Familial hyperinsulinism (HI) is a genetic disorder characterised by unregulated insulin secretion leading to hypoglycaemia. Mutations in either of the pancreatic ATP-sensitive potassium (KATP) channel-forming subunits, Kir6.2 or SUR1, can cause HI either by disrupting normal channel function or by interfering with membrane trafficking (Partridge et al. 2001; Huopio et al. 2002). The L147P mutation in the pore-forming Kir6.2 subunit produces a severe form of the disease (Thomas et al. 1996). Here we investigate the consequences of this mutation on the function and trafficking of Kir6.2.
The function of KATP channels was examined by two-electrode voltage clamp analysis of Xenopus oocytes injected with cRNA encoding the wild-type (WT) or L147P mutant Kir6.2 subunits together with SUR1.
Contrary to the WT channels, L147P-containing channels showed no currents in response to diazoxide and azide stimulation (n = 7), suggesting that L147P mutation destroys the channel function. To investigate if the loss of function is due to impaired trafficking to the cell surface, we have inserted a haemagglutinin A (HA) epitope into an extracellular loop of Kir6.2. The resultant construct was transfected into COS-7 cells together with SUR1, and the surface expression examined by immunocytochemistry (by staining for the HA epitope) and confocal microscopy. The results showed that the mutant channel was able to traffic to the cell surface. However, when the cells were permeabilised, unlike the WT channel, the mutant channels were distributed in punctate structures. To investigate the nature of the punctate structures COS-7 cells were co-transfected with the WT and mutant KATP channels together with GFP-tagged markers for various endocytic compartments. The cells were permeabilised and stained for Kir6.2 and examined by confocal microscopy. The data showed almost complete co-localisation of L147P Kir6.2 with GFP-Rab7, a marker of late endosomes/lysosomes. Such co-localisation was not apparent with the WT Kir6.2 containing channels.
In conclusion, these data suggest that the L147P mutation does not impair the ability of the channel to traffic to the cell surface. However, the mutation appears to alter the structure of the channel such that it not only impairs the channel function, but misdirects the channel to a late endosomal/lysosomal compartment. These effects explain why L147P mutation causes severe form of HI.
This work was funded by MRC and Emma and Leslie Reid Scholarship to AJS.