Peter Proks

Abstract:

Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive K(+) channel (K(ATP) channel), are a common cause of neonatal diabetes. We identified a novel KCNJ11 mutation, R50Q, that causes permanent neonatal diabetes (PNDM) without neurological problems. We investigated the functional effects this mutation and another at the same residue (R50P) that led to PNDM in association with developmental delay. Wild-type or mutant Kir6.2/SUR1 channels were examined by heterologous expression in Xenopus oocytes. Both mutations increased resting whole-cell currents through homomeric and heterozygous K(ATP) channels by reducing channel inhibition by ATP, an effect that was larger in the presence of Mg(2+). However the magnitude of the reduction in ATP sensitivity (and the increase in the whole-cell current) was substantially larger for the R50P mutation. This is consistent with the more severe phenotype. Single-R50P channel kinetics (in the absence of ATP) did not differ from wild type, indicating that the mutation primarily affects ATP binding and/or transduction. This supports the idea that R50 lies in the ATP-binding site of Kir6.2. The sulfonylurea tolbutamide blocked heterozygous R50Q (89%) and R50P (84%) channels only slightly less than wild-type channels (98%), suggesting that sulfonylurea therapy may be of benefit for patients with either mutation.

Abstract:

ATP-sensitive K+ (K(ATP)) channels are hetero-octamers of inwardly rectifying K+ channel (Kir6.2) and sulphonylurea receptor subunits (SUR1 in pancreatic beta-cells, SUR2A in heart). Heterozygous gain-of-function mutations in Kir6.2 cause neonatal diabetes, which may be accompanied by epilepsy and developmental delay. However, despite the importance of K(ATP) channels in the heart, patients have no obvious cardiac problems. We examined the effects of adenine nucleotides on K(ATP) channels containing wild-type or mutant (Q52R, R201H) Kir6.2 plus either SUR1 or SUR2A. In the absence of Mg2+, both mutations reduced ATP inhibition of SUR1- and SUR2A-containing channels to similar extents, but when Mg2+ was present ATP blocked mutant channels containing SUR1 much less than SUR2A channels. Mg-nucleotide activation of SUR1, but not SUR2A, channels was markedly increased by the R201H mutation. Both mutations also increased resting whole-cell K(ATP) currents through heterozygous SUR1-containing channels to a greater extent than for heterozygous SUR2A-containing channels. The greater ATP inhibition of mutant Kir6.2/SUR2A than of Kir6.2/SUR1 can explain why gain-of-function Kir6.2 mutations manifest effects in brain and beta-cells but not in the heart.

Abstract:

Type-2, or non-insulin-dependent diabetes mellitus is a serious disease that is now widespread throughout Western society. Glucose intolerance, or failure of glucose to stimulate insulin secretion, is a primary factor in the manifestation of this disease and is likely to be due to the failure of glucose metabolism to stimulate pancreatic beta-cell electrical activity, calcium influx, and insulin secretion. In this review we describe how ion channels regulate the electrical behaviour of the beta-cell and how the membrane potential depolarises in response to a rise in glucose metabolism. Central to these electrical events is the inhibition of ATP-sensitive potassium channel by ATP, and we summarise recent advances in our understanding of the properties of this ion channel in coupling beta-cell metabolism to electrical activity. We discuss the mechanism, specificity, and clinical implications of the pharmacological inhibition of KATP channels by sulphonyureas and other antidiabetic drugs. The roles of other ion channels in regulating electrical activity are considered, and also their potential use as targets for drug action in treating beta-cell disorders.

Abstract:

Recent studies have shown that heterozygous mutations in KCNJ11, which encodes Kir6.2, the pore-forming subunit of the ATP-sensitive potassium (K(ATP)) channel, cause permanent neonatal diabetes either alone (R201C, R201H) or in association with developmental delay, muscle weakness and epilepsy (V59G,V59M). Functional analysis in the absence of Mg2+, to isolate the inhibitory effects of ATP on Kir6.2, showed that both types of mutation reduce channel inhibition by ATP. However, in pancreatic beta-cells, K(ATP) channel activity is governed by the balance between ATP inhibition via Kir6.2 and Mg-nucleotide stimulation mediated by an auxiliary subunit, the sulphonylurea receptor SUR1. We therefore studied the MgATP sensitivity of KCNJ11 mutant K(ATP) channels expressed in Xenopus oocytes. In contrast to wild-type channels, Mg2+ dramatically reduced the ATP sensitivity of heterozygous R201C, R201H, V59M and V59G channels. This effect was predominantly mediated via the nucleotide-binding domains of SUR1 and resulted from an enhanced stimulatory action of MgATP. Our results therefore demonstrate that KCNJ11 mutations increase the current magnitude of heterozygous K(ATP) channels in two ways: by increasing MgATP activation and by decreasing ATP inhibition. They further show that the fraction of unblocked K(ATP) current at physiological MgATP concentrations correlates with the severity of the clinical phenotype.

Abstract:

ATP-sensitive potassium (K(ATP)) channels are found in a wide variety of cell types where they couple cell metabolism to electrical activity. In glucose-sensing tissues, these channels respond to fluctuating changes in blood glucose concentration, but in other tissues they are activated only under ischemic conditions or in response to hormonal stimulation. Although K(ATP) channels in different tissues have different regulatory subunits, in almost all cases (except vascular smooth muscle) the pore-forming subunit is the inwardly rectifying K(+) channel Kir6.2. This article reviews recent studies of Kir6.2, focussing on the relation between channel structure and function, and on naturally occurring mutations in Kir6.2 that lead to human disease. New insights into the location of the ATP-binding site, the permeation pathway for K(+), and the gating of the pore provided by homology modelling are discussed in relation to functional studies. Gain-of-function mutations in Kir6.2 cause permanent neonatal diabetes mellitus (PNDM) by reducing the ATP sensitivity of the K(ATP) channel and increasing the K(ATP) current, which is predicted to inhibit beta-cell electrical activity and insulin secretion. Mutations at specific residues, that cause a greater decrease in ATP sensitivity, are associated with additional neurological symptoms. The molecular mechanism underlying the differences in ATP sensitivity produced by these two classes of mutations is discussed. We speculate on how some mutations lead to neurological disease and why no obvious cardiac symptoms are observed. We also consider the implications of these studies for type-2 diabetes.