Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator.
Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2674-9., [PMID:9482946]

Abstract [show]
CFTR is a cyclic AMP (cAMP)-activated chloride (Cl-) channel and a regulator of outwardly rectifying Cl- channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl- channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3-1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36Cl- efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl- channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted alpha-helices 5 and 6, forms an essential part of the Cl- channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl- channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.

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No. Sentence Comment
28 For N-terminal truncation mutations, the M265V missense mutation and a silent mutation to create a unique SpeI site were introduced into the CFTR cDNA with a mutagenic oligonucleotide, 5Ј-GAC TAG TGA TTA CCT CAG AAG TGA TTG-3Ј. Login to comment
30 This created the #2c;259-M265V construct. Login to comment
98 ⌬259-M265V is identical to ⌬259-M265 but with methionine-265 changed to a valine, shifting the translation initiation codon downstream within the coding sequence. Login to comment
100 In sharp contrast, ⌬259-M265V does not produce any currents (Table 1.). Login to comment
103 No single channel events were observed from oocytes injected with the ⌬259-M265V construct, suggesting that this mutant either does not conduct Cl- or is not processed normally in oocytes. Login to comment
123 Cl- currents in CFTR cRNA-injected Xenopus oocytes cRNA injected Current, nA n P valueBasal cAMP-stimulated None -89.3 Ϯ 13.7 -82.7 Ϯ 13.5 9 NS Wild-type CFTR -117.2 Ϯ 27.7 -828.1 Ϯ 295.7 16 Ͻ0.001 ⌬259-M265 -133.4 Ϯ 27.6 -509.9 Ϯ 159.9 8 Ͻ0.01 ⌬259-M265V -106.2 Ϯ 32.1 -103.7 Ϯ 29. Login to comment
139 Importantly, ⌬259-M265V-transfected IB3-1 cell cultures and R334W͞R347P-transfected cultures also responded to cAMP in 36 Cl- efflux assays, despite the lack of intrinsic Cl- channel function in oocyte recordings (Table 2). Login to comment
158 In contrast, in IB3-1 cells transfected with ⌬259-M265V, currents were more strongly outwardly rectified and were completely inhibited by DIDS, with no underlying linear CFTR currents (Fig. 3). Login to comment
159 Consistent with oocyte expression and the Cl- efflux studies, these results showed that elimination of the first four ␣-helices of CFTR and mutation of methionine-265 to a valine eliminated CFTR`s ability to generate Cl- currents in both oocytes and IB3-1 cells. Login to comment
172 More importantly, results with T-N-R CFTR suggest that the region of CFTR important for regulatory interaction with ORCCs lies Table 2. cAMP-stimulated Cl- efflux in CFTR cDNA-transfected IB3-1 CF cells cDNA transfected n Cl- efflux, % lost per min Paired P valueBefore agonists After agonists Mock 42 33.01 Ϯ 3.12 29.53 Ϯ 2.22 NS Wild-type 37 22.99 Ϯ 1.47 46.51 Ϯ 6.53* Ͻ0.005 ⌬259-M265 30 21.85 Ϯ 1.43 47.67 Ϯ 5.95* Ͻ0.005 ⌬259-M265V 18 24.55 Ϯ 1.17 29.25 Ϯ 2.23** Ͻ0.05 TMD-1 (K370X) 24 16.63 Ϯ 1.80 53.51 Ϯ 9.50* Ͻ0.005 TMD-1 (K370EcoRV) 24 19.54 Ϯ 1.67 41.27 Ϯ 5.22* Ͻ0.005 T-N-R 18 19.21 Ϯ 1.89 28.05 Ϯ 3.35** Ͻ0.05 R334W-R347P 18 19.85 Ϯ 3.20 31.16 Ϯ 6.79** Ͻ0.05 R334W-R347P-TMD-1 18 23.12 Ϯ 2.60 26.26 Ϯ 3.42 NS The Before agonists value is the rate of 36Cl- efflux immediately prior to stimulation with cAMP agonists (2.5 ␮M forskolin, 250 ␮M CPT-cAMP, and 250 ␮M 8-bromo-cAMP). Login to comment
174 For mutants ⌬259-M265V, T-N-R, and R334W-R347P, the magnitude of cAMP stimulation is significantly less (P Ͻ 0.05, versus paired control value as denoted by two asterisks) than that for the wild type and other responding mutants [⌬259-M265, TMD-1 (K370X), TMD-1 (K370EcoRV), P Ͻ 0.005 as denoted by one asterisk], as determined by ANOVA followed by the Bonferroni ad hoc test. Login to comment
200 (B) ⌬259-M265V CFTR: Typical whole cell patch-clamp recordings of basal, cAMP-stimulated, and DIDS-inhibited cAMP-stimulated Cl- currents from a ⌬259-M265V-transfected cell. DIDS (500 ␮M) inhibited all of the current that was significantly outward rectified; no underlying linear current (CFTR current) was observed. Login to comment
