PROJECT SUMMARY The study of human islets isolated from the pancreas of infants with congenital hyperinsulinism (HI) during the previous funding cycle has afforded us the unique opportunity to examine the islet phenotype in KATPHI integrating function, metabolomics, and genomics. Our findings revealed a complex pathophysiology in which the primary KATP channel defect leads to secondary consequences affecting gene expression, fuel metabolism, and both the triggering and amplifying pathways of insulin secretion. However, many critical questions for addressing unmet needs for the treatment of HI and for the understanding of normal physiological mechanisms of insulin secretion remain unanswered. We are particularly interested in examining the role of two ion channels that are differentially expressed in HI islets in the normal regulation of insulin secretion and their potential role in the pathophysiology of HI: TMEM16A, a Ca2+-activated Cl– channel encoded by ANO1 which is markedly upregulated in KATPHI islets, and Kv7.1, encoded by KCNQ1, whose expression is markedly decreased in islets isolated from the pancreases of children with Beckwith Wiedemann syndrome and HI. In preliminary studies we found that pharmacological modulation of these channels alters insulin secretion. Our overall hypothesis is that both Kv7.1 and TMEM16A play critical roles in the termination of insulin secretion by keeping β-cell Vm hyperpolarized at rest and facilitating β-cell Vm repolarization after stimulation. To test this hypothesis, we propose two aims to examine the role of Kv7.1 and TMEM16A in the regulation of insulin secretion in normal and HI islets. To accomplish these aims we will use genetic and pharmacological approaches to modulate the activity of these channels in normal and HI human and mouse islets. We will examine: 1) the contribution of TMEM16A and Kv7.1 to β-cell Vm at resting and stimulated states; 2) the effect of TMEM16A and Kv7.1 activation and inhibition on cytosolic calcium and insulin secretion in normal human and mouse islets; 3) the effect of genetic inactivation of TMEM16A and Kv7.1 on glucose homeostasis in vivo and fuel-stimulated insulin secretion in vivo and in isolated islets using genetically modified mouse models. This study will expand our understanding of the pathophysiology of HI and will facilitate the identification of new genetic causes and potential new therapeutic targets for this devastating disease. The study may also have implications for the understanding of the mechanisms implicated in the progressive β-cell failure that leads to type 2 diabetes.