ABCMdb

PMID: 8663008

Sheppard DN, Travis SM, Ishihara H, Welsh MJ
Contribution of proline residues in the membrane-spanning domains of cystic fibrosis transmembrane conductance regulator to chloride channel function.
J Biol Chem. 1996 Jun 21;271(25):14995-5001., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser These residues are conserved across species, and mutations of two (P99L and P205S) are associated with cystic fibrosis. Login to comment 6 ABCC7 p.Pro99Leu The anion selectivity sequence of the mutants (Br- Cl- > I- ) resembled wild-type except for P99L (Br- Cl- ‫؍‬ I- ). Login to comment 9 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly Mutant channels were regulated like wild-type CFTR; however, single-channel conductance was decreased in the rank order: wild-type CFTR P99G > P99L P99A. Login to comment 23 ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser These residues are conserved across species and mutations of two (P99L and P205S) are associated with cystic fibrosis (CF) (12, 13). Login to comment 24 ABCC7 p.Pro99Leu ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser ABCC7 p.Pro205Ser Because P99L and P205S are associated with a milder clinical phenotype characterized by retention of some pancreatic function (termed pancreatic sufficiency) (2), these mutants may retain some residual Cl- channel function. Based on the above information, the aims of this study were to determine the contribution of proline residues located within the MSDs of CFTR to Cl- channel function and to understand how P99L and P205S cause a loss of Cl- channel function. Login to comment 76 ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser Cyclic AMP agonists activated whole cell currents in cells expressing each of the individual proline to alanine or glycine mutations and in cells expressing the CF-associated mutations, P99L and P205S. Login to comment 77 ABCC7 p.Pro99Leu ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly As an example, Fig. 2 shows data from studies of P99A, P99G, and P99L; qualitatively similar results were obtained with the Pro205 , Pro324 , and Pro1021 mutants (data not shown). Login to comment 78 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly As an example, Fig. 2 shows data from studies of P99A, P99G, and P99L; qualitatively similar results were obtained with the Pro205 , Pro324 , and Pro1021 mutants (data not shown). Login to comment 85 ABCC7 p.Pro99Leu However, the CF-associated mutation P99L had an altered anion selectivity sequence, Br- Ն Cl- ϭ I- (Fig. 3 and Table I). Login to comment 87 ABCC7 p.Pro99Leu However, the CF-associated mutation P99L had an altered anion selectivity sequence, Br2 $ Cl2 5 I2 (Fig. 3 and Table I). Login to comment 119 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly Reversal potentials (Erev) of baseline subtracted cAMP-activated whole cell currents were P99A, -30 Ϯ 1 mV (n ϭ 7); P99G, -34 Ϯ 2 mV (n ϭ 5); and P99L, -24 Ϯ 3 mV (n ϭ 6). Login to comment 122 ABCC7 p.Pro99Leu ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser ABCC7 p.Pro205Ala ABCC7 p.Pro205Gly ABCC7 p.Pro99Ala ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly ABCC7 p.Pro99Gly ABCC7 p.Pro1021Gly ABCC7 p.Pro1021Ala ABCC7 p.Pro324Ala ABCC7 p.Pro324Gly Mutant n Px/PCl Gx/GCl Br- Cl- IBr- ClI- CFTR 5 1.18 Ϯ 0.08 1.00 0.73 Ϯ 0.05 1.27 Ϯ 0.16 1.00 0.61 Ϯ 0.08 P99A 7 0.98 Ϯ 0.03 1.00 0.70 Ϯ 0.06 1.04 Ϯ 0.05 1.00 0.72 Ϯ 0.05 P99G 5 1.06 Ϯ 0.02 1.00 0.75 Ϯ 0.08 1.04 Ϯ 0.07 1.00 0.66 Ϯ 0.05 P99L 5 1.21 Ϯ 0.07 1.00 1.06 Ϯ 0.07 1.33 Ϯ 0.11 1.00 0.95 Ϯ 0.08 P205A 4 1.09 Ϯ 0.07 1.00 0.64 Ϯ 0.09 0.95 Ϯ 0.04 1.00 0.46 Ϯ 0.11 P205G 5 1.09 Ϯ 0.05 1.00 0.45 Ϯ 0.05 1.05 Ϯ 0.03 1.00 0.44 Ϯ 0.06 P205S 2 1.01 Ϯ 0.01 1.00 0.55 Ϯ 0.28 1.09 Ϯ 0.09 1.00 0.59 Ϯ 0.08 P324A 7 1.08 Ϯ 0.04 1.00 0.72 Ϯ 0.06 1.15 Ϯ 0.07 1.00 0.60 Ϯ 0.08 P324G 6 1.12 Ϯ 0.07 1.00 0.69 Ϯ 0.04 1.22 Ϯ 0.14 1.00 0.57 Ϯ 0.04 P1021A 3 1.15 Ϯ 0.17 1.00 0.73 Ϯ 0.11 1.17 Ϯ 0.10 1.00 0.47 Ϯ 0.19 P1021G 7 1.17 Ϯ 0.06 1.00 0.78 Ϯ 0.02 1.21 Ϯ 0.08 1.00 0.59 Ϯ 0.06 though for P99G the reduction was small, for P99A and P99L the effect was marked. Login to comment 123 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly Reversal potentials (Erev) of baseline subtracted cAMP-activated whole cell currents were P99A, 230 6 1 mV (n 5 7); P99G, 234 6 2 mV (n 5 5); and P99L, 224 6 3 mV (n 5 6). Login to comment 126 ABCC7 p.Pro99Leu ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser ABCC7 p.Pro205Ala ABCC7 p.Pro205Gly ABCC7 p.Pro99Ala ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly ABCC7 p.Pro99Gly ABCC7 p.Pro99Gly ABCC7 p.Pro1021Gly ABCC7 p.Pro1021Ala ABCC7 p.Pro324Ala ABCC7 p.Pro324Gly The