Identification and pharmacological correction of a membrane trafficking defect associated with a mutation in the sulfonylurea receptor causing familial hyperinsulinism

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a genetic dis order characterized by excess secretion of insulin and hypoglycemia. In mos t patients, the disease is caused by mutations in sulfonylurea receptor-1 ( SUR1), which, in association with Kir6.2, constitutes the functional ATP-se nsitive potassium (K-ATP) channel of the pancreatic beta -cell. Previous st udies reported that coexpression of the PHHI mutant R1394H-SUR1 with Kir6.2 in COS cells produces no functional channels. To investigate if the loss o f function could be due to impaired trafficking of mutant channels to the c ell membrane, we have cotransfected wild-type and mutant SUR1 subunits with Kir6.2 into HEK293 cells and examined their cellular localization by immun ofluorescent staining. Our results show that unlike the wildtype subunits, which showed fluorescence at the cell surface, the mutant subunits displaye d fluorescence in punctate structures. Co-immunostaining with antibodies ag ainst organelle-specific marker proteins identified these structures as the trans-Golgi network. Limited localization in clathrin-positive, but transf errin receptor-negative vesicles was also observed. The post-endoplasmic re ticulum localization suggests that the mutation does not impair the folding and assembly of the channels so as to cause its retention by the endoplasm ic reticulum. Diazoxide, a KATP channel opener drug that is used in the tre atment of PHHI, restored the surface expression in a manner that could be p revented by the channel blocker glibenclamide. When expressed in Xenopus oo cytes, R1394H-SUR1 formed functional channels with Kir6.2, indicating that the primary consequence of the mutation is impairment of trafficking rather than function. Thus, our data uncover a novel mechanism underlying the the rapeutic action of diazoxide in the treatment of PHHI, i.e. its ability to recruit channels to the membrane. Furthermore, this is the first report to describe a trafficking disorder effecting retention of mutant proteins in t he trans-Golgi network.