ABCMdb

ABCC8 p.Glu128*

None has been submitted yet.

Publications

PMID: 21321069 [PubMed] Pratt EB et al: "N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2."

No. Sentence Comment

35 In this study, we systematically replaced residues 74 and 128 with other amino acids (referred to as R74X and E128X) in full-length and mini-KATP channels to probe their structural and functional roles in the coupling of TMD0 to Kir6.2. Login to comment
70 Online supplemental material Fig. S1 shows the surface expression of R74X and E128X full-length KATP channels, as quantified by chemiluminescence after overnight pretreatment with 300 µM tolbutamide to rescue channel trafficking. Fig. S2 includes a protein sequence alignment performed by PRALINE (Simossis and Heringa, 2005) of TMD0 of SUR1 for human, hamster, dog, and zebrafish, and human SUR2. Login to comment
109 Figure 1. Expression studies of fSUR1 R74X and E128X KATP channels. Login to comment
114 (C) Representative immunoblots using anti-SUR1 antibody to detect expression of fSUR1 protein in cells cotransfected with Kir6.2 and either fSUR1 R74X (top) or E128X (bottom) cDNAs. Login to comment
116 Note the blots shown for R74X and E128X are from two separate experiments; therefore, signal intensity should be compared within each blot only. Login to comment
117 (D) Chemiluminescence assays performed to assess surface expression of E128X KATP channels. Login to comment
126 Figure 2. Functional studies of fSUR1 R74X and E128X KATP channels. Login to comment
127 (A) Representative traces from inside-out voltage clamp experiments performed in COSm6 cells transfected with WT Kir6.2 and WT, R74X (top), or E128X (bottom) SUR1. Login to comment
130 (B and C) ATP sensitivity expressed as half-maximal inhibitory concentration (IC50) for each R74X (B) and E128X (C) mutant. Login to comment
136 Broadly, E128X mutants showed greater surface expression than R74X mutants. Login to comment
137 Of the E128X channels tested, charge-reversal mutations (E128R and K) impeded surface expression most strongly (8 and 6% of WT, respectively). Login to comment
139 Next, ATP-induced inhibition of E128X-mutant channels was examined. Login to comment
144 Figure 3. Biochemical and immunostaining studies of TMD0 harboring select R74X or E128X mutations. Login to comment
145 (A and C) Representative immunoblots of FLAG-tagged SUR1 TMD0 constructs with either R74X (A) or E128X (C) mutations expressed in COSm6 cells. Login to comment
147 (B and D) Densitometry analysis was performed on the blots in A and C to quantify R74X (B) or E128X (D) fTMD0 protein levels relative to WT fTMD0. Login to comment
154 To further investigate how E128 contributes to KATP channel physiology, we analyzed E128X TMD0 proteins and the resulting mini-KATP channels when coexpressed with Kir6.2C36. Login to comment
158 We tested whether the same would be true for E128K mini-KATP channels, which would be a clear indication of each E128X mutant (Fig. S1), followed by a 2-h washout before recording. Login to comment
161 E128 mutations disrupt functional coupling between TMD0 and Kir6.2 in mini-KATP channels E128X substitutions affect KATP channel surface expression and ATP sensitivity in a pattern distinct from R74X substitutions. Login to comment
289 Role of E128 Full-length channels bearing E128X mutations are in general expressed at a higher level at the cell surface than R74X channels (Fig. 1). Login to comment