283 Summarized total whole cell currents (in nA) are presented as ICl- at -100 mV͞ICl- at -100 mV with n in parentheses: parental IB3-1, -101.9 Ϯ 12.1͞66.3 Ϯ 24.1 (8); nonresponders, -82.4 Ϯ 15.9͞57.2 Ϯ 14.7 (71); wild-type, -676.2 Ϯ 75.8͞878.9 Ϯ 76.0 (7); ⌬259-M265, -316.8 Ϯ 111.4͞653.7 Ϯ 63.3 (11); ⌬259-M265V, -206.9 Ϯ 52.3͞371.1 Ϯ 54.8 (8); TMD-1, -587.1 Ϯ 83.0͞582.5 Ϯ 84.8 (8); T-N-R, -289.3 Ϯ 27.3͞435.4 Ϯ 28.6 (6); dual arginine (Dual R), -177.5 Ϯ 39.8͞389.6 Ϯ 57.7 (8); and Dual R-TMD-1, -150.3 Ϯ 18.1͞147.3 Ϯ 15.3 (10). Login to comment
96 D259-M265V is identical to D259-M265 but with methionine-265 changed to a valine, shifting the translation initiation codon downstream within the coding sequence. Login to comment
102 No single channel events were observed from oocytes injected with the D259-M265V construct, suggesting that this mutant either does not conduct Cl2 or is not processed normally in oocytes. Login to comment
122 Cl2 currents in CFTR cRNA-injected Xenopus oocytes cRNA injected Current, nA n P value Basal cAMP-stimulated None 289.3 6 13.7 282.7 6 13.5 9 NS Wild-type CFTR 2117.2 6 27.7 2828.1 6 295.7 16 ,0.001 D259-M265 2133.4 6 27.6 2509.9 6 159.9 8 ,0.01 D259-M265V 2106.2 6 32.1 2103.7 6 29. Login to comment
138 Importantly, D259-M265V-transfected IB3-1 cell cultures and R334WyR347P-transfected cultures also responded to cAMP in 36 Cl2 efflux assays, despite the lack of intrinsic Cl2 channel function in oocyte recordings (Table 2). Login to comment
156 In contrast, in IB3-1 cells transfected with D259-M265V, currents were more strongly outwardly rectified and were completely inhibited by DIDS, with no underlying linear CFTR currents (Fig. 3). Login to comment
157 Consistent with oocyte expression and the Cl2 efflux studies, these results showed that elimination of the first four a-helices of CFTR and mutation of methionine-265 to a valine eliminated CFTR`s ability to generate Cl2 currents in both oocytes and IB3-1 cells. Login to comment
171 More importantly, results with T-N-R CFTR suggest that the region of CFTR important for regulatory interaction with ORCCs lies Table 2. cAMP-stimulated Cl2 efflux in CFTR cDNA-transfected IB3-1 CF cells cDNA transfected n Cl2 efflux, % lost per min Paired P value Before agonists After agonists Mock 42 33.01 6 3.12 29.53 6 2.22 NS Wild-type 37 22.99 6 1.47 46.51 6 6.53* ,0.005 D259-M265 30 21.85 6 1.43 47.67 6 5.95* ,0.005 D259-M265V 18 24.55 6 1.17 29.25 6 2.23** ,0.05 TMD-1 (K370X) 24 16.63 6 1.80 53.51 6 9.50* ,0.005 TMD-1 (K370EcoRV) 24 19.54 6 1.67 41.27 6 5.22* ,0.005 T-N-R 18 19.21 6 1.89 28.05 6 3.35** ,0.05 R334W-R347P 18 19.85 6 3.20 31.16 6 6.79** ,0.05 R334W-R347P-TMD-1 18 23.12 6 2.60 26.26 6 3.42 NS The Before agonists value is the rate of 36Cl2 efflux immediately prior to stimulation with cAMP agonists (2.5 mM forskolin, 250 mM CPT-cAMP, and 250 mM 8-bromo-cAMP). Login to comment
173 For mutants D259-M265V, T-N-R, and R334W-R347P, the magnitude of cAMP stimulation is significantly less (P , 0.05, versus paired control value as denoted by two asterisks) than that for the wild type and other responding mutants [D259-M265, TMD-1 (K370X), TMD-1 (K370EcoRV), P , 0.005 as denoted by one asterisk], as determined by ANOVA followed by the Bonferroni ad hoc test. Login to comment
199 (B) D259-M265V CFTR: Typical whole cell patch-clamp recordings of basal, cAMP-stimulated, and DIDS-inhibited cAMP-stimulated Cl2 currents from a D259-M265V-transfected cell. DIDS (500 mM) inhibited all of the current that was significantly outward rectified; no underlying linear current (CFTR current) was observed. Login to comment
281 Summarized total whole cell currents (in nA) are presented as ICl- at 2100 mVyICl- at 2100 mV with n in parentheses: parental IB3-1, 2101.9 6 12.1y66.3 6 24.1 (8); nonresponders, 282.4 6 15.9y57.2 6 14.7 (71); wild-type, 2676.2 6 75.8y878.9 6 76.0 (7); D259-M265, 2316.8 6 111.4y653.7 6 63.3 (11); D259-M265V, 2206.9 6 52.3y371.1 6 54.8 (8); TMD-1, 2587.1 6 83.0y582.5 6 84.8 (8); T-N-R, 2289.3 6 27.3y435.4 6 28.6 (6); dual arginine (Dual R), 2177.5 6 39.8y389.6 6 57.7 (8); and Dual R-TMD-1, 2150.3 6 18.1y147.3 6 15.3 (10). 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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No. Sentence Comment
72 These include A259CFTR and A259-M265V CFTR. Login to comment
76 The A259-M265V CFTR mutanthas its next initiation methionine at amino acid M281. Login to comment