ABCMdb

PMID: 21540180

Chang N, Liang T, Lin X, Kang Y, Xie H, Feng ZP, Gaisano HY
Syntaxin-1A interacts with distinct domains within nucleotide-binding folds of sulfonylurea receptor 1 to inhibit beta-cell ATP-sensitive potassium channels.
J Biol Chem. 2011 Jul 1;286(26):23308-18. Epub 2011 May 3., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC8 p.Lys719Met Only NBF1-WA (K719M) or NBF2-WA (K1385M) mutant no longer bound to Syn-1A; K1385M failed to disrupt Syn-1A-mediated inhibition of KATP channels. Login to comment 53 ABCC8 p.Asp854Asn ABCC8 p.Lys719Met Mutants on WA and WB motifs in these truncated NBF1 (K719M and D854N, respectively) and NBF2 (K1385M and D1506N, respectively) regions were also generated (see Fig. 5A) using QuikChange௡ site-directed mutagenesis according to the manufacturer`s instructions (Stratagene, La Jolla, CA). Login to comment 189 ABCC8 p.Asp854Asn ABCC8 p.Lys719Met The mutations were made by substitutions of lysine in WA and aspartic acid in WB with methionine and asparagine, respectively, and the consequent constructs are GST-NBF1-WA (K719M) (24), GST-NBF1-WB (D854N) (24, 25), GST-NBF2-WA (K1385M) (26), and GST-NBF2-WB (D1506N) (27). Login to comment 191 ABCC8 p.Asp854Asn ABCC8 p.Lys719Met ABCC8 p.Lys719Met The mutations were made by substitutions of lysine in WA and aspartic acid in WB with methionine and asparagine, respectively, and the consequent constructs are GST-NBF1-WA (K719M) (24), GST-NBF1-WB (D854N) (24, 25), GST-NBF2-WA (K1385M) (26), and GST-NBF2-WB (D1506N) (27). Login to comment 192 ABCC8 p.Lys719Met Because NBF1-WA (wt) domain did not disrupt Syn-1A/ SUR1 interactions on our FRET or patch clamp studies (Figs. 2-4 and supplemental Fig. S1), we did not further examine NBF1-WA (K719M). Login to comment 193 ABCC8 p.Lys719Met The WA motif mutations in NBF1-WA (K719M) (Fig. 5B, panel (i)) and NBF2-WA (K1385M) (Fig. 5B, panel (ii)) did not bind Syn-1A in contrast to its respective wild type proteins (Fig. 1B). Login to comment 194 ABCC8 p.Lys719Met Because NBF1-WA (wt) domain did not disrupt Syn-1A/ SUR1 interactions on our FRET or patch clamp studies (Figs. 2-4 and supplemental Fig. S1), we did not further examine NBF1-WA (K719M). Login to comment 198 ABCC8 p.Asp854Asn WB motif mutations in NBF1-WB (D854N) and NBF2-WB (D1506N) remained able to bind Syn-1A (Fig. 5B). Login to comment 199 ABCC8 p.Asp854Asn Both NBF1-WB (D854N) and NBF2-WB (D1506N) were also as effective as their wild type proteins in blocking Syn-1A inhibition of INS-1 beta-cell KATP channels (Fig. 5D and representative traces in supplemental Fig. S2) and in disrupting Syn-1A interactions with full-length SUR1 in the FRET assay (supplemental Fig. S3). Login to comment 200 ABCC8 p.Asp854Asn WB motif mutations in NBF1-WB (D854N) and NBF2-WB (D1506N) remained able to bind Syn-1A (Fig. 5B). Login to comment 201 ABCC8 p.Asp854Asn Both NBF1-WB (D854N) and NBF2-WB (D1506N) were also as effective as their wild type proteins in blocking Syn-1A inhibition of INS-1 beta-cell KATP channels (Fig. 5D and representative traces in supplemental Fig. S2) and in disrupting Syn-1A interactions with full-length SUR1 in the FRET assay (supplemental Fig. S3). Login to comment 246 ABCC8 p.Lys719Met Conserved lysine residues in WA motifs were substituted with methionine, generating NBF1-WA (K719M) and NBF2-WA (K1385M) truncated proteins. Login to comment 247 ABCC8 p.Asp854Asn Conserved aspartic acid residues were substituted with asparagines, generating NBF1-WB (D854N) and NBF2-WB (D1506N) truncated proteins. Login to comment 249 ABCC8 p.Asp854Asn ABCC8 p.Lys719Met ABCC8 p.Lys719Met Panel (i), NBF-1: WA motif mutant NBF1-WA (K719M) could not bind Syn-1A, whereas WB motif mutant NBF1-WB (D854N) remained able to bind Syn-1A. Full-length NBF1, WT NBF1-WA, and WT NBF1-WB are positive controls. Login to comment 250 ABCC8 p.Asp854Asn Conserved aspartic acid residues were substituted with asparagines, generating NBF1-WB (D854N) and NBF2-WB (D1506N) truncated proteins. Login to comment 252 ABCC8 p.Asp854Asn ABCC8 p.Lys719Met Panel (i), NBF-1: WA motif mutant NBF1-WA (K719M) could not bind Syn-1A, whereas WB motif mutant NBF1-WB (D854N) remained able to bind Syn-1A. Full-length NBF1, WT NBF1-WA, and WT NBF1-WB are positive controls. Login to comment 257 ABCC8 p.Asp854Asn FRET studies shown in supplemental Fig. S3 demonstrated that WB motif mutants including NBF1-WB (D854N) and NBF2-WB (D1506N), like their corresponding wild type proteins, disrupted Syn-1A interactions with SUR1. Login to comment 259 ABCC8 p.Asp854Asn WB motif mutants NBF1-WB (D854N) and NBF2-WB (D1506N), like their WT proteins, remained able to block Syn-1A inhibition of KATP channels. Login to comment 261 ABCC8 p.Asp854Asn FRET studies shown in supplemental Fig. S3 demonstrated that WB motif mutants including NBF1-WB (D854N) and NBF2-WB (D1506N), like their corresponding wild type proteins, disrupted Syn-1A interactions with SUR1. Login to comment 263 ABCC8 p.Asp854Asn WB motif mutants NBF1-WB (D854N) and NBF2-WB (D1506N), like their WT proteins, remained able to block Syn-1A inhibition of KATP channels. Login to comment