Project Summary The training plan outlined in this proposal focuses on elucidating the molecular mechanisms by which the two-pore domain K+ channel TALK-1 modulates β-cell function and contributes to β-cell failure during the pathogenesis of diabetes. TALK-1 is the most highly expressed K+ channel in β-cells where, in mice, it limits electrical activity, [Ca2+]c influx, and insulin secretion1. However, its function in human β-cells is unknown. Interestingly, TALK-1 is not only expressed on the plasma membrane, but is also functionally expressed on the endoplasmic reticulum (ER) membrane, where it provides a countercurrent to enhance [Ca2+]ER release. Importantly, a non-synonymous polymorphism (rs1535500) in TALK-1 results in an increased risk for T2DM and a mutation in TALK-1 causes neonatal diabetes. rs1535500 results in increased TALK-1 activity, and we have recently shown that the mutation (R13Q) that causes neonatal diabetes results in enhanced [Ca2+]ER release. We therefore predict that the neonatal mutation results in enhanced TALK-1 activity on the ER membrane, thereby enhancing [Ca2+]ER release, resulting in ER stress and β-cell dysfunction. Furthermore, [Ca2+]ER and mitochondrial Ca2+ ([Ca2+]mito) are tightly linked through the mitochondrial associated membrane (MAM)3. Therefore, alterations in TALK-1 modulation of β-cell [Ca2+]ER is predicted to affect the function of β-cell mitochondria. Indeed, preliminary data has shown that TALK-1 expression reduces intracellular ATP levels and increases the production of mitochondrial production of reactive oxygen species in response to stress. Together, this elutes to an intracellular role for TALK-1 that when perturbed, contributes to the pathogenesis of diabetes. The goal of this study is to elucidate these β-cell specific intracellular roles and understand how they become perturbed in diabetes. We hypothesize that TALK-1 control of β-cell [Ca2+]ER handling modulates mitochondrial function and metabolism as well as contributes to the ER stress response under conditions associated with diabetes. To test this hypothesis, we will determine how TALK-1 affects β-cell function by utilizing a novel floxed KCNK16 mouse crossed with either a β-cell specific Ins1cre, or conditional Ins1creERT2 to specifically ablate TALK- 1 channels in mouse β-cells. We will also transduce human islets with an adenovirus containing an insulin promoter driving a dominant-negative TALK-1 subunit to investigate the role of TALK-1 on human β-cell function (Aim 1). These findings will be extended to detailed studies of how the rs15355500 polymorphisms and R13Q mutations in TALK-1 contribute to β-cell failure during the pathogenesis of diabetes. (Aim 2). Successful completion of the proposed research will advance our understanding of the fundamental mechanisms modulating β-cell failure. Through this fellowship application, I will develop 1) a novel understanding of the mechanisms of β-cell TALK-1 channels and how they contribu...