Membrane ion channels and diabetes.

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.

A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice.

AIMS/HYPOTHESIS: C57BL/6J mice exhibit impaired glucose tolerance. The aims of this study were to map the genetic loci underlying this phenotype, to further characterise the physiological defects and to identify candidate genes. METHODS: Glucose tolerance was measured in an intraperitoneal glucose tolerance test and genetic determinants mapped in an F2 intercross. Insulin sensitivity was measured by injecting insulin and following glucose disposal from the plasma. To measure beta cell function, insulin secretion and electrophysiological studies were carried out on isolated islets. Candidate genes were investigated by sequencing and quantitative RNA analysis. RESULTS: C57BL/6J mice showed normal insulin sensitivity and impaired insulin secretion. In beta cells, glucose did not stimulate a rise in intracellular calcium and its ability to close KATP channels was impaired. We identified three genetic loci responsible for the impaired glucose tolerance. Nicotinamide nucleotide transhydrogenase (Nnt) lies within one locus and is a nuclear-encoded mitochondrial proton pump. Expression of Nnt is more than sevenfold and fivefold lower respectively in C57BL/6J liver and islets. There is a missense mutation in exon 1 and a multi-exon deletion in the C57BL/6J gene. Glucokinase lies within the Gluchos2 locus and shows reduced enzyme activity in liver. CONCLUSIONS/INTERPRETATION: The C57BL/6J mouse strain exhibits plasma glucose intolerance reminiscent of human type 2 diabetes. Our data suggest a defect in beta cell glucose metabolism that results in reduced electrical activity and insulin secretion. We have identified three loci that are responsible for the inherited impaired plasma glucose tolerance and identified a novel candidate gene for contribution to glucose intolerance through reduced beta cell activity.

Properties of BK(Ca) channels formed by bicistronic expression of hSloalpha and beta1-4 subunits in HEK293 cells.

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels are sensitive to both voltage and internal [Ca(2+)] and are found in many tissues. Their physiological roles range from causing relaxation of smooth muscle to regulating the frequency of action potential firing. There is considerable variation between different tissues in their Ca(2+)- and voltage-dependence. Much of this variation results from the association of the pore-forming alpha subunit (hSloalpha) with different beta subunits leading to altered channel properties. Since hSloalpha alone produces functional BK(Ca) channels, we have used a bicistronic expression method to ensure that both alpha and beta subunits are expressed, with the beta subunit being in excess. Using this method we have investigated the effect of four beta subunits (beta1 to beta4) on cloned BK(Ca) channels. The four beta subunits were individually cloned into a vector that had hSloalpha cDNA inserted downstream of an internal ribosome entry site. The constructs were transiently transfected into HEK293 cells together with a construct that expresses green fluorescent protein, as a marker for transfection. Fluorescent cells expressed BK(Ca) channels whose currents were recorded from inside-out or outside-out patches. The currents we measured using this expression system were similar to those expressed in Xenopus oocytes by Brenner et al. (Brenner, R., Jegla, T.J., Wickenden, A., Liu, Y., Aldrich, R.W. 2000. Cloning and functional expression of novel large-conductance calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4. J. Biol. Chem.275:6453-6461.)

A residue in the intracellular vestibule of the pore is critical for gating and permeation in Ca2+-activated K+ (BKCa) channels.

1. We have used patch clamp to record large-conductance Ca2+-activated K+ (BKCa) currents from a human embryonic kidney cell line (HEK293) expressing wild-type and mutant hSlo channels. 2. When we mutated F380 in the S6 region, thought to contribute to the intracellular vestibule of the pore, to isoleucine (F380I), very little channel activity was recorded. In contrast, mutation to tyrosine (F380Y) resulted in significant voltage-dependent currents. 3. The unitary conductances of F380I, F380Y and wild-type channels were 92 +/- 6 pS (n = 3), 166 +/- 5 pS (n = 3) and 294 +/- 5 pS (n = 5), respectively. 4. Both mutant and wild-type hSlo channels were sensitive to 100 nM iberiotoxin. 5. The F380Y mutant produced channels that were active at negative membrane potentials, even in the absence of Ca2+. 6. We conclude that this conserved residue within BKCa channels may line the conduction pathway and forms a key element of the gating mechanism.

Block of cloned BKCa channels (rSlo) expressed in HEK 293 cells by N-methyl d-glucamine.

We have investigated the conductance properties of large-conductance Ca2+-activated K+ (BKCa) channels formed by stable expression of the rSlo gene in HEK 293 cells. Single-channel recordings were obtained from inside-out patches excised into solution containing 100 microM Ca2+ to ensure a relatively high open probability over the range of membrane potentials studied (-120 to +100 mV). The unitary conductance of these channels at +80 mV was 221.6+/-5.4 pS in symmetrical 140 mM K+. Decreasing the K+ concentration on either side of the membrane, while maintaining ionic strength by adding N-methyl d-glucamine (NMDG+), reduced the unitary conductance. The reduction in conductance was greater when internal K+ was lowered by replacement with NMDG+. However, if sucrose was used as the internal K+ substitute instead of NMDG+ the reduction in unitary conductance was similar to that seen on reducing external K+. A rate-theory model whereby NMDG+ produces a very rapid block of the BKCa channel from the inside, but not the outside, is able to describe our results.