ABCMdb

PMID: 23695995

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., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC8 p.Glu203Lys 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 ABCC8 p.Glu203Lys ABCC8 p.Glu203Lys 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 ABCC8 p.Glu203Lys ABCC8 p.Glu203Lys 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 ABCC8 p.Ala116Pro ABCC8 p.Glu128Lys ABCC8 p.Phe27Ser ABCC8 p.Phe686Ser ABCC8 p.Arg495Gln ABCC8 p.Glu203Lys ABCC8 p.Glu203Lys ABCC8 p.Glu203Lys 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 ABCC8 p.Glu203Lys 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 21 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser Positions of SUR1-E203 and Kir6.2-Q52 residues (open squares) as well as the two TMD0 trafficking mutations F27S and A116P (open circles) are indicated. Login to comment 25 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser ABCC8 p.Glu203Lys E203K//Q52E mutation pair in correcting the processing defect of F27S and A116P. Login to comment 26 ABCC8 p.Phe27Ser ABCC8 p.Glu203Lys ABCC8 p.Glu203Lys ABCC8 p.Glu203Cys 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 ABCC8 p.Glu203Lys ABCC8 p.Glu203Cys 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 30 ABCC8 p.Phe27Ser Although the precise mechanism underlying our findings remains to be determined, the charge-dependence of the effect of Q52-Kir6.2 mutation on F27S-SUR1 processing leads us to speculate that the negative charge at this position may not shown). Login to comment 31 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser ABCC8 p.Glu203Lys ABCC8 p.Glu203Lys 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 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser ABCC8 p.Glu203Lys 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 ABCC8 p.Glu203Lys 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 ABCC8 p.Phe27Ser ABCC8 p.Glu203Lys 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 ABCC8 p.Glu203Lys 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 ABCC8 p.Glu203Lys 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 39 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser ABCC8 p.Glu203Cys ABCC8 p.Glu203Cys 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 ABCC8 p.Phe27Ser ABCC8 p.Glu203Cys We attempted to test this hypothesis by treating cells expressing F27S/E203C//Q52C with H2 O2 . Login to comment 41 ABCC8 p.Phe27Ser While we were able to observe a crosslinked SUR1-Kir6.2 species on immunoblots within 10 min of H2 O2 exposure, no significant correction of the F27S processing defect was detected even after 30 min or overnight H2 O2 exposure (data not shown). Login to comment 44 ABCC8 p.Phe27Ser ABCC8 p.Glu203Lys Next, we tested the role of individual E203K-SUR1 or Q52E-Kir6.2 mutations in F27S-SUR1 mutant processing. Login to comment 45 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser ABCC8 p.Glu203Lys ABCC8 p.Glu203Lys 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 46 ABCC8 p.Phe27Ser Moreover, we found that while Q52D-Kir6.2 similarly improved the processing and surface expression of F27S-SUR1, Q52K-Kir6.2 did not (Fig. 3B). Login to comment 47 ABCC8 p.Ala116Pro Similar observations were made for the A116P mutation (data Figure 2. Login to comment 48 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser ABCC8 p.Glu203Lys The E203K//Q52E mutation pair suppresses the processing defect caused by the F27S or A116P SUR1 mutation. Login to comment