ABCMdb

PMID: 11278813

Hammerle MM, Aleksandrov AA, Riordan JR
Disease-associated mutations in the extracytoplasmic loops of cystic fibrosis transmembrane conductance regulator do not impede biosynthetic processing but impair chloride channel stability.
J Biol Chem. 2001 May 4;276(18):14848-54. Epub 2001 Feb 6., 2001-05-04 [PubMed]

Sentences

No. Mutations Sentence Comment

14 ABCC7 p.Arg117His However, one relatively common missense mutation in EL1, R117H, was shown earlier to reduce chloride conductance (2). Login to comment 75 ABCC7 p.Arg117His ABCC7 p.Arg117Leu ABCC7 p.Arg117Cys ABCC7 p.Glu217Gly ABCC7 p.Ser108Phe ABCC7 p.Pro111Leu ABCC7 p.Tyr109Cys ABCC7 p.Thr908Asn ABCC7 p.Arg117Pro ABCC7 p.Pro1013Leu ABCC7 p.Pro111Ala ABCC7 p.Glu116Lys ABCC7 p.Asp110His TABLE I Oligonucleotide primers used to generate mutations Mutation Primer S108F GGAAGAATCATAGCTTtCTATGACCCGGATAAC Y109C AGAATCATAGCTTCCTgTGACCCGGATAACAAG D110H ATCATAGCTTCCTATcACCCGGATAACAAGGAG P111A ATAGCTTCCTATGACgCGGATAACAAGGAGGAA P111L ATAGCTTCCTATGACCtGGATAACAAGGAGGAA E116K CCGGATAACAAGGAGaAACGCTCTATCGCGATT R117C GATAACAAGGAGGAAtGCTCTATCGCGATTTAT R117H GATAACAAGGAGGAACaCTCTATCGCGATTTAT R117L GATAACAAGGAGGAACtCTCTATCGCGATTTAT R117P GATAACAAGGAGGAACcCTCTATCGCGATTTAT E217G ATGGGGCTAATCTGGGgGTTGTTACAGGCGTCT T908N TATGCAGTGATTATCAaCAGCACCAGTTCGTAT P1013L GTCGCAGTTTTACAACtCTACATCTTTGTTGCA FIG. 2. Login to comment 80 ABCC7 p.Gly1127Glu tion reported in a patient in EL6, G1127E, was not expressed. Login to comment 86 ABCC7 p.Glu116Lys However, in the case of E116K the surface signal is disproportionally weaker relative to the immunoblot indicating that it could be somewhat impeded in transport to the cell surface. Login to comment 91 ABCC7 p.Ser108Phe ABCC7 p.Thr908Asn Exceptions are S108F, which matures as efficiently as the wild type, and T908N, which matures even more efficiently. Login to comment 99 ABCC7 p.Glu217Gly For example, E217G in EL2 reduces the efflux rate greatly. Login to comment 100 ABCC7 p.Pro1013Leu On the other hand, P1013L in EL5 exhibits rates as high as wild type. Single Channel Properties of EL-Fig. 4 contains representative recordings of single channels in planar lipid bilayers with which membrane vesicles isolated from cells expressing each of the variants and the wild type were fused. Login to comment 104 ABCC7 p.Ser108Phe The behavior of the most N-terminal mutant in EL1, S108F, indicates that substitution of the small hydroxyl amino acid with the larger aromatic phenylalanine results in a channel with no stable open state. Login to comment 117 ABCC7 p.Ser108Phe ABCC7 p.Tyr109Cys ABCC7 p.Asp110His A, squares, wild type; circles, S108F; triangles, Y109C; diamonds, D110H; crosses, wild type without stimulation. Login to comment 118 ABCC7 p.Pro111Leu ABCC7 p.Pro111Ala ABCC7 p.Glu116Lys B, squares, P111A; circles, P111L; triangles, E116K. Login to comment 119 ABCC7 p.Arg117His ABCC7 p.Arg117Leu ABCC7 p.Arg117Cys ABCC7 p.Arg117Pro C, squares, R117C; circles, R117H; triangles, R117L; diamonds, R117P. Login to comment 120 ABCC7 p.Glu217Gly ABCC7 p.Thr908Asn ABCC7 p.Pro1013Leu D, squares, E217G; circles, T908N; triangles, P1013L. Login to comment 122 ABCC7 p.Ser108Phe ABCC7 p.Tyr109Cys The substitution of the aromatic tyrosine in the adjacent position by the small thiol residue (Y109C) also results in a very unstable open state, but the tracing is different from that of S108F. Login to comment 126 ABCC7 p.Asp110His Replacing the aspartate residue in the next position by histidine (D110H) resulted in a much more normal looking channel with a major mean conductance of about 8.5 pS. Login to comment 130 ABCC7 p.Pro111Leu P111L appears very much like wild type with a mean conductance of 10.3 pS. Login to comment 134 ABCC7 p.Pro111Ala