ABCMdb

PMID: 22311976

Wang F, Olson EM, Shyng SL
Role of Derlin-1 protein in proteostasis regulation of ATP-sensitive potassium channels.
J Biol Chem. 2012 Mar 23;287(13):10482-93. Epub 2012 Feb 6., [PubMed]

Sentences

No. Mutations Sentence Comment

119 ABCC8 p.Ala116Pro A116P- and F1388-SUR1 are mutations identified from patients with congenital hyperinsulinism (13,36). Login to comment 121 ABCC8 p.Ala116Pro We infected INS-1 cells with adenoviruses carrying Kir6.2 and A116P or F1388 f-SUR1 and performed co-immunoprecipitation experiments using FLAG-antibody agarose beads. Login to comment 123 ABCC8 p.Ala116Pro D, INS-1 cells infected with recombinant adenoviruses to express exogenous WT f-SUR1 alone (lane 2), WT f-SUR1 and Kir6.2 (lane 3), mutant A116P f-SUR1 and Kir6.2 (lane 4), or mutant èc;F1388 f-SUR1 and Kir6.2 (lane 5). Login to comment 136 ABCC8 p.Ala116Pro A116P- and èc;F1388-SUR1 are mutations identified from patients with congenital hyperinsulinism (13, 36). Login to comment 138 ABCC8 p.Ala116Pro We infected INS-1 cells with adenoviruses carrying Kir6.2 and A116P or èc;F1388 f-SUR1 and performed co-immunoprecipitation experiments using FLAG antibody-agarose beads. Login to comment 171 ABCC8 p.Ala116Pro ABCC8 p.Cys26Ser We therefore studied three SUR1 mutations, C26S, A116P and F1388, predicted to have folding defects in the extracellular (ER-luminal), transmembrane and cytosolic domains based on current topology model of SUR1 (Supplemental Figure 3) (43). Login to comment 175 ABCC8 p.Ala116Pro In the A116P mutant, knockdown of Derlin-1 even led to appearance of the mature upper band. Login to comment 176 ABCC8 p.Ala116Pro Surface biotinylation experiments further confirmed expression of the A116P mutant channel in the plasma membrane (Figure 6C, D). Login to comment 188 ABCC8 p.Ala116Pro ABCC8 p.Cys26Ser We therefore studied three SUR1 mutations, C26S, A116P, and èc;F1388, predicted to have folding defects in the extracellular (ER-luminal), transmembrane and cytosolic domains based on the current topology model of SUR1 (supplemental Fig. 3) (43). Login to comment 190 ABCC8 p.Ala116Pro The amount of p97 detected in the immunoprecipitates was much higher for truncated SUR1 that had significantly reduced protein levels (a.a. 1-1022 and a.a. 1-607), reminiscent of that observed for the misfolding mutant A116P and F1388 (Figure 1D). Login to comment 227 ABCC8 p.Ala116Pro ABCC8 p.Cys26Ser A, HEK293 cells were transfected with Kir6.2 and WT, C26S, A116P, or èc;F1388 SUR1 along with Derlin-1 shRNA or the scramble control. Login to comment 228 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Cys26Ser Curiously, while a clear increase in the immature SUR1 was observed in all three mutants, only A116P was able to exit the ER and reach the cell surface upon Derlin-1 knockdown, despite that A116P had overall lower protein levels than C26S and F1388. Login to comment 229 ABCC8 p.Ala116Pro Knockdown of Derlin-1 increased the intensity of the core-glycosylated SUR1 in all cases and also the complex-glycosylated SUR1 in WT and A116P. Login to comment 231 ABCC8 p.Ala116Pro C, surface biotinylation was performed in HEK293 cells transfected with A116P SUR1 and Kir6.2 together with Derlin-1 shRNA or the scramble plasmid. Login to comment 250 ABCC8 p.Ala116Pro In the A116P mutant, knockdown of Derlin-1 even led to the appearance of the mature upper band. Login to comment 251 ABCC8 p.Ala116Pro Surface biotinylation experiments further confirmed expression of the A116P mutant channel in the plasma membrane (Fig. 6, C and D). Login to comment 264 ABCC8 p.Ala116Pro The amount of p97 detected in the immunoprecipitates was much higher for truncated SUR1 that had significantly reduced protein levels (aa 1-1022 and aa 1-607), reminiscent of that observed for the misfolding mutant A116P and èc;F1388 (Fig. 1D). Login to comment 317 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Cys26Ser Curiously, although a clear increase in the immature SUR1 was observed in all three mutants, only A116P was able to exit the ER and reach the cell surface upon Derlin-1 knockdown, despite the fact that A116P had overall lower protein levels than C26S and èc;F1388. Login to comment