ABCMdb

ABCC8 p.Glu203Lys

Predicted by SNAP2:	A: N (66%), C: N (61%), D: N (87%), F: N (57%), G: N (66%), H: N (66%), I: D (53%), K: N (78%), L: D (53%), M: D (53%), N: N (78%), P: N (66%), Q: N (87%), R: N (82%), S: N (78%), T: N (78%), V: N (66%), W: D (59%), Y: N (57%),
Predicted by PROVEAN:	A: N, C: D, D: N, F: D, G: N, H: N, I: D, K: N, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: D, W: D, Y: D,

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[hide] Engineered interaction between SUR1 and Kir6.2 tha... J Gen Physiol. 2012 Aug;140(2):175-87. doi: 10.1085/jgp.201210803. Epub 2012 Jul 16. Pratt EB, Zhou Q, Gay JW, Shyng SL
Engineered interaction between SUR1 and Kir6.2 that enhances ATP sensitivity in KATP channels.
J Gen Physiol. 2012 Aug;140(2):175-87. doi: 10.1085/jgp.201210803. Epub 2012 Jul 16., [PMID:22802363]

Abstract [show]
The ATP-sensitive potassium (K(ATP)) channel consisting of the inward rectifier Kir6.2 and SUR1 (sulfonylurea receptor 1) couples cell metabolism to membrane excitability and regulates insulin secretion. Inhibition by intracellular ATP is a hallmark feature of the channel. ATP sensitivity is conferred by Kir6.2 but enhanced by SUR1. The mechanism by which SUR1 increases channel ATP sensitivity is not understood. In this study, we report molecular interactions between SUR1 and Kir6.2 that markedly alter channel ATP sensitivity. Channels bearing an E203K mutation in SUR1 and a Q52E in Kir6.2 exhibit ATP sensitivity approximately 100-fold higher than wild-type channels. Cross-linking of E203C in SUR1 and Q52C in Kir6.2 locks the channel in a closed state and is reversible by reducing agents, demonstrating close proximity of the two residues. Our results reveal that ATP sensitivity in K(ATP) channels is a dynamic parameter dictated by interactions between SUR1 and Kir6.2.

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Sentences [show]
No. Sentence Comment
24 Channels bearing an E203K mutation in SUR1 and a Q52E in Kir6.2 exhibit ATP sensitivity &#e07a;100-fold higher than wild-type channels. Login to comment
58 We show that coexpression of a Kir6.2 with the mutation Q52E in the N-terminal domain and a SUR1 with the mutation E203K in the cytoplasmic region immediately following TMD0 yielded channels with ATP sensitivity nearly 100-fold higher than WT channels. Login to comment
102 Error bars represent SEM, and some are smaller than the symbols. (K199E, R202E, E203K, and K205E) and then coexpressed with WT-, Q52E-, or Q52R-Kir6.2. Login to comment
108 However, the most striking change of ATP sensitivity indicative of functional interaction was seen in channels formed by Q52E-Kir6.2 and E203K-SUR1. Login to comment
109 The Q52E-Kir6.2//E203K-SUR1 channels exhibited a marked A SUR1 mutation functionally interacts with Q52E of Kir6.2 to enhance ATP sensitivity To identify a residue or residues in SUR1 that might interact with Q52E or Q52R to influence ATP sensitivity, we performed mutagenesis of SUR1, focusing on the following charged residues: K199, R202, E203, and K205. Login to comment
114 These residues were mutated to oppositely charged amino acids Figure 2.ߓ Screening of SUR1 residues identifies the E203K mutation that when combined with Q52E in Kir6.2 gives rise to channels with extremely high ATP sensitivity. Login to comment
117 Only 1 mM ATP was tested in the cases of E203K-SUR1// Q52R-Kir6.2 and K205E-SUR1// Q52R-Kir6.2 because of extremely low ATP sensitivity. Login to comment
