ABCMdb

PMID: 25637631

Devaraneni PK, Martin GM, Olson EM, Zhou Q, Shyng SL
Structurally distinct ligands rescue biogenesis defects of the KATP channel complex via a converging mechanism.
J Biol Chem. 2015 Mar 20;290(12):7980-91. doi: 10.1074/jbc.M114.634576. Epub 2015 Jan 30., [PubMed]

Sentences

No. Mutations Sentence Comment

113 ABCC8 p.Tyr230Ala Point mutations in the proposed site A (S1238Y) or site B (Y230A) (Fig. 2B) render channels less sensitive to GBC block, and combining both mutations completely abolishes channel inhibition by GBC (39-41). Login to comment 116 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala First, we asked whether CBZ rescue of TMD0 trafficking mutants is disrupted by GBC binding mutations, Y230A, S1238Y, or both (Y230A/S1238Y). Login to comment 117 ABCC8 p.Phe27Ser The F27S SUR1-TMD0 mutation previously shown to respond to GBC and CBZ rescue (22) was used as an example. Login to comment 120 ABCC8 p.Phe27Ser By contrast, only the immature band was observed in cells co-expressing F27S-SUR1 and Kir6.2, a defect that was efficiently corrected by GBC and CBZ. Login to comment 135 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala KATP Channel Pharmacological Chaperones MARCH 20, 2015ߦVOLUME 290ߦNUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 7983 expected, the rescue effect of GBC was attenuated by Y230A or S1238Y and abolished by Y230A/S1238Y. Login to comment 136 ABCC8 p.Phe27Ser Remarkably, both single binding mutations alone and the double binding mutation also rendered CBZ ineffective in rescuing F27S (Fig. 2C). Login to comment 141 ABCC8 p.Tyr230Ala As shown in Fig. 2D, Y230A rendered channels less sensitive to CBZ inhibition. Login to comment 143 ABCC8 p.Tyr230Ala Combining Y230A and S1238Y completely prevented CBZ from inhibiting channel activity. Login to comment 147 ABCC8 p.Tyr230Ala B, topology model of SUR1 showing the location of GBC binding site A mutation S1238Y and site B mutation Y230A. Login to comment 148 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser TMD0 trafficking mutations F27S, A116P, and V187D used in the study as well as the ER retention motif RKR are also shown. Login to comment 149 ABCC8 p.Tyr230Ala C, trafficking-defectiveSUR1mutantcontainingTMD0mutationF27SincombinationwithGBCbindingmutationY230A(left),S1238Y(middle),ordoublebinding mutation Y230A/S1238Y (right) was co-expressed with Kir6.2 and subjected to drug treatment (0.1% DMSO vehicle (V), 5 òe;M GBC (G), or 10 òe;M CBZ (C)) overnight (16 h) as indicated. Login to comment 150 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser For comparison, WT control (WT-SUR1 af9; WT-Kir6.2) and F27S in WT-SUR1 background (F27S-SUR1 af9; WT-Kir6.2) with or without drugtreatmentwasincludedineachblot.Tubulinblotsservedasloadingcontrols.Thefilledandopencirclesinthisandsubsequentblotsindicatethecomplex- and core-glycosylated SUR1 proteins, respectively. Login to comment 152 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala COSm6 cells were transfected with Kir6.2 and WT-SUR1, Y230A-SUR1, S1238Y-SUR1, or Y230A/S1238Y-SUR1. Login to comment 157 ABCC8 p.Tyr230Ala Note that the inhibition was not reversible for WT and the Y230A mutant but was reversible for the S1238Y mutant. Login to comment 163 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro For this set of experiments, SUR1-TMD0 trafficking mutations A116P and V187D, which we have shown previously to respond to GBC and CBZ rescue (20, 22, 23, 41, 44), were used as examples. Login to comment 164 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro In SUR1RKR3AAA bearing A116P or V187D expressed without Kir6.2, both exhibited only the core-glycosylated lower band, in contrast to WT-SUR1RKR3AAA, which showed both lower and upper bands; treatment with CBZ failed to correct the mutant SUR1 processing defects (Fig. 3A). Login to comment 167 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Interestingly, we noted that CBZ treatment significantly enhanced the core-glycosylated A116P- and V187D- SUR1RKR3AAA band intensity even in the absence of Kir6.2. Login to comment 168 ABCC8 p.Ala116Pro A likely explanation is that CBZ protects the misfolded SUR1 proteins against ER-associated degradation, which would be consistent with our previous metabolic pulse-chase study showing that GBC also slows down the degradation rate of A116P-SUR1 expressed alone without Kir6.2 (20). Login to comment 176 ABCC8 p.Tyr230Ala The Y230A/S1238Y double mutation in SUR1, which abolished the ability of both drugs to rescue the trafficking defects of TMD0-SUR1 mutations also rendered the drugs unable to correct the processing defect of SUR1 coexpressed with I296L-Kir6.2 (Fig. 3D). Login to comment 189 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser Using F27S-SUR1, we found that whereas processing of the mutant SUR1 in cells co-expressing WT-Kir6.2 was fully normalized by both drugs, as evidenced by the abundant upper band, this was not the case when F27S-SUR1 was co-expressed with èc;N30-Kir6.2, where the upper band in the drug-treated cells was barely detectable (Fig. 4A). Login to comment 216 ABCC8 p.Phe27Ser A, effects of Kir6.2 Nand C-terminal deletions on pharmacological rescue of trafficking-defective SUR1-TMD0 mutant F27S. Login to comment 217 ABCC8 p.Phe27Ser Shown are SUR1 blots of cells expressing F27S-SUR1 along with WT-, èc;N30-, or èc;C25-Kir6.2 and treated overnight with DMSO (0.1%) (V), 5 òe;M GBC (G), or 10 òe;M CBZ (C). Login to comment 219 ABCC8 p.Phe27Ser C, Kir6.2 blots from cells co-expressing WTor F27S-SUR1 and WTor èc;N30-Kir6.2 and treated with DMSO, 5 òe;M GBC, or 10 òe;M CBZ. Login to comment 243 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser D, Kir6.2Y12AzF cross-links to F27S-SUR1 only in cells treated overnight with 5 òe;M GBC to rescue the trafficking defect caused by F27S. Login to comment 249 ABCC8 p.Phe27Ser We then tested whether pharmacological chaperones will induce cross-linking of Kir6.2 to the TMD0-SUR1 trafficking mutant F27S. Login to comment 307 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser Our observation that in the absence of pharmacological chaperones, F27S-SUR1 fails to confer cross-linking suggests that F27S disrupts SUR1-Kir6.2 interactions, perhaps due to misfolding of TMD0. Login to comment 312 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser It is worth noting that we have recently found that substitution of glutamine at the N-terminal amino acid position 52 of Kir6.2 by glutamate or aspartate suppresses the processing defect caused by F27S or A116P mutations in the TMD0 of SUR1 (67), consistent with a model of coupled conformational maturation between SUR1 and Kir6.2. 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