ABCC8 p.Glu128Trp
| Predicted by SNAP2: | A: D (66%), C: D (59%), D: D (85%), F: D (71%), G: D (85%), H: D (80%), I: D (75%), K: D (71%), L: D (75%), M: D (66%), N: D (85%), P: D (91%), Q: D (75%), R: D (80%), S: D (80%), T: D (80%), V: D (75%), W: D (80%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, C: D, D: N, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: N, R: D, S: D, T: D, V: D, W: D, Y: D, |
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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
23 Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP2.
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ABCC8 p.Glu128Trp 21321069:23:36
status: NEW37 In contrast, E128K disrupts functional coupling between TMD0 and Kir6.2 by abrogating the effects of SUR1 on channel response to PIP2. Moreover, the mutation E128W leads to spontaneous current inactivation that can be prevented or reversed by PIP2.
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ABCC8 p.Glu128Trp 21321069:37:158
status: NEW215 Inactivation of E128W channels could be recovered by exposure to high concentrations of ATP (1, 3, or 5 mM) in a time-dependent manner, such that lengthening the time of ATP ex-posure resulted in more current when the patch was reexposed to nucleotide-free solution (Fig. 8, A and B).
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ABCC8 p.Glu128Trp 21321069:215:16
status: NEW216 Further, exposure of the E128W patches to PIP2 slowed or reversed the inactivation as well as potentiated the ATP-induced "resetting" of channel activity (Fig. 8 C).
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ABCC8 p.Glu128Trp 21321069:216:25
status: NEW217 These results indicate that E128W causes KATP channel inactivation by destabilizing PIP2-KATP channel interactions, and that ATP exposure re- est-ablishes the interactions as seen in the transient channel activity when the inhibitory effect of ATP is removed.
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ABCC8 p.Glu128Trp 21321069:217:28
status: NEW225 The E128W mutation in SUR1 causes inactivation in full-length channels that can be recovered by PIP2 and by exposure and subsequent removal of ATP Additional evidence supporting the involvement of E128 in mediating channel response to PIP2 came from the intriguing phenotype observed with the E128W mutation.
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ABCC8 p.Glu128Trp 21321069:225:4
status: NEWX
ABCC8 p.Glu128Trp 21321069:225:293
status: NEW243 Although the exact location of R74 with respect to the membrane has not Figure 8. E128W causes KATP channel inactivation that can be recovered by ATP exposure and subsequent removal of ATP and can be reversed by PIP2.
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ABCC8 p.Glu128Trp 21321069:243:89
status: NEW249 (B) The ability of high concentrations (1-5 mM) of ATP to reset the E128W KATP channel is illustrated by this scatter plot.
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ABCC8 p.Glu128Trp 21321069:249:68
status: NEW254 (C) Representative trace showing that exposure of the E128W KATP channels to 5 µM PIP2 (striped bars) decreased and reversed inactivation as well as increased the efficacy of ATP (black lines, 5 mM) to reset the channel.
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ABCC8 p.Glu128Trp 21321069:254:54
status: NEW270 (B) The E128W mutation destabilizes the channel in the SUR1-Kir6.2-coupled, PIP2-bound open state, leading to channel inactivation.
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ABCC8 p.Glu128Trp 21321069:270:8
status: NEW280 A novel KATP channel inactivation mechanism revealed by the E128W mutation and structural implications The importance of E128 in the structural and functional integrity of KATP channels is further accentuated by the inactivation phenotype of the E128W mutant.
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ABCC8 p.Glu128Trp 21321069:280:60
status: NEWX
ABCC8 p.Glu128Trp 21321069:280:246
status: NEW282 First, current inactivation induced by E128W is distinct from rundown.
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ABCC8 p.Glu128Trp 21321069:282:39
status: NEW283 Second, inactivation caused by E128W is prevented or reversed by PIP2.
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ABCC8 p.Glu128Trp 21321069:283:31
status: NEW287 That inactivation induced by E128W is overcome by PIP2 lends strong support to our proposal that E128 plays a critical role in the stabilization of Kir6.2-PIP2 interactions by SUR1.
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ABCC8 p.Glu128Trp 21321069:287:29
status: NEW288 In addition, the ATP-dependent recovery from inactivation in the E128W mutant suggests that conformational changes in Kir6.2 tertiary structure of R74K TMD0 and its ability to assemble with Kir6.2 to form channels may be contingent upon the SUR1 structures downstream of TMD0.