conductance for P99G was 7.31 Ϯ 0.24 pS (n ϭ 5), not significantly different from wild type (p ϭ 0.26). Login to comment 127 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala In contrast, the conductances of P99A and P99L were significantly decreased at 4.66 Ϯ 0.25 pS (n ϭ 5, p Ͻ 0.0001) and 4.97 Ϯ 0.24 pS (n ϭ 5, p Ͻ 0.0001), respectively. Login to comment 129 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala Because the single-channel current amplitudes of P99A and P99L were much reduced and because in most cases the patches of membrane contained large numbers of channels, we could not accurately measure single-channel kinetics. Login to comment 130 ABCC7 p.Pro99Gly The conductance for P99G was 7.31 6 0.24 pS (n 5 5), not significantly different from wild type (p 5 0.26). Login to comment 131 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala In contrast, the conductances of P99A and P99L were significantly decreased at 4.66 6 0.25 pS (n 5 5, p , 0.0001) and 4.97 6 0.24 pS (n 5 5, p , 0.0001), respectively. Login to comment 133 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala Because the single-channel current amplitudes of P99A and P99L were much reduced and because in most cases the patches of membrane contained large numbers of channels, we could not accurately measure single-channel kinetics. Login to comment 145 ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser ABCC7 p.Pro205Ala ABCC7 p.Pro205Gly ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly ABCC7 p.Pro1021Gly ABCC7 p.Pro1021Ala ABCC7 p.Pro324Ala ABCC7 p.Pro324Gly The number of cells responding to cAMP agonists with Cl- current activation relative to the total number of cells tested for each construct was: CFTR (8/16; 50%), P99A (11/12; 92%), P99G (9/19; 47%), P99L (10/19; 53%), P205A (7/12; 58%), P205G (5/9; 56%), P205S (7/20; 35%), P324A (9/18; 50%), P324G (9/22; 41%), P1021A (8/18; 44%), and P1021G (7/16; 44%). Login to comment 149 ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser ABCC7 p.Pro205Ala ABCC7 p.Pro205Gly ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly ABCC7 p.Pro1021Gly ABCC7 p.Pro1021Ala ABCC7 p.Pro324Ala ABCC7 p.Pro324Gly The number of cells responding to cAMP agonists with Cl2 current activation relative to the total number of cells tested for each construct was: CFTR (8/16; 50%), P99A (11/12; 92%), P99G (9/19; 47%), P99L (10/19; 53%), P205A (7/12; 58%), P205G (5/9; 56%), P205S (7/20; 35%), P324A (9/18; 50%), P324G (9/22; 41%), P1021A (8/18; 44%), and P1021G (7/16; 44%). Login to comment 172 ABCC7 p.Pro99Ala A, regulation of P99A by phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (75 nM) and intracellular MgATP (0.88 mM). Login to comment 173 ABCC7 p.Pro99Ala Representative recording are from an excised inside-out membrane patch from a HeLa cell transiently expressing P99A. Login to comment 176 ABCC7 p.Pro99Leu ABCC7 p.Pro99Gly Similar results were observed with P99G and P99L; n Ͼ 5 for each mutant. Login to comment 177 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly B, representative single-channel recordings are from excised inside-out membrane patches from HeLa cells transiently expressing wild-type CFTR, P99A, P99G, and P99L. Login to comment 178 ABCC7 p.Pro99Ala Representative recording are from an excised inside-out membrane patch from a HeLa cell transiently expressing P99A. Login to comment 180 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly C, single-channel I-V relationships of CFTR (circles), P99A (squares), P99G (triangles), and P99L (inverted triangles. Login to comment 181 ABCC7 p.Pro99Leu ABCC7 p.Pro99Gly Similar results were observed with P99G and P99L; n . Login to comment 183 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly B, representative single-channel recordings are from excised inside-out membrane patches from HeLa cells transiently expressing wild-type CFTR, P99A, P99G, and P99L. Login to comment 186 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly C, single-channel I-V relationships of CFTR (circles), P99A (squares), P99G (triangles), and P99L (inverted triangles. Login to comment 192 ABCC7 p.Pro574His ABCC7 p.Ala455Glu In this regard they are similar to the CF mutations A455E and P574H that disrupt processing but generate channels that retain significant activity (23). Login to comment 194 ABCC7 p.Pro99Leu When Pro99 was mutated to leucine, the channel lost its ability to discriminate between Cl- and I- . Login to comment 198 ABCC7 p.Pro574His ABCC7 p.Ala455Glu In this regard they are similar to the CF mutations A455E and P574H that disrupt processing but generate channels that retain significant activity (23). Login to comment 199 ABCC7 p.Pro99Leu ABCC7 p.Pro99Ala ABCC7 p.Pro99Gly Substitution of alanine, glycine, and leucine at Pro99 decreased single-channel conductance in the rank order: wild-type CFTR Ն P99G Ͼ P99L Ն P99A. Login to comment 200 ABCC7 p.Pro99Leu When Pro99 was mutated to leucine, the channel lost its ability to discriminate between Cl2 and I2 . Login to comment 202 ABCC7 p.Pro99Gly Glycine, which is found in hinge regions and like proline is not favored in ␣-helices, can substitute for proline at this residue because the single-channel conductance of P99G does not differ from wild type. Login to comment 204 ABCC7 p.Pro99Cys Because P99C did not react with sulfhydryl-specific reagents, Akabas and collaborators concluded that Pro99 does not line the channel pore (7). Login to comment 205 ABCC7 p.Pro99Gly Substitution of alanine, glycine, and leucine at Pro99 decreased single-channel conductance in the rank order: wild-type CFTR $ P99G . Login to comment 206 ABCC7 p.Pro99Leu ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser ABCC7 p.Pro99Ala Implications for Cystic Fibrosis-P99L and P205S are CF mutations located in MSD1 that are associated with a milder (pancreatic sufficiency) clinical phenotype (12, 13). Login to comment 207 ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser Our studies of the processing and function of P99L and P205S explain why these mutants generate less Cl- current than wild-type CFTR. Login to comment 208 ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser Loss of Cl- channel function caused by P205S was predominantly a result of defective protein processing; whereas that caused by P99L was a consequence of both defective protein processing and altered Cl- channel function. Login to comment 210 ABCC7 p.Pro99Gly Glycine, which is found in hinge regions and like proline is not favored in a-helices, can substitute for proline at this residue because the single-channel conductance of P99G does not differ from wild type. Login to comment 212 ABCC7 p.Pro205Ser ABCC7 p.Pro99Cys This suggests that the defective processing of the P205S mutant observed in HeLa cells likely accounts for the loss of Cl- channel function in patients bearing this mutation. Login to comment 214 ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser Implications for Cystic Fibrosis-P99L and P205S are CF mutations located in MSD1 that are associated with a milder (pancreatic sufficiency) clinical phenotype (12, 13). Login to comment 215 ABCC7 p.Arg117His ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser The present results complement and extend our previous study of mild CF mutants located in MSD1 (R117H, R334W, and R347P). Login to comment 216 ABCC7 p.Pro99Leu ABCC7 p.Pro205Ser Loss of Cl2 channel function caused by P205S was predominantly a result of defective protein processing; whereas that caused by P99L was a consequence of both defective protein processing and altered Cl2 channel function. Login to comment 217 ABCC7 p.Pro574His ABCC7 p.Ala455Glu ABCC7 p.Pro205Ser The mechanism of dysfunction of P205S resembles that of the nucleotide-binding domain 1 pancreatic sufficiency mutants A455E and P574H, which are misprocessed (23). Login to comment 218 ABCC7 p.Pro99Leu Interestingly, P99L forms a Cl- channel with altered pore properties and is also misprocessed. Login to comment 220 ABCC7 p.Pro205Ser This suggests that the defective processing of the P205S mutant observed in HeLa cells likely accounts for the loss of Cl2 channel function in patients bearing this mutation. Login to comment 221 ABCC7 p.Pro99Leu We thank Dr. M. Schwartz (Department of Clinical Genetics, University Hospital, Copenhagen, Denmark) for generously providing details of the clinical phenotype of the patient with the P99L mutation prior to publication. Login to comment 223 ABCC7 p.Arg117His ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro The present results complement and extend our previous study of mild CF mutants located in MSD1 (R117H, R334W, and R347P). Login to comment 225 ABCC7 p.Pro574His ABCC7 p.Ala455Glu ABCC7 p.Pro205Ser The mechanism of dysfunction of P205S resembles that of the nucleotide-binding domain 1 pancreatic sufficiency mutants A455E and P574H, which are misprocessed (23). Login to comment 226 ABCC7 p.Pro99Leu Interestingly, P99L forms a Cl2 channel with altered pore properties and is also misprocessed. Login to comment 230 ABCC7 p.Pro99Leu We thank Dr. M. Schwartz (Department of Clinical Genetics, University Hospital, Copenhagen, Denmark) for generously providing details of the clinical phenotype of the patient with the P99L mutation prior to publication. Login to comment