Qing Zhou 1 , Carmit Avnon Ziv 2 , Benjamin Glaser 2 , David H. Zangen 2 , and Show-Ling Shyng 1

1. Identification of a heterozygous sulfonylurea receptor 1 mutation that exerts a strong dominant-negative effect on KATP channel response to MgADP Qing Zhou1, CarmitAvnon Ziv2, Benjamin Glaser2, David H. Zangen2, and Show-Ling Shyng1 1Center for Research on Occupation & Environmental Toxicology Oregon Health & Science University, Portland OR, 97239; 2Division of Pediatric Endocrinology, Department of Pediatrics, Hadassah Hebrew University Medical Center, Jerusalem, Israel 2 5 The 1508AS insertion mutant is expressed at the cell surface under both homozygous and simulated heterozygous condition The dominant negative effect was diminished when the ratio of Mutant:WT SUR1 cDNA that was contransfected COS cells was 1:4 or lower Abstract ATP-sensitive potassium (KATP) channels couple cell metabolism to cell excitability thus mediating a range of physiological responses to metabolic stress. In pancreatic β-cells, KATP channels regulate insulin secretion according to plasma glucose concentrations. Mutations in the channel genes ABCC8 encoding the regulatory sulfonylurea receptor 1 or KCNJ11 encoding the pore-forming inwardly rectifying potassium channel Kir6.2 that lead to loss of channel function are causes of congenital hyperinsulinism, characterized by inappropriate insulin secretion despite severe hypoglycemia. The disease-causing mutations can be recessively inherited, which are usually associated with severe disease phenotype, or dominantly inherited, which are commonly associated with less severe disease phenotype and are clinically responsive to the KATP channel opener diazoxide. The most prominent channel gating defects caused by mutations identified in congenital hyperinsulinism is loss of channel response to the stimulatory effect of MgADP and diazoxide. Here, we have identified a heterozygous in-frame insertion mutation in exon 37 of the ABCC8 gene that results in duplication of two amino acids ala-ser at position 1508 in the second nucleotide binding fold 2 (NBF2) from a patient with severe congenital hyperinsulinism unresponsive to diazoxide. Functional characterization of mutant channels reconstituted in COS cells show that the mutation does not disrupt surface expression of the channel but abolishes channel response to MgADP and diazoxide. Strikingly, in simulated heterozygous expression condition, the mutant SUR1 subunit exhibited a strong dominant negative effect on WT SUR1 subunit. This clinical and in-vitro strong dominant negative effect is distinct from other heterozygous mutations reported previously present an interesting case for understanding the structural mechanisms underlying channel response to MgADP and diazoxide. A B A B A Western Blots of WT or mutant SUR1 co-expressed with Kir6.2 in COSm6. The lower band is the core-glycosylated form and upper band is the complex-glycosylated form. B Cell surface expression quantified using chmiluminescence assays (n=3). Expressions of the mutant and WT:mutant (1:1) heterozygous are normalized to that of WT. 3 The 1508AS insertion mutant expressed in COS cells showed no activity upon metabolic inhibition in Rb+ efflux assay and has no response to MgADP stimulation and greatly reduced response to diazoxide A Schematic of the distribution of the different channel types expected in homomeric and heteromeric states. The red and black circles show mutant and WT SUR1, respectively. BAveraged response of channels to MgADP stimulation.Inside-out voltage clamps were excised from COSm6 cells transfected with different ratio of WT and mutant cDNAs. we set up this experiment to see at which point the mutant starts to lose it's dominant negative effect. This data might help correlate with the mRNA expression data which obtained in patients who are carriers of the mutation but do not show symptoms. A B A COSm6 cells expressing WT or mutant channels were subjected to 86Rb+ efflux assay. B Representative inside-out patch clamp recordings showing MgADP and diazoxide responses in WT and mutant channels. Future work We will design a couple of tandem constructs that can express KATP channels whose subunit compositions of WT and mutant are predetermined. The MgADP and diazoxide stimulation experments will be performed by using these constructs to 1 4 The 1508AS insertion mutation exerts a strong dominant-negative effect over WT SUR1 function in simulated heterozygous expression condition A B transfect COSm6 cells and excise inside-out patchs. The red, black and white circles show mutant, WT and Kir6.2, respectively. Inter-subunit linkers are indicated by – (amino acid sequence is QQQQQXXQQQQQ) Conclusions A Representative inside-out voltage clamp current traces showing homomeric WT and heteromeric (WT+mutant) channels response to MgADP and diazoxide. BAveraged response of channels to MgADP stimulation. The current in 0.1 mmol/l ATP/0.5 mmol/l ADP or 0.1 mmol/l ATP/0.3 mmol/l diazoxide was expressed as • AS 1508 insertion mutation is an SUR1 mutation, which has dominant negative effect and can cause severe congenital hyperinsulinism unresponsive to diazoxidein homozygous and heterozygous states. • The mutation does not disrupt surface expression of the channel but abolishes channel response to MgADP and diazoxide. • In simulated heterozygous expression condition, the mutant SUR1 subunit exhibited a strong dominant negative effect on WT SUR1 subunit. percentage of those observed in K-INT solution. The values of both WT:mutant and mutant alone are significantly lower than that of WT (P <0.001). This research was funded by: NIH R01-DK057699 and R01-DK066485 to SLS