ABCMdb

ABCC7 p.Lys536Gln

ClinVar:	c.1606A>T , p.Lys536* ? , not provided
CF databases:	c.1606A>G , p.Lys536Glu (CFTR1) ? ,
Predicted by SNAP2:	A: N (57%), C: D (66%), D: D (71%), E: D (59%), F: D (75%), G: N (53%), H: N (72%), I: N (57%), L: N (61%), M: D (63%), N: N (66%), P: D (66%), Q: N (78%), R: N (87%), S: N (57%), T: N (57%), V: N (57%), W: D (80%), Y: N (53%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, L: D, M: D, N: N, P: N, Q: N, R: N, S: N, T: N, V: D, W: D, Y: D,

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[hide] Folding and rescue of a cystic fibrosis transmembr... J Biol Chem. 2010 Aug 27;285(35):27033-44. Epub 2010 Jun 15. Da Paula AC, Sousa M, Xu Z, Dawson ES, Boyd AC, Sheppard DN, Amaral MD
Folding and rescue of a cystic fibrosis transmembrane conductance regulator trafficking mutant identified using human-murine chimeric proteins.
J Biol Chem. 2010 Aug 27;285(35):27033-44. Epub 2010 Jun 15., 2010-08-27 [PMID:20551307]

Abstract [show]
Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys(584) interacts with residue Leu(581) in human CFTR Glu(584) interacts with Phe(581) in mouse CFTR. Introduction of the murine residue (Phe(581)) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR.

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No. Sentence Comment
124 Thus, we identified six residues in 12b-NBD1 (E527Q, E528Q, S531T, K536Q, I539T, and K584E) and 12 residues in 114c-NBD2 (T1263I, P1290T, K1302Q, Y1307N, Q1309K, S1311K, R1325K, V1338T, C1344Y, L1367I, D1394G, and E1409D) (see supplemental Fig. 1 and supplemental Table 1, A and B). Login to comment
150 For example, compare the data for S531T and K536Q in Fig. 2, C and D. Taken together and consistent with the WB data (Fig. 2, A and B, and Table 1), our iodide efflux data suggest that most of the NBD1 and NBD2 mutants exhibited channel activity, arguing that they reach the cell surface. Login to comment
187 TABLE 1 Summary information of CFTR point mutants analyzed in present study CFTR variants Clinical dataa Band C/band Bb (؎S.E., n ‫؍‬ 5) Processingc Normalized processingd Normalized iodide efflux functione (؎S.E., n ‫؍‬ 6) Iodide efflux to processed proteinf % % % % peak intensity % WT-CFTR -g 83 Ϯ 3 77 100 100 Ϯ 8 - Murine - 86 Ϯ 5 66 86 74 Ϯ 4 86 Ϯ 4 E527Q Mild CF 64 Ϯ 5 49 63 46 Ϯ 4 73 Ϯ 4 E528Q - 86 Ϯ 5 79 102 135 Ϯ 16 132 Ϯ 10 S531T - 87 Ϯ 6 81 105 71 Ϯ 5 67 Ϯ 5 K536Q - 69 Ϯ 3 42 54 51 Ϯ 4 94 Ϯ 3 I539T Revertant 112 Ϯ 5 81 105 49 Ϯ 6 46 Ϯ 5 L581F - 118 Ϯ 3 83 107 72 Ϯ 5 67 Ϯ 3 L581F/K584E - 125 Ϯ 2 77 100 100 Ϯ 12 100 Ϯ 8 T1263I Mild CF 75 Ϯ 3 76 98 31 Ϯ 8 31 Ϯ 5 P1290T Asymptomatic 87 Ϯ 3 82 106 92 Ϯ 10 86 Ϯ 6 K1302Q - 72 Ϯ 3 77 100 37 Ϯ 2 37 Ϯ 2 Y1307N - 82 Ϯ 2 76 98 70 Ϯ 5 71 Ϯ 3 Q1309K - 79 Ϯ 4 77 100 26 Ϯ 2 26 Ϯ 3 S1311K - 73 Ϯ 4 72 93 33 Ϯ 7 35 Ϯ 5 R1325K - 64 Ϯ 6 78 101 47 Ϯ 2 46 Ϯ 4 V1338T - 88 Ϯ 2 77 100 37 Ϯ 11 37 Ϯ 6 C1344Y - 71 Ϯ 4 76 98 86 Ϯ 4 87 Ϯ 4 L1367I - 72 Ϯ 5 80 103 36 Ϯ 5 34 Ϯ 5 D1394G - 78 Ϯ 4 86 111 93 Ϯ 12 83 Ϯ 8 E1409D - 70 Ϯ 3 70 90 43 Ϯ 5 47 Ϯ 4 a Data from the CFTR mutation database. Login to comment

