ABCMdb

PMID: 7542778

Fulmer SB, Schwiebert EM, Morales MM, Guggino WB, Cutting GR
Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents.
Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6832-6., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Login to comment 5 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as measured by chloride efflux. Login to comment 7 ABCC7 p.Ala455Glu While CFTrR bearing either mutation could function as a chloride channel, only CFTR bearing A455E retained the function of regulating the ORCC. Login to comment 26 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu To determine whether disease-associated mutations affect the regulatory function of CFTR, the functional consequences of two mutations, A455E and G551D, were studied. Login to comment 27 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu To determine whether disease-associated mutations affect the regulatory function of CFTR, the functional consequences of two mutations, A455E and G551D, were studied. Login to comment 28 ABCC7 p.Gly551Asp When G551D/ AF508 compound heterozygotes were compared with sex- and age-matched AF508 homozygotes, there was no difference in severity of pancreatic or pulmonary disease (15). Login to comment 29 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu When G551D/ AF508 compound heterozygotes were compared with sex- and age-matched AF508 homozygotes, there was no difference in severity of pancreatic or pulmonary disease (15). Login to comment 30 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu However, comparison of A455E/AF508 compound heterozygotes with AF508 homozygotes revealed significantly better lung function in the heterozygotes, as measured by mean yearly decline in the volume of air forcibly expired in 1 s [forced expiratory volume in 1 s (FEV1); P < 0.0005] (16, 17). Login to comment 31 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu Since the ORCC appears to be regulated by CFTR in respiratory epithelium, we studied the function of CFTR bearing either A455E or G551D in a Abbreviations: CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; ORCC, outwardly rectifying chloride channel; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; DIDS, 4,4'-diisothiocyanostil- bene-2,2'-disulfonic acid; IBMX, 3-isobutyl-1-methylxanthine; RSV, Rous sarcoma virus. Login to comment 32 ABCC7 p.Ala455Glu Our data suggest that CFTR bearing A455E can function as a cAMP-regulated chloride channel and also maintains its capacity to regulate the ORCC. Login to comment 33 ABCC7 p.Ala455Glu Our data suggest that CFTR bearing A455E can function as a cAMP-regulated chloride channel and also maintains its capacity to regulate the ORCC. Login to comment 36 ABCC7 p.Ala455Glu The A455E mutation was created using the oligonucleotide 5'- GTTGTTGGAGGTTGCTGG-3'. Login to comment 37 ABCC7 p.Ala455Glu The A455E mutation was created using the oligonucleotide 5'- GTTGTTGGAGGTTGCTGG-3'. Login to comment 45 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu The mutations, A455E and G551D, were created in pBQ4.7 (a generous gift from J. Rommens and L.-C. Tsui; The Hospital for Sick Children, Toronto) then shuttled to the pAvS6.CFTR or pRSV-CFTR by using Kpn2I and Hpa I (BRL) restriction sites common to all vectors. Login to comment 46 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu The mutations, A455E and G551D, were created in pBQ4.7 (a generous gift from J. Rommens and L.-C. Tsui; The Hospital for Sick Children, Toronto) then shuttled to the pAvS6.CFTR or pRSV-CFTR by using Kpn2I and Hpa I (BRL) restriction sites common to all vectors. Login to comment 68 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu As seen in Fig. 1, oocytes injected with wild-type, G551D-, or A455E-CFTR generated cAMP-stimulated Cl-currents significantly greater than basal level. Login to comment 69 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu As seen in Fig. 1, oocytes injected with wild-type, G551D-, or A455E-CFTR generated cAMP-stimulated Cl- currents significantly greater than basal level. Login to comment 71 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu The currents in A455E-CFTR-injected oocytes were about 67% of wild-type, suggesting that A455E-CFTR forms a cAMP-activated chloride channel in Xenopus oocytes. Login to comment 72 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu The currents in A455E-CFTR-injected oocytes were about 67% of wild-type, suggesting that A455E-CFTR forms a cAMP-activated chloride channel in Xenopus oocytes. Login to comment 76 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu |== I Control ~~ Fornkon +IBMX * Control Wild-type G551D A455E FIG. 1. Bar graph summary of Cl-currents (Icl) measured at a clamped voltage of -90 mV before and after stimulation with forskolin (10 ,uM) and IBMX (1 mM) for Xenopus oocytes injected with water (Control; n = 9), wild-type CFTR cRNA (n = 16), G551D-CFTR cRNA (n = 6), or A455E-CFTR cRNA (n = 6). Login to comment 77 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu |==I Control ~~ Fornkon +IBMX * Control Wild-type G551D A455E FIG. 1. Bar graph summary of Cl- currents (Icl) measured at a clamped voltage of -90 mV before and after stimulation with forskolin (10 ,uM) and IBMX (1 mM) for Xenopus oocytes injected with water (Control; n = 9), wild-type CFTR cRNA (n = 16), G551D-CFTR cRNA (n = 6), or A455E-CFTR cRNA (n = 6). Login to comment 85 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu IB3-1 cells were transfected with the RSV vector containing wild-type CFTR, G551D-CFTR, or A455E-CFTR. Login to comment 86 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu IB3-1 cells were transfected with the RSV vector containing