119 (B) Representative traces from WT or Q52E-Kir6.2//E203K-SUR1 KATP channels exposed to various concentrations of ATP or nucleotide-free Kint/EDTA control solution; note difference in ATP concentrations used. Login to comment
121 (C) Dose-response data illustrating the mean currents in several ATP concentrations relative to maximum in nucleotide-free solution for WT and Q52E-Kir6.2//E203K-SUR1 KATP channels. Login to comment
122 Best fit curves were generated using the Hill equation (IC50: 11 &#b1; 1 &#b5;M for WT and 140 &#b1; 5 nM for Q52E-Kir6.2// E203K-SUR1). Login to comment
124 For comparison, the best fit curve for Q52E-Kir6.2 (see Fig. 1) and mean currents for the three ATP concentrations tested on E203K-SUR1 channels (see A) were also included. Login to comment
125 Error bars represent SEM, and some are smaller than the symbols. used to determine whether increased ATP sensitivity seen in the Q52E-Kir6.2//WT-SUR1 and Q52E-Kir6.2// E203K-SUR1 channels was related to decreased channel Po (Fig. 3). Login to comment
127 The fold increase of current from baseline was calculated (Fig. 3 B), with Q52E-Kir6.2//WT-SUR1 and Q52E-Kir6.2//E203K-SUR1 showing increased response com- paredwithWT(4.4&#b1;0.7,3.7&#b1;0.9vs.1.3&#b1;0.1,respectively, after second exposure), whereas the E203K-SUR1 response was less than WT (1.1 &#b1; 0.1). Login to comment
128 Intrinsic Po was estimated using these values (PIP2 method) as well as via noise analysis; estimates from the two methods are compared in Fig. 3 C. Q52E-Kir6.2//WT-SUR1 and Q52E-Kir6.2//E203K-SUR1 mutant channels had significantly reduced Po compared with WT (0.28 &#b1; 0.05, 0.36 &#b1; 0.06 vs. 0.76 &#b1; 0.04 using the PIP2 method and 0.16 &#b1; 0.04, 0.31 &#b1; 0.04 vs. 0.76 &#b1; 0.03 using noise analysis, respectively). Login to comment
129 Estimates of E203K Po are 0.92 &#b1; 0.02 and 0.83 &#b1; 0.02 using the PIP2 and noise analysis methods. Login to comment
130 The analyses revealed that although there is a 10-fold difference in IC50 between Q52E-Kir6.2//WT-SUR1 and Q52E-Kir6.2// E203K-SUR1, their estimated intrinsic Po values are not significantly different. Login to comment
131 This suggests that the unusually high ATP sensitivity of Q52E-Kir6.2//E203K-SUR1 can only be partially accounted for by changes in Po and/or channel-PIP2 interactions (see Discussion). Login to comment
133 The result suggests that E203K of SUR1 interacts with Q52E of Kir6.2 either directly or indirectly to increase ATP sensitivity. Login to comment
138 Both methods were Figure 3.ߓ Changes in intrinsic Po do not fully account for high Q52E-Kir6.2// E203K-SUR1 ATP sensitivity. Login to comment
146 Number in each dataset for PIP2 method is given in B (right); n for NA method is 19, 18, 17, and 19 for WT, E203K-SUR1, Q52E-Kir6.2, and Q52E-Kir6.2// E203K-SUR1, respectively. Login to comment
153 Addition of H2O2 to channels already closed by 1 mM ATP also induced cross-linking as indicated by the greatly reduced channel activity after washout of H2O2 and ATP and subsequent increase in activity with Cross-linking between Q52C of Kir6.2 and E203C of SUR1 stabilizes channels in a closed state To test whether E203K of SUR1 and Q52E of Kir6.2 are in close proximity to interact directly, we substituted both residues with cysteines and performed cross-linking experiments. Login to comment
