ABCMdb

PMID: 7694298

Smit LS, Wilkinson DJ, Mansoura MK, Collins FS, Dawson DC
Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.
Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9963-7., [PubMed]

Sentences

No. Mutations Sentence Comment

39 ABCC7 p.Val1475Met The full-length constructs contained a valine-for-methionine substitution at amino acid 1475 (V1475M). Login to comment 60 ABCC7 p.Asp1370Asn ABCC7 p.Gly551Ala ABCC7 p.Lys1250Gln ABCC7 p.Gly1349Ala For this reason, expression levels for easily activated constructs (wild type, G551A, G1349A, K1250Q, and D1370N) were adjusted by reducing the amount of injected RNA so that the maximum Cl- conductance was similar to that attained by less sensitive constructs. Login to comment 66 ABCC7 p.Gly551Ser As previously reported (18), G551S, a mutation associated with mild disease (34), exhibited a moderate reduction in sensitivity (K1l2 = 1.1 mM IBMX) compared to wild-type (Kl2 = 0.3 mM). Login to comment 67 ABCC7 p.Gly1349Ser The analogous substitution in NBF2 (G1349S), although not identified in patients, produced a virtually identical reduction in sensitivity (K1l2 = 1.1 mM IBMX). Login to comment 68 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser ABCC7 p.Gly551Ala ABCC7 p.Gly1349Ala G551D, associated with severe CF (35, 36), and G1349D, also a CF mutation (37), both exhibited a dramatic reduction in sensitivity (K1l2 = 2.5 0 0 wt (12) 100 E .E CO) NBF1 A A G551A c O G551S V v G551 D NBF2 (8) A-A G1349A (9) * * G1349S (6) '-V G1349D (4) (6) (8) 0.2 0.5 1 IBMX, mM FIG. 2. Login to comment 77 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser To explore further the relative contributions of the two domains, we measured the activation of Cl- currents in oocytes expressing the double mutants G551S/G1349S and G551D/G1349D. Login to comment 86 ABCC7 p.Gly551Ser In NBF1 the substitution resulted in a moderate reduction in the sensitivity of CFTR to activation (K1l2 = 0.8 mM), roughly equivalent to that seen with G551S. Login to comment 89 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys1250Arg ABCC7 p.Lys464Arg ABCC7 p.Lys464Arg Alanine and arginine substitutions at lysine-464 and -1250 were associated with sensitivities similar to those observed with the glutamine substitutions (K464A or K464R, Kil2 = 0.8 mM; K1250A or K1250R, K,12 < 0.02 mM). Login to comment 92 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser ABCC7 p.Gly1349Ser ABCC7 p.Lys464Gln The dose- 100- g 80-E C3) ° 60- V ai) X 40- E co 20 NBF1 O O G551S (9) v-v G551D (6) 0 Owt (12) T 6o NBF1 + NBF2 O--O G551S + G1349S (7) *--* G551 D + G 1 349D (5) Oz0 6 0 / T/ * , * /1 ° T 0 r / / / 3 _ -- ........ 0.02 0.05 0.2 0.5 1 2 IBMX, mM O-O wt (12) V-V K464Q ( 4) 1OOT 9 80E 0I-) I00160- -0 . Login to comment 93 ABCC7 p.Lys1250Gln a) 40 E (1'- 20- Y + F-V K1250Q ( 4) 0 T~ ~ ~~~ /,/ I I I I T/T /V10 T/ v ~~~I I~~~~~~~~~ V V O ° ~~~~~T/ Q .y 1 0.02 0.05 0.2 0.5 1 IBMX, mM 2 5 FIG. 4. Login to comment 99 ABCC7 p.Gly551Asp In NBF1, this substitution resulted in a dramatic reduction in sensitivity (K1l2 = 2.5 mM), roughly equivalent to that seen with AF508 (18) or G551D, whereas in NBF2, the identical substitution produced an increase in sensitivity to forskolin and IBMX (Kql2 = 0.02 mM). Login to comment 104 ABCC7 p.Gly1349Asp Even in the case of a mutation that severely compromised activation, e.g. G1349D, current-voltage plots were indistinguishable from wild type. Login to comment 107 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Ser Both glycines were replaced by serines (G551S + G1349S) or aspartates (G551D + G1349D), and the dose-response relationships were constructed as in Fig. 2. Login to comment 108 ABCC7 p.Asp1370Asn A 80+ 60 + 40+- 20 + wt rl% - 7 1 k (12) t rd:\z-Za uab/ZN k b) A-- K A D1370N ( 5) T/ z T A T I I 0 f I~0A I~~~~~~ T t u A~~~~ 0.2. ,0.02 0.05 0.2 0.5 IBMX, mM 2 45 FIG. 5. Login to comment 119 ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp Anderson and Welsh (28) found that in detached patches from transfected cells, mutation of the conserved glycine (G551S or G1349D) dramatically reduced the value ofthe open probability (PO) in the presence ofPKA and MgATP. Login to comment 129 ABCC7 p.Lys1250Met Of particular interest is the observation (28) that mutation of the Walker A lysine in NBF2 to methionine (K1250M) produced a 4-fold reduction in the apparent Kil2 for the activation of CFTR by ATP but also completely eliminated inhibition by 1 mM ADP. Login to comment