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ABCC8 p.Glu128Trp 21321069:288:65
status: NEW310 Finally, our discovery of the E128W inactivation mutation provides novel insight into the structural relationship between channel subunits as a function of ATP and PIP2.
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ABCC8 p.Glu128Trp 21321069:310:30
status: NEW347 The similar gating properties seen in E128W and Kir6.2 inactivation mutations predicted to alter inter-Kir6.2 subunit interfaces imply that these mutations disrupt normal channel gating via a converging mechanism that is sensitive to both PIP2 and ATP.
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ABCC8 p.Glu128Trp 21321069:347:38
status: NEW352 The finding is especially provocative when considered in the context of the SUR1-Kir6.2 KATP channel complex because it opens up the possibility that SUR1 can modulate Kir6.2 conduction primarily through cytoplasmic domain interfaces, such as that mediated by E128. We propose that in KATP channels, SUR1 stabilizes a Kir6.2 structure in the PIP2-bound open state via cytoplasmic interactions, and that the inactivation phenotype of E128W represents transition from a SUR1-coupled conformation to Kir6.2 structures lacking the stabilizing effect of SUR1, as seen in Kir6.2C36.
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ABCC8 p.Glu128Trp 21321069:352:433
status: NEW353 ATP binding is envisioned to cause a conformational switch in Kir6.2, such that channels can enter a PIP2-bound open state once ATP is removed.Thus,inthecaseofKir6.2inactivationmutations, ATP reactivates the channel by reestablishing Kir6.2 subunit interface (Lin et al., 2003), and in the case of E128W, by reestablishing SUR1-Kir6.2 interactions necessary for stabilizing channel opening, albeit only briefly (see Fig. 9 B).
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ABCC8 p.Glu128Trp 21321069:353:298
status: NEW354 Increasing the concentration of PIP2 in the membrane is expected to shift the equilibrium toward the PIP2-bound open state by mass action, thereby slowing and reversing inactivation as well as boosting the ATP-induced resetting of the channel in both Kir6.2 and E128W-SUR1 inactivation mutants (Figs.
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ABCC8 p.Glu128Trp 21321069:354:262
status: NEW[hide] ATP activates ATP-sensitive potassium channels com... Channels (Austin). 2011 Jul-Aug;5(4):314-9. doi: 10.4161/chan.5.4.16510. Epub 2011 Jul 1. Pratt EB, Shyng SL
ATP activates ATP-sensitive potassium channels composed of mutant sulfonylurea receptor 1 and Kir6.2 with diminished PIP2 sensitivity.
Channels (Austin). 2011 Jul-Aug;5(4):314-9. doi: 10.4161/chan.5.4.16510. Epub 2011 Jul 1., [PMID:21654216]
Abstract [show]
ATP-sensitive potassium (K(ATP)) channels are inhibited by ATP and activated by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Both channel subunits Kir6.2 and sulfonylurea receptor 1 (SUR1) contribute to gating: while Kir6.2 interacts with ATP and PIP(2), SUR1 enhances sensitivity to both ligands. Recently, we showed that a mutation, E128K, in the N-terminal transmembrane domain of SUR1 disrupts functional coupling between SUR1 and Kir6.2, leading to reduced ATP and PIP(2) sensitivities resembling channels formed by Kir6.2 alone. We show here that when E128K SUR1 was co-expressed with Kir6.2 mutants known to disrupt PIP(2) gating, the resulting channels were surprisingly stimulated rather than inhibited by ATP. To explain this paradoxical gating behavior, we propose a model in which the open state of doubly mutant channels is highly unstable; ATP binding induces a conformational change in ATP-unbound closed channels that is conducive to brief opening when ATP unbinds, giving rise to the appearance of ATP-induced stimulation.
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None has been submitted yet.
No. Sentence Comment
40 Moreover, another mutation at the same site, E128W, causes rapid destabilization of channel activity that is reversed by PIP2 .
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ABCC8 p.Glu128Trp 21654216:40:45
status: NEW86 The ATP-induced conformational change referred to here is analogous to the previously reported effect of ATP on several mutations that cause spontaneous channel inactivation, including E128W in SUR1 and R192E, R301E and R314E in Kir6.2.14,23 In the inactivation mutants, exposure to high concentrations of ATP followed by subsequent washout to remove the inhibitory effect of ATP recovers channels from inactivation and allows channels to open briefly before they inactivate again (this inactivation phenomenon can be seen in the R192E// E128K mutant following removal of ATP, Fig. 2A, middle).
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ABCC8 p.Glu128Trp 21654216:86:185
status: NEW