ABCMdb

ABCC8 p.Arg74*

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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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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
78 Surface expression for every R74X-mutant tested, except the charge-conserving R74K mutation, was significantly reduced to <40% of WT (Fig. 1 B). 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
119 R74 mutations in TMD0 constructs reduce steady-state protein levels and disrupt surface expression of mini-KATP channels Assessing the specific effects of R74X mutations on TMD0 is possible using a truncated SUR1 construct (amino acids 1-198). Login to comment
120 Steady-state levels of total cellular fTMD0 harboring R74X were assessed via Western blot. 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
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
156 Also in contrast to R74X, E128K fTMD0 was detected at the cell surface of COSm6 cells when cotransfected with Kir6.2C36 (Fig. 3 E), indicating that the formation of mini-KATP channels is suitable for electrophysiological analysis. 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
257 nately, however, R74X TMD0 mutants failed to express at the cell surface when coexpressed with Kir6.2C36, making it difficult to address the question of whether the gating defects in full-length channels are caused by changes in the functional coupling between TMD0 and Kir6.2 or are a consequence of altered influences by SUR1 structures outside TMD0. Login to comment
261 Thus, the here comes into play when hypothesizing the structural changes R74X substitutions have on TMD0 conformation. Login to comment
263 Accordingly, with the exception of lysine, all other amino acids used in the R74X screen (A, H, C, W, F, Y, L, D, or E) are predicted to shift the boundary of TM1 and TM2 (as well as TM3 for R74D and R74E) by TOPCONS (Fig. S2). Login to comment
264 The decrease in full-length KATP channel surface expression of R74X mutants (Fig. 1) and steady-state protein levels of TMD0 (Fig. 3) is consistent with these substitutions having an effect on protein structure and stability. Login to comment
265 Sulfonylureas acting as chemical chaperones partially overcome the trafficking defect in R74X full-length KATP channels, with the exception of R74D (Fig. S1). 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