Substitution of the proline with the smaller aliphatic alanine residue (P111A) produced a channel with much more rapid kinetics and a reduced conductance of 9.2 pS even at 21 °C (Fig. 4). Login to comment 135 ABCC7 p.Glu116Lys The mutation five residues further in the C-terminal direction resulted in a charge reversal (E116K). Login to comment 136 ABCC7 p.Ser108Phe This mutant displays properties quite similar to S108F; channel openings occur but cannot be maintained. Login to comment 138 ABCC7 p.Arg117His The R117H mutant was reported previously to exhibit a small reduction in both current amplitude and open time (2). Login to comment 142 ABCC7 p.Arg117Leu ABCC7 p.Arg117Cys ABCC7 p.Ser108Phe ABCC7 p.Glu116Lys Both R117C and R117L had very unstable open states like S108F and E116K with the cysteine substitution able to maintain openings FIG. 4. Login to comment 148 ABCC7 p.Arg117Pro R117P also displayed only transient openings. Login to comment 149 ABCC7 p.Arg117His Thus the more frequent R117H mutation seems to compromise open-channel structure less than the other three substitutions at this position. Login to comment 152 ABCC7 p.Glu217Gly Strikingly, the single missense mutation (E217G) in the short EL2 also results in channels with only transient rather than stable openings, perhaps implying that this loop is involved also in stabilizing open pore structure. Login to comment 153 ABCC7 p.Thr908Asn The T908N mutation in EL4, the second of the two larger extracytoplasmic loops of CFTR, has a somewhat similar effect on the single channel behavior as those in EL1 that cause a very unstable open state. Login to comment 156 ABCC7 p.Thr908Asn As seen in the T908N trace, there are in fact relatively long openings with several brief closings within to generate burstlike behavior. Login to comment 162 ABCC7 p.Pro1013Leu The final disease-associated mutant analyzed, P1013L, in the fifth exterior loop exhibited robust wild-type-like behavior, and consistent with its 36 Cl efflux ability, it has a Po of 0.5, higher than the wild type. Login to comment 171 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg117Leu ABCC7 p.Arg117Cys ABCC7 p.Glu217Gly ABCC7 p.Ser108Phe ABCC7 p.Pro111Leu ABCC7 p.Tyr109Cys ABCC7 p.Thr908Asn ABCC7 p.Arg117Pro ABCC7 p.Pro1013Leu ABCC7 p.Pro111Ala ABCC7 p.Glu116Lys ABCC7 p.Asp110His For example a nucleotide binding domain mutation, G551D, precludes virtually all TABLE II Relative charge transport capacity of mutants Mutants S108F Y109C D110H P111L P111A E116K R117H R117C R117L R117P E217G T908N P1013L Imutant/Iwt 100% 11 15 27 173 105 12 80 27 5 11 10 48 170 FIG. 5. Login to comment 176 ABCC7 p.Arg117His Of missense mutations identified in codons for residues in extracytoplasmic loops, only R117H, which occurs relatively frequently in patients, has been studied in any detail (2). Login to comment 183 ABCC7 p.Glu116Lys The charge-reversal mutation, E116K, at the immediately preceding position had a remarkably similar effect to that of the most perturbing substitutions of Arg-117. Login to comment 186 ABCC7 p.Pro111Leu The P111L variant in contrast to all the other EL1 mutants differed from wild type only at elevated temperatures. Login to comment 188 ABCC7 p.Glu217Gly The E217G mutant in EL2 had a similar effect. Login to comment 190 ABCC7 p.Thr908Asn The T908N mutation in EL4, which is novel in that it results in the introduction of an additional consensus site for N-glycosylation that is used (17), also alters channel gating. Login to comment 191 ABCC7 p.Pro1013Leu Thus although the P1013L change in EL5 did not seem to compromise channel activity except at higher temperatures, all of the others analyzed seriously detracted from the ability of CFTR to sustain a stable open pore. Login to comment