LQT1-associated mutations increase KCNQ1 proteasomal degradation independently of Derlin-1.
Mutations in the potassium channel KCNQ1 that determine retention of the mutated proteins in the endoplasmic reticulum (ER) are associated with the autosomal dominant negative Romano-Ward LQT1 cardiac syndrome. In the present study, we have analyzed the consequences and the potential molecular mechanisms involved in the ER retention of three Romano-Ward mutations located in KCNQ1 N terminus (Y111C, L114P, and P117L). We showed that the mutant KCNQ1 proteins exhibited reduced expression levels with respect to wild-type (WT)-KCNQ1. Radiolabeling pulse-chase experiments revealed that the lower expression levels did not result from reduced rate of synthesis. Instead, using a combination of Western blot and pulse-chase experiments, we showed that the mutant channel Y111C-KCNQ1, used as a model, was ubiquitinated and degraded in the proteasome more rapidly (t((1/2)) = 82 min) than WT-KCNQ1 channel (t((1/2)) = 113 min). On the other hand, KCNQ1 degradation did not appear to involve the GTP-dependent pathway. We also showed that KCNE1 stabilized both wild-type and Y111C proteins. To identify potential actors involved in KCNQ1 degradation, we studied the implication of the ER-resident protein Derlin-1 in KCNQ1 degradation. We showed that although KCNQ1 and Derlin-1 share the same molecular complex and co-immunoprecipitate when co-expressed in HEK293FT cells, Derlin-1 did not affect KCNQ1 steady state expression and degradation. These data were confirmed in T84 cells that express endogenous KCNQ1 and Derlin-1. Small interfering RNA knock-down of Derlin-1 did not modify KCNQ1 expression level, and no interaction between endogenous KCNQ1 and Derlin-1 could be detected.
Peroz
David
D
INSERM, UMR915, Nantes, France.
Dahimène
Shehrazade
S
Baró
Isabelle
I
Loussouarn
Gildas
G
Mérot
Jean
J
D019943:Amino Acid Substitution; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D051657:KCNQ1 Potassium Channel / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D029597:Romano-Ward Syndrome / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000235:genetics
C487709:DERL1 protein, human; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D006160:Guanosine Triphosphate; D046988:Proteasome Endopeptidase Complex