ABCC8 p.Glu128*
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[hide] N-terminal transmembrane domain of SUR1 controls g... J Gen Physiol. 2011 Mar;137(3):299-314. Epub 2011 Feb 14. Pratt EB, Tewson P, Bruederle CE, Skach WR, Shyng SL
N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2.
J Gen Physiol. 2011 Mar;137(3):299-314. Epub 2011 Feb 14., [PMID:21321069]
Abstract [show]
Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (P(o)) and bursting patterns of activity observed in full-length K(ATP) channels. However, the nature of TMD0-Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in K(ATP) channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-K(ATP) channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced P(o), exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP(2)) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP(2). Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP(2). These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP(2). Moreover, they uncover a novel mechanism of K(ATP) channel inactivation involving aberrant functional coupling between SUR1 and Kir6.2.
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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.
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ABCC8 p.Glu128* 21321069:35:110
status: NEW70 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.
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ABCC8 p.Glu128* 21321069:70:78
status: NEW109 Figure 1. Expression studies of fSUR1 R74X and E128X KATP channels.
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ABCC8 p.Glu128* 21321069:109:54
status: NEW114 (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.
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ABCC8 p.Glu128* 21321069:114:160
status: NEW116 Note the blots shown for R74X and E128X are from two separate experiments; therefore, signal intensity should be compared within each blot only.
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ABCC8 p.Glu128* 21321069:116:34
status: NEW117 (D) Chemiluminescence assays performed to assess surface expression of E128X KATP channels.
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ABCC8 p.Glu128* 21321069:117:71
status: NEW126 Figure 2. Functional studies of fSUR1 R74X and E128X KATP channels.
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ABCC8 p.Glu128* 21321069:126:54
status: NEW127 (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.
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ABCC8 p.Glu128* 21321069:127:143
status: NEW130 (B and C) ATP sensitivity expressed as half-maximal inhibitory concentration (IC50) for each R74X (B) and E128X (C) mutant.
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ABCC8 p.Glu128* 21321069:130:106
status: NEW136 Broadly, E128X mutants showed greater surface expression than R74X mutants.
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ABCC8 p.Glu128* 21321069:136:9
status: NEW137 Of the E128X channels tested, charge-reversal mutations (E128R and K) impeded surface expression most strongly (8 and 6% of WT, respectively).
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ABCC8 p.Glu128* 21321069:137:7
status: NEW139 Next, ATP-induced inhibition of E128X-mutant channels was examined.
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ABCC8 p.Glu128* 21321069:139:32
status: NEW144 Figure 3. Biochemical and immunostaining studies of TMD0 harboring select R74X or E128X mutations.
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ABCC8 p.Glu128* 21321069:144:89
status: NEW145 (A and C) Representative immunoblots of FLAG-tagged SUR1 TMD0 constructs with either R74X (A) or E128X (C) mutations expressed in COSm6 cells.
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ABCC8 p.Glu128* 21321069:145:97
status: NEW147 (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.
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ABCC8 p.Glu128* 21321069:147:94
status: NEW154 To further investigate how E128 contributes to KATP channel physiology, we analyzed E128X TMD0 proteins and the resulting mini-KATP channels when coexpressed with Kir6.2C36.
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ABCC8 p.Glu128* 21321069:154:84
status: NEW158 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.
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ABCC8 p.Glu128* 21321069:158:113
status: NEW161 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.
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ABCC8 p.Glu128* 21321069:161:89
status: NEW289 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).
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ABCC8 p.Glu128* 21321069:289:42
status: NEW