wild-type CFTR, G551D-CFTR, or A455E-CFTR. Login to comment 91 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu IB3-1 cells g+ DIDS were transiently transfected with + Glyb wild-type CFTR (A), G551D-CFTR (B), or A455E-CFTR (C). Login to comment 92 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu IB3-1 cells g+ DIDS were transiently transfected with + Glyb wild-type CFTR (A), G551D-CFTR (B), or A455E-CFTR (C). Login to comment 96 ABCC7 p.Gly551Asp Fifteen seconds after addition of CPT-cAMP, efflux rates from cells transfected with wild-type CFTR (41.7% &#b1; 6.0% of Cl- lost per min; n = 12) or G551D-CFTR (40.8% &#b1; 4.5%; n = 6) were significantly greater (P < 0.05) than that of mock-transfected cells (28.9% &#b1; 4.4%; n = 12). Login to comment 97 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu Fifteen seconds after addition of CPT-cAMP, efflux rates from cells transfected with wild-type CFTR (41.7% ± 6.0% of Cl- lost per min; n = 12) or G551D-CFTR (40.8% ± 4.5%; n = 6) were significantly greater (P < 0.05) than that of mock-transfected cells (28.9% ± 4.4%; n = 12). Login to comment 98 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu Thirty seconds after CPT-cAMP addition, efflux rates from cells transfected with wild-type CFTR (47.8% ± 12.3%; n = 12) and A455E-CFTR (38.1% ± 6.5%; n = 6) were significantly greater (P < 0.05) than that of mock-transfected cells (26.4% ± 5.5%; n = 12). Login to comment 99 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu These results indicate that both A455E-CFTR and G551D-CFTR produce cAMP-mediated chloride secretion in IB3-1 cells. Login to comment 110 ABCC7 p.Gly551Asp The results for IB3-1 cells expressing G551D-CFTR are shown in Fig. 2B. Currents activated by CPT-cAMP were lower than in wild-type CFTR-expressing cells (note current scales) and had a linear I-V relationship. Login to comment 111 ABCC7 p.Gly551Asp The results for IB3-1 cells expressing G551D-CFTR are shown in Fig. 2B. Currents activated by CPT-cAMP were lower than in wild-type CFTR-expressing cells (note current scales) and had a linear I-V relationship. Login to comment 114 ABCC7 p.Ala455Glu A455E-CFTR-expressing cells showed results similar to wild type, although the induced currents were lower (Fig. 2C). Login to comment 115 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu A455E-CFTR-expressing cells showed results similar to wild type, although the induced currents were lower (Fig. 2C). Login to comment 116 ABCC7 p.Ala455Glu The I-Vrelationship for A455E-CFTR-expressing cells was slightly outwardly rectified. Login to comment 120 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu The similarity between the currents of wild-type CFTR and A455E-CFTR and their differences from G551D-CFTR currents were evident from examination ofthe outwardly rectified versus linear current component for each (Fig. 3). Login to comment 121 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu The similarity between the currents of wild-type CFTR and A455E-CFTR and their differences from G551D-CFTR currents were evident from examination ofthe outwardly rectified versus linear current component for each (Fig. 3). Login to comment 122 ABCC7 p.Ala455Glu Wild-type CFTR-expressing and A455E-CFTR-expressing cells also showed a significant increase (P < 0.05) in outwardly rectified current. Login to comment 123 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu Wild-type CFTR-expressing and A455E-CFTR-expressing cells also showed a significant increase (P < 0.05) in outwardly rectified current. Login to comment 124 ABCC7 p.Ala455Glu Taken together, these results indicated that although an outwardly rectified, DIDS-sensitive current contributed to the whole cell currents in wild-type CFTR-expressing and A455E-CFTR-expressing cells, it did not con- 0 c) .., 500 A 400 300 200 100 0 500 ( < 400 0. Login to comment 128 ABCC7 p.Gly551Asp (C) G551D-CFTR (Nonexp, n = 12; Exp, n = 5). Login to comment 129 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu (C) G551D-CFTR (Nonexp, n = 12; Exp, n = 5). Login to comment 130 ABCC7 p.Ala455Glu (D) A455E-CFTR (Nonexp, n = 15; Exp, n = 6). Login to comment 131 ABCC7 p.Gly551Asp Error bars indicate 1 SEM. tribute to the whole cell current in G551D-CFTR-expressing cells. Login to comment 132 ABCC7 p.Gly551Asp Error bars indicate 1 SEM. tribute to the whole cell current in G551D-CFTR-expressing cells. Login to comment 134 ABCC7 p.Gly551Asp One of these mutations, G551D, has been extensively studied with respect to its effect upon processing of CFTR and its function as a chloride channel. Login to comment 135 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp One of these mutations, G551D, has been extensively studied with respect to its effect upon processing of CFTR and its function as a chloride channel. Login to comment 136 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp CFTR bearing the G551D mutation is properly glycosylated and localized in the cell membrane (24-26). Login to comment 137 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Our finding that a partially functional chloride channel can be formed by G551D-CFTR in oocytes and epithelial cells is consistent with previous functional studies of this mutant protein. Login to comment 138 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Drumm et al. (27) found that G551D-CFTR expressed in Xenopus oocytes is functional when 1-5 mM IBMX is used; our experiments inXenopus oocytes used 1 mM IBMX. Login to comment 139 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp When stably transduced with a recombinant retrovirus, mouse L cells expressing G551D-CFTR generated currents in response to high concentrations of