ABCMdb

PMID: 9482946

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., [PubMed]

Sentences

No. Mutations Sentence Comment

28 ABCC7 p.Met265Val 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 ABCC7 p.Met265Val ABCC7 p.Met265Val This created the #2c;259-M265V construct. Login to comment 41 ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro For null the construct, R334W͞R347P in the transmembrane domain (TMD)-1 background (R334W͞ R347P-TMD-1), the identical mutations were introduced along with a silent NcoI site (as above) as well as a stop codon and an EcoRV site slightly downstream with a longer mutagenic oligonucleotide, 5Ј-GGA ATC ATC CTC TGG AAA ATA TTC ACC ACC ATC TCA TTC TGC ATT GTT CTG CCC ATG GCG GTC ACT CGG CAA TTT CCA TGG GCT GTA CAA ACA TGG TAT GAC TCT CTT GGA GCA ATA AAC TAA ATA CAG GAT ATC TTA C-3Ј. Login to comment 96 ABCC7 p.Met265Val 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 98 ABCC7 p.Met265Val ABCC7 p.Met265Val ⌬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 ABCC7 p.Met265Val In sharp contrast, ⌬259-M265V does not produce any currents (Table 1.). Login to comment 102 ABCC7 p.Met265Val 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 103 ABCC7 p.Met265Val 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 114 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro Finally, dual Cl2 conduction mutations, R334W and R347P (referred to as dual arginine CFTR) were made. Login to comment 115 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro Finally, dual Cl- conduction mutations, R334W and R347P (referred to as dual arginine CFTR) were made. Login to comment 122 ABCC7 p.Met265Val 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 123 ABCC7 p.Arg334Trp ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro ABCC7 p.Met265Val 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 124 ABCC7 p.Arg334Trp ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro 9 NS TMD-1 -321.5 Ϯ 75.6* -587.7 Ϯ 145.5 10 Ͻ0.05 T-N-R -110.7 Ϯ 27.6 -316.3 Ϯ 35.7 8 Ͻ0.05 R334W-R347P 107.4 Ϯ 16.1 -104.7 Ϯ 17.3 6 NS R334W-R347P- TMD-1 -75.9 Ϯ 20.1 -117.3 Ϯ 22.4 6 NS Current values are shown for all mutants immediately before and 5 min after stimulation with cAMP agonists [forskolin, 10 ␮M; 3-isobutyl-1-methylxanthine (IBMX), 1 mM] at the -90-mV clamped voltage. Login to comment 138 ABCC7 p.Met265Val 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 139 ABCC7 p.Met265Val 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 156 ABCC7 p.Met265Val 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 ABCC7 p.Met265Val 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 158 ABCC7 p.Met265Val 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 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Met265Val 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 161 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro Likewise, cAMP-stimulated whole cell Cl- currents generated by R334W-R347P (dual arginine) CFTR in transfected IB3-1 cells were strongly outwardly rectified and blocked fully by DIDS. Login to comment 169 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro Insertion of both R334W and R347P mutations into a TMD-1 background eliminated its ability to generate Cl2 currents, as shown in Table 1, and its ability to activate ORCCs, as demonstrated by the complete lack of any currents when this construct was expressed in IB3-1 cells (Fig. 3). Login to comment 170 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro Insertion of both R334W and R347P mutations into a TMD-1 background eliminated its ability to generate Cl- currents, as shown in Table 1, and its ability to activate ORCCs, as demonstrated by the complete lack of any currents when this construct was expressed in IB3-1 cells (Fig. 3). Login to comment 171 ABCC7 p.Arg334Trp ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro ABCC7 p.Lys370* ABCC7 p.Met265Val 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 172 ABCC7 p.Arg334Trp ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro ABCC7 p.Lys370* ABCC7 p.Met265Val 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 173 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Lys370* ABCC7 p.Met265Val 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 174 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Lys370* ABCC7 p.Met265Val 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 199 ABCC7 p.Met265Val ABCC7 p.Met265Val (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 200 ABCC7 p.Met265Val ABCC7 p.Met265Val (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 209 ABCC7 p.Gly551Asp Those mutations that are expected to cause the most severe disease are those, such as G551D, that both drastically affect the ability of CFTR to move Cl2 effectively itself and also eliminate its ability to regulate other channels such as the ORCC. Login to comment 210 ABCC7 p.Gly551Asp Those mutations that are expected to cause the most severe disease are those, such as G551D, that both drastically affect the ability of CFTR to move Cl- effectively itself and also eliminate its ability to regulate other channels such as the ORCC. Login to comment 281 ABCC7 p.Met265Val 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 283 ABCC7 p.Met265Val 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