We have employed an amino derivative of the imidazoline ligand, efaroxan, to isolate imidazoline binding proteins from solubilised extracts of rat brain, by affinity chromatography. A number of proteins were specifically retained on the affinity column and one of these was immunoreactive with an antiserum raised against the ion conducting pore component of the ATP-sensitive potassium channel. Patch clamp experiments confirmed that, like its parent compound, amino-efaroxan blocks ATP-sensitive potassium channels in human pancreatic L Lcells and can stimulate the insulin secretion from these cells. The results reveal that a member of the ion conducting pore component family is strongly associated with imidazoline binding proteins in brain and in the endocrine pancreas.z 1999 Federation of European Biochemical Societies.
ATP-sensitive K؉ channels (K ATP channels) couple -cell metabolism to electrical activity and thereby play an essential role in the control of insulin secretion. Gain-of-function mutations in Kir6.2 (KCNJ11), the pore-forming subunit of this channel, cause neonatal diabetes. We investigated the effect of the most common neonatal diabetes mutation (R201H) on -cell electrical activity and insulin secretion by stable transfection in the INS-1 cell line. Expression was regulated by placing the gene under the control of a tetracycline promoter. Transfection with wildtype Kir6.2 had no effect on the ATP sensitivity of the K ATP channel, whole-cell K ATP current magnitude, or insulin secretion. However, induction of Kir6.2-R201H expression strongly reduced K ATP channel ATP sensitivity (the halfmaximal inhibitory concentration increased from ϳ20 mol/l to ϳ2 mmol/l), and the metabolic substrate methyl succinate failed to close K ATP channels or stimulate electrical activity and insulin secretion. Thus, these results directly demonstrate that Kir6.2 mutations prevent electrical activity and insulin release from INS-1 cells by increasing the K ATP current and hyperpolarizing the -cell membrane. This is consistent with the ability of the R201H mutation to cause neonatal diabetes in patients. The relationship between K ATP current and the membrane potential reveals that very small changes in current amplitude are sufficient to prevent hormone secretion. Diabetes 55: [3075][3076][3077][3078][3079][3080][3081][3082] 2006 N eonatal diabetes is a rare inherited form of diabetes that manifests within the first 6 months of life (1,2). Approximately 50% of cases of neonatal diabetes result from heterozygous mutations in KCNJ11, the gene encoding Kir6.2, which constitutes the pore-forming subunit of the ATPsensitive K ϩ channel (K ATP channel) (1-11). Functional studies of these mutations in heterologous systems have shown that they result in a reduction in the ability of ATP to close the K ATP channel (10 -16).In pancreatic -cells, a proportion of K ATP channels is open at substimulatory glucose concentrations (17). The resulting K ϩ efflux holds the -cell membrane at a hyperpolarized potential, preventing electrical activity and insulin secretion (18). Elevation of the plasma glucose concentration stimulates glucose uptake and metabolism by the -cell, causing an increase in ATP and an accompanying decrease in MgADP. These changes in adenine nucleotide concentrations close K ATP channels and, as a consequence, elicit membrane depolarization, opening of voltage-gated Ca 2ϩ channels, and Ca 2ϩ -dependent electrical activity. This leads to increased Ca 2ϩ influx and insulin release (18). Mutations that render the K ATP channel less sensitive to inhibition by ATP are therefore predicted to prevent -cell depolarization, electrical activity, and insulin secretion in response to increased oxidative metabolism. The observation that a glucose challenge fails to elicit a rise in plasma C-peptide or insulin in patients carrying neo...
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