ABCMdb

PMID: 10570926

Matsuo M, Tucker SJ, Ashcroft FM, Amachi T, Ueda K
NEM modification prevents high-affinity ATP binding to the first nucleotide binding fold of the sulphonylurea receptor, SUR1.
FEBS Lett. 1999 Sep 24;458(3):292-4., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC8 p.Cys717Ser However, when the cysteine residue in the Walker A motif of the first nucleotide binding fold (NBF1) of SUR1 was replaced with serine (C717S), photoaffinity labeling was not inhibited by 100 WWM NEM. These results suggest that NBF1 of SUR1 has a NEM-sensitive structure similar to that of NBF1 of MDR1, a multidrug transporter, and confirm NBF1 as the high-affinity ATP binding site on SUR1. Login to comment 25 ABCC8 p.Lys719Met Hamster SUR1 (K719M) cDNA was generously provided by Dr Susumu Seino (Chiba University, Japan). Login to comment 27 ABCC8 p.Cys717Ser The C717S and K1385M mutations used in this study were made in rat SUR1. Login to comment 58 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser We therefore examined the e¡ects of NEM on 8-azido-ATP binding to a mutant form of SUR1, in which the cysteine residue within the Walker A motif of NBF1 was replaced with serine (C717S). Login to comment 59 ABCC8 p.Cys717Ser Membranes containing equal amounts of the wild-type and the C717S mutant form of SUR1 were treated with 100 WM NEM, and photoa¤nity labeling with 5 WM 8-azido-[Q-32 P]- ATP was then examined (Fig. 3). Login to comment 60 ABCC8 p.Cys717Ser Both wild-type and C717S SUR1 were photoa¤nity labeled to the same extent in the absence of NEM. Login to comment 61 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser However, by contrast to wild-type SUR1, photoa¤nity labeling of C717S SUR1 was una¡ected by pretreatment with NEM. Login to comment 63 ABCC8 p.Lys719Met However, the K719M mutant form of SUR1 (in which the lysine residue within the Walker A motif of NBF1 was replaced with methionine) was not photoa¤nity labeled either in the absence or in the presence of NEM. These results indicate that cysteine-717 within NBF1 of SUR1 is responsible for inhibition of high-a¤nity 8-azido-ATP binding by NEM, and suggest that NBF1 of SUR1 has a NEM-sensitive structure similar to that of NBF1 of MDR1. Login to comment 64 ABCC8 p.Asp854Asn ABCC8 p.Lys719Met We have reported previously that mutations in either the Walker A or B motifs of NBF1, K719M and D854N, abolish Fig. 1. Login to comment 73 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser The cysteine residue within the Walker A motif of NBF of SUR1 was replaced with serine in the C717S mutant form. Login to comment 77 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser The inhibition of this high-a¤nity 8-azido-ATP binding to SUR1 by NEM and the lack of inhibition found with the C717S mutation now con'rms that NBF1 is the high-a¤nity ATP binding site identi'ed on SUR1. Login to comment 85 ABCC8 p.Lys719Met Acknowledgements: We thank Dr S. Seino (Chiba University, Japan) for providing hamster SUR1 (K719M) cDNA and the antibody against SUR1. Login to comment