185 The Q52K-Kir6.2 and E203-SUR1 (WT-SUR1) charge pair does not recapitulate the high ATP sensitivity observed in Q52E-Kir6.2//E203K-SUR1 channels Based on the high ATP sensitivity observed in the Q52E-Kir6.2//E203K-SUR1 channels and the evidence supporting physical proximity between the two residues, one Figure 5.ߓ Cross-linking does not occur in WT, WT-Kir6.2// E203C-SUR1, or Q52C-Kir6.2//WT-SUR1 channels. Login to comment
200 As expected, addition of the E128K mutation in SUR1 also diminished the ability of Q52E-Kir6.2//E203K-SUR1 channels to respond to PIP2 stimulation (Fig. 7 B; also see Fig. 3 [A and B] for comparison). Login to comment
206 However, no change in channel activity was observed in Q52C-Kir6.2//E128C-SUR1 than WT (Fig. 6), in contrast to the near 100-fold increase in ATP sensitivity observed in Q52E-Kir6.2// E203K-SUR1 (Fig. 2). Login to comment
207 These results suggest that the specific interaction between Q52E-Kir6.2 and E203K-SUR1 that results in a super ATP-sensitive channel is likely dependent on the structural context surrounding the mutant amino acid pair. Login to comment
221 Error bars represent SEM, and some are smaller than the symbols. for Q52E-Kir6.2//E128K-SUR1 and Q52E-Kir6.2// E128K/E203K-SUR1 channels was closer to that of WT-Kir6.2//E128K-SUR1 (and Kir6.2&#e044;C channels [Pratt et al., 2009]) than that of either Q52E-Kir6.2//WT-SUR1 or Q52E-Kir6.2//E203K-SUR1 (Fig. 7, C and D). Login to comment
224 E128K-SUR1 also suppressed the effects of Q52E-Kir6.2 and Q52E-Kir6.2//E203K-SUR1 on ATP sensitivity. Login to comment
231 (B) E128K in SUR1 abolishes the ability of Q52E-Kir6.2//E203K-SUR1 channels to be stimulated by PIP2. Login to comment
235 (C and D) Inhibition by ATP was tested in channels composed of E128K-SUR1 with Q52E-Kir6.2, Q52R-Kir6.2, or Q52E-Kir6.2//E203K-SUR1. Login to comment
244 Channels formed by Q52E-Kir6.2 and E203K-SUR1 are not activated by metabolic inhibition ATP inhibition and MgADP stimulation are two primary physiological mechanisms that regulate KATP channel activity in &#e062; cells. Login to comment
248 Because Q52E-Kir6.2//WT-SUR1 and Q52E-Kir6.2//E203K-SUR1 channels also show reduced intrinsic Po (Fig. 3 C), which could reduce efflux, we included the F55L-Kir6.2//WT-SUR1 mutant that exhibits &#e07a;10-fold reduced intrinsic Po but normal nucleotide sensitivities and surface expression for comparison (Lin et al., 2006b). Login to comment
250 In contrast, both Q52E-Kir6.2//WT-SUR1 and Q52E-Kir6.2//E203K-SUR1 mutants had greatly reduced activity close to untransfected control cells (Fig. 8 C), despite surface expression levels similar to WT (Fig. 8, A and B). Login to comment
256 A wealth of structure-function evidence supports the view that although the Kir6.2 tetramer harbors the ATP-binding sites for channel Figure 8.ߓ Increased ATP sensitivity in channels formed by E203K-SUR1 and Q52E-Kir6.2 compromise their ability to open in response to metabolic inhibition. Login to comment
260 Mutations at Q52E-Kir6.2 and/or E203K-SUR1 do not disrupt protein trafficking. Login to comment
262 Mutations at Q52E-Kir6.2 or E203K-SUR1 or both do not affect the number of channels at the cell surface. Login to comment
267 Channels composed of Q52E-Kir6.2 with and without E203K-SUR1 show significantly diminished efflux compared with controls, WT and F55L-Kir6.2; the latter control was included because it has decreased intrinsic Po (&#e07a;0.11) but normal ATP sensitivity and surface expression. Login to comment