IBMX (25). Login to comment 140 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp In the absence of IBMX, G551D-CFIR generated whole-cell currents in IB3-1 cells that were a similar magnitude to those attributed solely to the chloride channel of wild-type CFTR (see Fig. 2A and B). Login to comment 141 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp However, G551D-CFTR expressed by a vaccinia virus system in HeLa cells did not generate a chloride conductance (24). Login to comment 142 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The lack of G551D-CFTR function may be attributed to differences in the cell type or expression system used in the latter study. Login to comment 143 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp There is a striking difference between the I-V relationships of wild-type CFTR and G551D-CFTR in the absence of any channel blockers. Login to comment 144 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The I-V relationship for wild-type CFTR is outwardly rectified, whereas that for G551D-CFTR is linear. Login to comment 145 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Currents generated by either G551D-CFTR or wild-type CFTR were sensitive to glybenclamide; however, this compound was a less potent inhibitor of G551D-CFTR currents and appeared to block in a voltage-dependent manner. Login to comment 147 ABCC7 p.Gly551Asp The G551D mutation could cause structural changes in the first nucleotide-binding domain that alter both ATP and glybenclamide interactions with CFTR, thereby accounting for the decreased function of this mutant protein and its altered response to glybenclamide. Login to comment 148 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The G551D mutation could cause structural changes in the first nucleotide-binding domain that alter both ATP and glybenclamide interactions with CFTR, thereby accounting for the decreased function of this mutant protein and its altered response to glybenclamide. Login to comment 149 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp In support of this concept, the G551D mutation significantly decreased the ATP binding of a recombinant protein containing a portion of the first nucleotide binding domain of CFTR (28). Login to comment 150 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu On the other hand, DIDS, a compound that blocks the ORCC, inhibited a significant fraction of wild-type CFTR currents but had no effect on G551D-CFTR currents. Login to comment 151 ABCC7 p.Ala455Glu The A455E mutation also alters CFTR function. Login to comment 152 ABCC7 p.Ala455Glu Therefore, it is difficult to predict the molecular consequences of the A455E mutation. Login to comment 153 ABCC7 p.Ala455Glu Therefore, it is difficult to predict the molecular consequences of the A455E mutation. Login to comment 156 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu They found that A455E alters the processing of CFTR, and, when properly processed, A455E-CFTR functions as a chloride channel (30). Login to comment 157 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu They found that A455E alters the processing of CFTR, and, when properly processed, A455E-CFTR functions as a chloride channel (30). Login to comment 158 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu In this study, A455E-CFTR-generated currents were about the same fraction (about two-thirds) of wild-type CFTR in both mammalian and Xenopus systems. Login to comment 159 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu This result indicates that if A455E causes a processing defect, it is not ameliorated by expression in Xenopus oocytes at 25°C. A455E-CFTR expressed in IB3-1 cells has both chloride channel and regulatory functions intact. Login to comment 160 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu Therefore, our data are consistent with the concept that A455E could cause a processing defect, which leads to decreased amounts of A455E-CFTR at the cell surface. Login to comment 161 ABCC7 p.Ala455Glu A455E-CFTR that reaches the cell membrane appears to function similar to wild-type CFTR. Login to comment 162 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu Patients carrying A455E have been shown to be pancreatic sufficient, a situation that occurs with other partially functional mutants (31), but A455E also appears to be associated with mild lung disease (16, 17). Login to comment 163 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu Patients carrying A455E have been shown to be pancreatic sufficient, a situation that occurs with other partially functional mutants (31), but A455E also appears to be associated with mild lung disease (16, 17). Login to comment 164 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu This study demonstrates that A455E does not abolish the function of CFTR as a regulator, in particular as a regulator of the ORCC. Login to comment 165 ABCC7 p.Gly551Asp However, CFTR bearing G551D, a mutation that is associated with severe pancreatic and pulmonary aspects of CF, does not function as a regulator and functions minimally as a chloride channel. Login to comment 166 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu The authors thank Dr. I. McIntosh for creating the G551D and A455E mutations, S. Allen for excellent technical assistance, J. Staton for secretarial assistance, and Genetic Therapy, Inc., for the gift of the pAvS6 vectors. Login to comment 167 ABCC7 p.Gly551Asp ABCC7 p.Ala455Glu The authors thank Dr. I. McIntosh for creating the G551D and A455E mutations, S. Allen for excellent technical assistance, J. Staton for secretarial assistance, and Genetic Therapy, Inc., for the gift of the pAvS6 vectors. Login to comment