Abstract
ATP-sensitive K+ (KATP) channels composed of potassium inward-rectifier type 6.2 and sulfonylurea receptor type 1 subunits (Kir6.2/SUR1)4 are expressed in various cells in the brain and endocrine pancreas where they couple metabolic status to membrane potential. In β-cells, increases in cytosolic [ATP/ADP]c inhibit KATP channel activity, leading to membrane depolarization and exocytosis of insulin granules. Mutations in ABCC8 (SUR1) or KCNJ11 (Kir6.2) can result in gain or loss of channel activity and cause neonatal diabetes (ND) or congenital hyperinsulinism (CHI), respectively. SUR1 is reported to be a Mg2+-dependent ATPase. A prevailing model posits that ATP hydrolysis at SUR1 is required to stimulate openings of the pore. However, recent work shows nucleotide binding, without hydrolysis, is sufficient to switch SUR1 to stimulatory conformations. The actions of nucleotides, ATP and ADP, on ND (SUR1E1506D) and CHI (SUR1E1506K) mutants, without Kir6.2, were compared to assess both models. Both substitutions significantly impair hydrolysis in SUR1 homologs. SUR1E1506D has greater affinity for MgATP than wildtype; SUR1E1506K has reduced affinity. Without Mg2+, SUR1E1506K has a greater affinity for ATP4− consistent with electrostatic attraction between ATP4−, unshielded by Mg2+, and the basic lysine. Further analysis of ND and CHI ABCC8 mutants in the second transmembrane and nucleotide-binding domains (TMD2 and NBD2) found a relation between their affinities for ATP (±Mg2+) and their clinical phenotype. Increased affinity for ATP is associated with ND; decreased affinity with CHI. In contrast, MgADP showed a weaker relationship. Diazoxide, known to reduce insulin release in some CHI cases, potentiates switching of CHI mutants from non-stimulatory to stimulatory states consistent with diazoxide stabilizing a nucleotide-bound conformation. The results emphasize the greater importance of nucleotide binding vs. hydrolysis in the regulation of KATP channels in vivo.
Highlights
Neuroendocrine ATP-sensitive K+ channels, (SUR1/Kir6.2)4, couple cell metabolism with K+ efflux, and membrane potential, in various neurons and endocrine cells including pancreatic islet α, β, and δ-cells whose secretory hormones maintain glucose homeostasis
SPECIFIC MUTATIONS HAVE LITTLE TO NO EFFECT ON THE AFFINITY OF INWARD FACING, NUCLEOTIDE-FREE CONFORMATIONS OF SUR1 FOR [3H]GBC To assess the effect of the ABCC8/SUR1 mutations used in this study on GBC binding the dissociation constants, KG, were determined in saturation binding assays in the absence of nucleotides www.frontiersin.org and Mg2+
The main finding of this study is the direct correlation between the apparent affinities of mutant SURs for MgATP and their associated clinical phenotype, i.e., neonatal diabetes mellitus (ND) vs. congenital hyperinsulinism (CHI)
Summary
Neuroendocrine ATP-sensitive K+ channels, (SUR1/Kir6.2), couple cell metabolism with K+ efflux, and membrane potential, in various neurons and endocrine cells including pancreatic islet α, β-, and δ-cells whose secretory hormones maintain glucose homeostasis. These channels are regulated, in part, by the ATP/ADP ratio. Both nucleotides bind the Kir6.2 pore to reduce the open channel probability (Po), while Mg-nucleotide interactions with SUR1 antagonize this effect. In pancreatic β-cells, increased glucose metabolism and the subsequent rise in [ATP/ADP]C reduces KATP channel activity leading to downstream insulin release. CHI can be due to mutations that disturb trafficking and/or assembly reducing the number of functional channels at the plasma membrane [1,2,3]
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