Peter Proks

Abstract:

OBJECTIVE: Activating mutations in the KCNJ11 and ABCC8 genes encoding the Kir6.2 and SUR1 subunits of the pancreatic ATP-sensitive K(+) channel are the most common cause of permanent neonatal diabetes. In contrast to KCNJ11, where only dominant heterozygous mutations have been identified, recessively acting ABCC8 mutations have recently been found in some patients with neonatal diabetes. These genes are co-located on chromosome 11p15.1, centromeric to the imprinted Beckwith-Wiedemann syndrome (BWS) locus at 11p15.5. We investigated a male with hemihypertrophy, a condition classically associated with neonatal hyperinsulinemia and hypoglycemia, who developed neonatal diabetes at age 5 weeks. RESEARCH DESIGN AND METHODS: The KCNJ11 and ABCC8 genes and microsatellite markers on chromosome 11 were analyzed in DNA samples from the patient and his parents. RESULTS: A paternally inherited activating mutation (N72S) in the ABCC8 gene was identified in the proband. The mutation was present at 70% in the patient's leukocytes and 50% in buccal cells. Microsatellite analysis demonstrated mosaic segmental paternal uniparental isodisomy (UPD) of 11pter-11p14 in the proband that encompassed the ABCC8 gene and the BWS locus. CONCLUSIONS: We report a patient with neonatal diabetes, hemihypertrophy, and relatively high birth weight resulting from telomeric segmental paternal UPD of chromosome 11, which unmasks a recessively acting gain-of-function mutation in the ABCC8 gene and causes deregulation of imprinted genes at the BWS locus on 11p15.5.

Abstract:

Oocytes from the Xenopus laevis represent one of the most widely used expression systems for functional characterization of ion channels. Their large size facilitates both injection of heterologous cRNA and subsequent electrophysiological recordings of ion channel currents. Furthermore, Xenopus oocytes translate cRNA very efficiently, resulting in the generation of a large number of ion channels in the plasma membrane. In this chapter, we outline methods for oocyte preparation and maintenance and describe procedures for patch-clamping of oocytes, with a special focus on the macropatch technique. We discuss some common problems associated with patch-clamping of oocytes and their use as an expression system for ion channels.

Abstract:

Activating mutations in the genes encoding the ATP-sensitive potassium (K(ATP)) channel subunits Kir6.2 and SUR1 are a common cause of neonatal diabetes. Here, we analyse the molecular mechanism of action of the heterozygous mutation F132L, which lies in the first set of transmembrane helices (TMD0) of SUR1. This mutation causes severe developmental delay, epilepsy and permanent neonatal diabetes (DEND syndrome). We show that the F132L mutation reduces the ATP sensitivity of K(ATP) channels indirectly, by altering the intrinsic gating of the channel. Thus, the open probability is markedly increased when Kir6.2 is co-expressed with mutant TMD0 alone or with mutant SUR1. The F132L mutation disrupts the physical interaction between Kir6.2 and TMD0, but does not alter the plasmalemma channel density. Our results explain how a mutation in an accessory subunit can produce enhanced activity of the K(ATP) channel pore (formed by Kir6.2). They also provide further evidence that interactions between TMD0 of SUR1 and Kir6.2 are critical for K(ATP) channel gating and identify a residue crucial for this interaction at both physical and functional levels.

Abstract:

Heterozygous activating mutations in the KCNJ11 gene encoding the pore-forming Kir6.2 subunit of the pancreatic beta cell K(ATP) channel are the most common cause of permanent neonatal diabetes (PNDM). Patients with PNDM due to a heterozygous activating mutation in the ABCC8 gene encoding the SUR1 regulatory subunit of the K(ATP) channel have recently been reported. We studied a cohort of 59 patients with permanent diabetes who received a diagnosis before 6 mo of age and who did not have a KCNJ11 mutation. ABCC8 gene mutations were identified in 16 of 59 patients and included 8 patients with heterozygous de novo mutations. A recessive mode of inheritance was observed in eight patients with homozygous, mosaic, or compound heterozygous mutations. Functional studies of selected mutations showed a reduced response to ATP consistent with an activating mutation that results in reduced insulin secretion. A novel mutational mechanism was observed in which a heterozygous activating mutation resulted in PNDM only when a second, loss-of-function mutation was also present.