273 Although Q52E-Kir6.2//E203K-SUR1 channels are highly sensitive to ATP inhibition, charge swap at the two positions as in Q52K-Kir6.2//WT(E203)-SUR1 channels does not recapitulate the same markedly increased ATP sensitivity. Login to comment
276 Interestingly, the effect of Q52R-Kir6.2, Q52E-Kir6.2, or the combined Q52E-Kir6.2//E203K-SUR1 mutation on ATP sensitivity is dependent on another SUR1 residue, E128, in the short intracellular loop between transmembrane helices 3 and 4 of TMD0. Login to comment
280 Our observations that when E128K-SUR1 was combined with Q52R-Kir6.2, Q52E-Kir6.2, or Q52E-Kir6.2//E203K-SUR1, the resulting channels all had PIP2 and ATP sensitivity closer to that of WT-Kir6.2//E128K-SUR1 (or Kir6.2&#e044;C) further support this notion. Login to comment
288 First, to our knowledge, the Q52E-Kir6.2//E203K-SUR1 channel is the first mutant reported to have such a profound increase in ATP sensitivity. Login to comment
293 We found the Po of Q52E-Kir6.2//E203K-SUR1 channels is indeed lower compared with WT channels (Fig. 3 C); however, decreased Po alone cannot account for the marked increase in ATP sensitivity of the channel because Q52E-Kir6.2//WT-SUR1 channels, despite having similarly reduced Po, are &#e07a;15-fold less sensitive to ATP inhibition. Login to comment
297 Although it has been suggested that SUR1 increases channel sensitivity to ATP by participating in ATP binding (Babenko and Bryan, 2003; Babenko, 2005) direct evidence is lacking, and the single E203K mutation in SUR1 had little effect on channel ATP sensitivity. Login to comment
301 Together, the evidence led us to propose a scenario wherein the intersubunit interaction between E203K-SUR1 and Q52E-Kir6.2 allosterically stabilizes the channel complex in an ATP-bound closed state to increase the channel`s apparent ATP sensitivity. Login to comment
345 In the Q52E-Kir6.2//E203K-SUR1 channels, the ATP sensitivity is so high it was impossible to find a concentration of MgATP or MgADP within the physiological range that would antagonize the inhibitory effect of the nucleotides. Login to comment

[hide] Engineered Kir6.2 mutations that correct the traff... Channels (Austin). 2013 Jul-Aug;7(4):313-7. Epub 2013 May 21. Zhou Q, Pratt EB, Shyng SL
Engineered Kir6.2 mutations that correct the trafficking defect of K(ATP) channels caused by specific SUR1 mutations.
Channels (Austin). 2013 Jul-Aug;7(4):313-7. Epub 2013 May 21., [PMID:23695995]

Abstract [show]
KATP channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. The molecular interactions between SUR1 and Kir6.2 that govern channel gating and biogenesis are incompletely understood. In a recent study, we showed that a SUR1 and Kir6.2 mutation pair, E203K-SUR1 and Q52E-Kir6.2, at the SUR1/Kir6.2 interface near the plasma membrane increases the ATP-sensitivity of the channel by nearly 100-fold. Here, we report the finding that the same mutation pair also suppresses channel folding/trafficking defects caused by select SUR1 mutations in the first transmembrane domain of SUR1. Analysis of the contributions from individual mutations, however, revealed that the correction effect is attributed largely to Q52E-Kir6.2 alone. Moreover, the correction is dependent on the negative charge of the substituting amino acid at the Q52 position in Kir6.2. Our study demonstrates for the first time that engineered mutations in Kir6.2 can correct the biogenesis defect caused by specific mutations in the SUR1 subunit.

