ABCMdb

ABCC8 p.Glu203Cys

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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No. Sentence Comment
25 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. Login to comment
86 Fig. S3 shows that Q52C-Kir6.2//E203C-SUR1 channels have WT-like ATP sensitivity in the absence of cross-linking. Fig. S4 shows that control channels do not cross-link in the presence of an inhibitory concentration of ATP plus oxidizing agent; it also shows that Tris(2-carboxyethyl)phosphine (TCEP) is an effective reducing agent to reverse the cross-linking within Q52C-Kir6.2//E203C-SUR1 channels. Login to comment
152 These observations indicate that cross-linking between Q52C-Kir6.2 and E203C-SUR1 causes the channel to enter an ATP-independent closed state. 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
155 In inside-out patches, Q52C-Kir6.2//E203C-SUR1 channels exhibited ATP sensitivity indistinguishable from that of WT channels (Fig. S3). Login to comment
158 The residual currents could represent uncross-linked channels caused Figure 4.ߓ Reversible cysteine cross-linking is observed between Q52C-Kir6.2 and E203C-SUR1. Login to comment
159 Representative traces of inside-out patch voltage-clamp recordings from COSm6 cell transfected with Q52C-Kir6.2 and E203C-SUR1. Login to comment
162 (A) Current from Q52C-Kir6.2//E203C-SUR1 channels rapidly decreases to a plateau level in the presence of H2O2 and can be subsequently restored when DTT is applied. Login to comment
163 This pattern of activity suggests that the proximity of Q52C-Kir6.2 and E203C-SUR1 is close enough to allow intersubunit disulfide bond formation. Login to comment
164 (B) Cross-linking between Q52C-Kir6.2 and E203C-SUR1 can also occur from the ATP-bound, closed state as indicated by current decline with H2O2 in the presence of saturating ATP concentrations. Login to comment
166 (C) Cross-linking between Q52C-Kir6.2 and E203C-SUR1 locks channels in a PIP2-insensitive closed state. Login to comment
173 As shown in Fig. 4 C (and Fig. S4), PIP2 failed to recover the activity of cross-linked Q52C-Kir6.2//E203C-SUR1 channels; however, after exposure to DTT, channel activity was readily increased by PIP2. Login to comment
174 These results indicate that cross-linking E203C of SUR1 with Q52C of Kir6.2 induces channel closure and stabilizes the channel in a closed state that cannot be activated by PIP2. Login to comment
177 Occasionally we observed a decrease in current amplitude upon H2O2 exposure that differed from Q52C-Kir6.2//E203C-SUR1 channels in that it followed a much slower time course and was readily reversed upon returning to Kint/EDTA or exposure to PIP2 (Fig. 5 A, right). Login to comment
178 Similar findings were made with WTKir6.2//E203C-SUR1 channels (n = 5 with three patches showing little effect by H2O2 and two patches showing decreased currents that were recovered by PIP2; Fig. 5 B). 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
186 (A-C) Representative traces of inside-out patch voltage-clamp recordings from COSm6 cell transfected with control WT (A), WT-Kir6.2// E203C-SUR1 (B), or Q52C-Kir6.2//WT-SUR1 (C) channels. Login to comment
190 Occasional decreases in current were observed with H2O2 exposure, but always slower and less robust than compared with Q52C-Kir6.2// E203C-SUR1 channels (Fig. 4). Login to comment
289 Second, the Q52C-Kir6.2// E203C-SUR1 cross-linking results provide direct physical and functional evidence for a molecular interaction between SUR1 and Kir6.2 that controls channel activity. Login to comment
300 That cross-linking of E203C-SUR1 and Q52C-Kir6.2 induces channel closure in the absence of ATP and renders channels refractory to PIP2 stimulation demonstrates that closure of the Kir6.2 pore complex can be achieved by simply altering molecular interactions between SUR1 and Kir6.2 without inhibitory ligands. 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
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
39 Close physical proximity of the two residues is further supported by the observation that in inside-out patch-clamp recording of E203C-SUR1// Q52C-Kir6.2 channels, application of the oxidizing reagent H2 O2 to induce disulfide bond formation locked the channels in a closed state that was reversible by the reducing agent dithiothreotol.21 Given this, we considered the possibility that cross-linking of E203C//Q52C may rescue the folding/assembly defect caused by F27S- or A116P-SUR1 by stabilizing the mutant SUR1-Kir6.2 interface at this location. Login to comment
40 We attempted to test this hypothesis by treating cells expressing F27S/E203C//Q52C with H2 O2 . Login to comment