Grant: $418,750 - National Institutes of Health - Aug. 17, 2009
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Award Description: The long-term objective of this program is to understand how mutations in the ABCC8 gene, that encodes the regulatory subunit of the neuroendocrine-type ATP-sensitive potassium (KATP) channel, cause Neonatal Diabetes (ND). The neuroendocrine-type KATP channels couple glucose-dependent metabolism with the excitability of pancreatic beta-cells and certain neurons, thus controlling insulin secretion and glucose homeostasis. These channels are complexes of four pore-forming Kir6.2 subunits, encoded by the KCNJ11 gene, and four regulatory SUR1 subunits, encoded by the ABCC8 gene. The probability of a KATP channel being open (Po) is determined by a balance of the Mg-dependent stimulatory action of nucleotides interacting with the nucleotide-binding domains (NBDs) of the ATP-Binding Cassette protein SUR1 and the Mg-independent inhibitory action of ATP binding to the KATP pore. ABCC8 mutations abolishing the Mg-ADP/ATP-dependent stimulatory action of SUR1 on the ATP-inhibited Kir6.2 have been discovered in infants with hyperinsulinemic hypoglycemia that oversecrete insulin, implying that gain-of-Po mutations in ABCC8 could be diabetogenic. In collaboration with an international network for the study of ND for genetic diagnosis, we discovered multiple novel mutations in ABCC8 in patients with Transient and Permanent ND, including ND with neurological abnormalities. The genetic evidence for the causal role of these and more recently identified ND-ABCC8 mutations is compelling. ND-ABCC8 mutations map to SUR1 domains which may regulate the Po. Our detailed analysis of several ND-SUR1/Kir6.2 channels indicates they have increased on-cell activity which can hyperpolarize pancreatic beta-cells. These mutant channels show a markedly elevated Po when exposed to physiologic Mg-nucleotides in isolated membrane fragments. We hypothesized that the increase in the basal Po in intact cells is a common diabetogenic effect of ND-ABCC8 mutations. We propose to determine the principal mechanisms by which ND-SUR1 significantly increase the Po of KATP channels under physiologic Mg-nucleotide conditions. We have demonstrated previously that SUR1 subunits have three regulatory effects on the KATP pore: Beyond stimulating the pore in the presence of MgATP/ADP, SUR1 controls the maximal Po in the absence of ligands, and increases the apparent affinity of Kir6.2 for inhibitory ATP. Therefore we hypothesize there are three principal mechanisms by which NDSUR1 can hyperactive the channel: 1) an increase in the Mg-nucleotide dependent stimulatory action of SUR1, 2) an increase in the maximal nucleotide-independent Po, and 3) a decrease in the sensitivity of the channel to inhibitory ATP with no change in ligand-independent gating kinetics. We will test our hypotheses using the patch-clamp method, single-channel kinetics analysis, affinity photolabeling, and structural modeling. The specific aims of the revised application are to complete the verification that our ND-SUR1 significantly increase the Po of KATP channels in intact mammalian cells, and to reveal principal biophysical mechanisms of KATP hyperactivation by ND-SUR1 subunits. Public Health Relevance Statement: As the pandemic of diabetes continues one in three children born in 2000 will be diagnosed with diabetes in their lifetime. The difficulty of treating this disorder stems in part from our insufficient understanding of the biophysical mechanisms of key regulators of insulin secretion. The proposed studies of sulfonylurea receptors and KATP channels will deepen our understanding of diabetogenic mechanisms of metabolism-excitation uncoupling to allow better treatment of neonatal diabetes and other KATP channelopathies.
Project Description: Over the last several months we progressed toward both specific aims of the revised DK77827-01A2: i) to complete the verification that Neonatal Diabetes (ND) mutations in ABCC8/KCNJ11 channels increase the mean open probability (Po) in mammalian cells, and ii) to reveal the biophysical mechanisms of KATP hyperactivation. We focused on severe ND mutations which map to domains hypothesized to control the maximal Po. Dr. Babenko has processed and analyzed nearly all records of macroscopic and single-channel currents through severe ND-SUR1/Kir6.2 channels. Initial findings have been reported (Abstract from Biophys J 2009. 94(1), on-line): ?How do mutations in the L0-linker of ABCC8 produce neonatal diabetes?? Andrey P. Babenko. Numerous mutations in ABCC8 (SUR1), the neuroendocrine-type regulatory subunit of KATP channels, cause Neonatal Diabetes (ND). Many of these mutations compromising insulin release cluster around the first ND mutation identified in the SUR1 L0-linker, L213R. Here we show the ND-SUR1 increased the on-cell open channel probability, Po, not the density of Kir6.2-based channels. Single-channel kinetics analyses demonstrate that the increase in the Po is due to a ligand-independent stabilization of the active (burst) state. A similar diabetogenic effect was reproduced by deletions in the Kir6.2 N-terminus that partners with the L0-linker controlling the slow(inter-burst) gating transitions. These findings illustrate the physiologic significance of our original model of SUR1/Kir6.2 coupling where the L0-linker plays a key role in controlling the maximal Po. We started similar analysis to test that ND mutations in the M0 helix produce diabetes through a similar mechanism. The PI processed ~50% of records of macroscopic and unitary currents through V59X mutants, including those reported to cause ND with epilepsy and developmental delay. Our next goal is to complete this analysis before the 54th Annual Biophysical Meeting.
Jobs Summary: These funds allowed to partly compensate Dr. Babenko, M.D., Ph.D. for his work on this project and retain this nationally acclaimed scientist at PNDRI as its Principal Scientist-PI. (Total jobs reported: 1)
Project Status: Less Than 50% Completed
This award's data was last updated on Aug. 17, 2009. Help expand these official descriptions using the wiki below.