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Sentences [show]
No. Sentence Comment
2 In a recent study, we showed that a SUR1 and Kir6.2 mutation pair, E203K-SUR1 and Q52E-Kir6.2, at theSUR1/Kir6.2interfaceneartheplasma membrane increases the ATP-sensitivity of the channel by nearly 100-fold. Login to comment
10 In addition, the cytoplasmic loop L0 immediately following TMD0 interacts with the N-terminal cytoplasmic domain of Kir6.2 to modulate channel gating.12,17-20 Recently, we identified an engineered interaction between SUR1- E203K and Kir6.2-Q52E (denoted as E203K//Q52E; hereinafter "//" separates mutations in SUR1 and Kir6.2, and "/" separates mutations within the same subunit) that increased the channelߛs sensitivity to ATP by nearly 100-fold.21 E203 Addendum to: Pratt EB, Zhou Q, Gay JW, Shyng SL. Engineered interaction between SUR1 and Kir6.2 that enhances ATP sensitivity in KATP channels. Login to comment
14 Results and Discussion To test if the interaction between E203K-SUR1 and Q52E-Kir6.2 affects the biogenesis of channels with previously identified SUR1-TMD0 trafficking mutations,25,26 we placed several such mutations on the E203K//Q52E background and assessed channel processing efficiency by western blots. Login to comment
16 Of the three TMD0 mutations tested, F27S and A116P showed a clear upper band in addition to the lower immature band in the E203K//Q52E background; by contrast, the same trafficking mutations placed in the background without the E203K//Q52E mutations only exhibited the lower band (Fig. 2), indicating the proteins were retained in the ER as reported previously.25,26 Another TMD0 mutation, E128K, as well as three other previously identified, congenital hyperinsulinism-causing SUR1 trafficking mutations outside of TMD0 (R495Q, F686S and L1350Q),25 however, showed no improvement in their processing efficiency when combined with E203K//Q52E (data not shown). Login to comment
17 These results led us to conclude that the E203K//Q52E mutations can overcome the folding and trafficking defects caused by some but not all TMD0 mutations. Login to comment
25 E203K//Q52E mutation pair in correcting the processing defect of F27S and A116P. Login to comment
26 This scenario differs somewhat from that observed for gating regulation whereby E203K-SUR1 does not affect channel ATP-sensitivity and Q52E-Kir6.2 increases ATP-sensitivity by ~5-fold but E203K//Q52E increases ATP-sensitivity by ~100-fold.21 Moreover, while crosslinking of E203C//Q52C induces channel closure21 it does not appear to rescue the trafficking defect caused by F27S, at least under the experimental conditions we have tested. Login to comment
27 Together these observations argue that the electrostatic interactions between E203K//Q52E or crosslinking between E203C//Q52C needed to observe a profound change in gating are not required for the trafficking defect rescue. Login to comment
31 Note in the case of Q52K-Kir6.2, the pairing with E203 residue in SUR1 would represent a reverse-switch of charge at the two positions in relation to the E203K//Q52E mutation pair, and yet unlike E203K//Q52E, E203//Q52K failed to correct the trafficking defect caused by F27S and A116P. Login to comment
32 These results suggest that correction of the trafficking defects of F27S and A116P in the E203K//Q52E background is unlikely a consequence of electrostatic interactions between amino acids at the 203-SUR1 and 52-Kir6.2 positions, and that a negatively charged amino acid at position 52 of Kir6.2 is the major driving factor for expression rescue. Login to comment
34 Our study identifies several mutations, including E203K// Q52E, Q52E-Kir6.2 and Q52D-Kir6.2, that can significantly improve the processing and surface expression of channels harboring specific TMD0 mutations. Login to comment
36 Analysis of the contribution from individual mutations revealed that the processing defect caused by F27S-SUR1 is little affected by E203K-SUR1 but is significantly alleviated by the Q52E and D mutations in Kir6.2 alone. Login to comment
37 In fact, the Q52E- or Q52D-Kir6.2 mutations alone were nearly as effective as the E203K//Q52E has been found to increase channel sensitivity to ATP inhibition by nearly 100-fold in our recent study.21 The markedly increased ATP sensitivity is likely due to close electrostatic interactions between the two oppositely charged mutant residues. Login to comment
38 Mutation of Q52E-Kir6.2 alone only increased ATP-sensitivity by ~5-fold whereas E203K-SUR1 did not change ATP-sensitivity significantly. Login to comment
44 Next, we tested the role of individual E203K-SUR1 or Q52E-Kir6.2 mutations in F27S-SUR1 mutant processing. Login to comment
45 Surprisingly, while the E203K-SUR1 mutation had little effect on F27S-SUR1 processing, co-expression of F27S-SUR1 with Q52E-Kir6.2 was sufficient to increase the upper F27S-SUR1 band, and surface F27S-SUR1 detected by surface biotinylation was nearly as abundant as F27S/E203K//Q52E (Fig. 3A). Login to comment
48 The E203K//Q52E mutation pair suppresses the processing defect caused by the F27S or A116P SUR1 mutation. Login to comment