[hide] Cystic fibrosis-type mutational analysis in the AT... J Biol Chem. 1994 Aug 12;269(32):20575-83. Hoof T, Demmer A, Hadam MR, Riordan JR, Tummler B
Cystic fibrosis-type mutational analysis in the ATP-binding cassette transporter signature of human P-glycoprotein MDR1.
J Biol Chem. 1994 Aug 12;269(32):20575-83., 1994-08-12 [PMID:7914197]

Abstract [show]
Members of the ATP-binding cassette transporter superfamily such as the P-glycoproteins (MDR) and the cystic fibrosis transmembrane conductance regulator (CFTR) share conserved sequence motifs in their nucleotide binding fold that are the major targets for CFTR mutations in patients with cystic fibrosis. Cystic fibrosis-type mutations were introduced at analogous positions into the human MDR1 gene. Heterologous expression of wild-type or mutated MDR1 revealed similar mRNA transcript levels in Chinese hamster ovary K1 recipients, but the subsequent processing was defective for all mutations that give rise to severe cystic fibrosis in the case of CFTR. Functional multidrug transporter MDR1, however, was obtained when amino acid substitutions were introduced into a less conserved position of the ATP-binding cassette transporter signature (codon 536 in MDR1). The profile of cross-resistance and chemosensitization was modulated in these codon 536 variants, which suggests that this region is involved in the drug transport function of P-glycoprotein.

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26 20575 CF-type Mutations in MDRl TABLEI Oligonucleotides used in RTIPCR assays, mutagenesis,and sequencing Sequence Primer' Applicationb 5'-GAGGTGAAGAAGGGCCAGACG-3' mdrlP3175s* P 5'-TTCTGGATGGTGGACAGGCGGTGA-3' mdrlP3716a*P 5'-GTGCAGAGTGGGCAGACGGTG-3' mdrl-1249s 5'-TTACGAACTGTAGACAAACGATGAG-3' 5'-GAGGAGCAGCTTATG-3' 5'-GTGGTTCAGGTGGCT-3' mdrl-1702s S 5'-GAAAACATTCGCTATGGCCGTGAAAATG-3' mdrl-1456s S 5'-GCAGCTGATGAATCC-3' mdrl-1918s S 5"GTGGTGGGCAGgaCAGAGGATCGC-3' mdrl-1595sM (K536Q) 5'-GTTGAGTGGTGuCAGAAGCAGAG-3' mdrl-1590sM (G534D) 5'-GTGGTGGGCAGwCAGAGGATCGC-3' mdrl-1595sM (K536R) 5"CCAGTTGAGGGGTGGGCAG-3' mdrl-1587sM(S532R) 5'-GAAAACATTCGsGCCGTGAAAATG-3' mdrl-1486sM (AY490) 5'-GGCAAGGGCATCCTGGCTGCAGA-3' alh79s* P 5'-TAACGGGCCAGAACATTGGCATT-3' alh521a* P p, s p, s S mdrl-1781a mdrl-1076s The number indicates the positionof the first primer nucleotide in thecorresponding EMBL file cDNA sequences of rabbit aldolase A (ald), human MDRl (mdrl),or CHOMDRl (mdrlc);s, sense direction; a, antisense direction tocDNA sequence. Login to comment
90 The localization ofAY490, S532R, G534D, K536R, K536Q, and AY490-K536Q in the N-terminal half of MDRl is shown in Fig. 1. Login to comment
44 The double mutant AY490-K536Q was constructed by digestion of the pBSmdrl single mutants AY490 and K536Q with ApaI and religationof mutated fragments. Login to comment
97 All G418-preselected clones grew in the presence of the lowest concentrations of 50 ng of adriamycidml or 100 ng of colchicine/ml (Fig. 21, but only the introduction of wild-type MDRl, K536Q MDRl, or K536R MDRl intoCHO K1 recipients hadconferred resistance toboth drugs above the base-line level of the antisense MDRl transfection control (Fig. 2). Login to comment
161 Autoradiogram of 75-pg gel-separatedmembraneproteins of wild-type (A),K536Q (B),and K536R MDRls CHO K1 cells (C) that had been photolabeled with["'Iliodomycin in thepresence of (from left to right in eachpanel) 0,15,and 300 nM vinblastine. Login to comment
190 The phenotype of multidrug resistance and collateral sensitivity was retained in the K536Q and K536R MDRl variants. Login to comment
192 K536Q P-glycoprotein was 2-5-fold less drug resistant thanwild type (which also explains the inefficacyof the K536Q substitution in the AY490 MDRl mutant), but K536R P-glycoprotein was more active than wild type in CHO K1 transfectants. Login to comment