The training plan outlined in this proposal focuses on elucidating the molecular mechanisms by which the two-pore domain K+ channel TALK-1 modulates ?-cell function and contributes to ?-cell failure during the pathogenesis of diabetes. TALK-1 is the most highly expressed K+ channel in ?-cells where, in mice, it limits electrical activity, [Ca2+]c influx, and insulin secretion1. However, its function in human ?-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 ?-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 ?-cell [Ca2+]ER is predicted to affect the function of ?-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 ?-cell specific intracellular roles and understand how they become perturbed in diabetes. We hypothesize that TALK-1 control of ?-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 ?-cell function by utilizing a novel floxed KCNK16 mouse crossed with either a ?-cell specific Ins1cre, or conditional Ins1creERT2 to specifically ablate TALK- 1 channels in mouse ?-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 ?-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 ?-cell failure during the pathogenesis of diabetes.
(Aim 2). Successful completion of the proposed research will advance our understanding of the fundamental mechanisms modulating ?-cell failure. Through this fellowship application, I will develop 1) a novel understanding of the mechanisms of ?-cell TALK-1 channels and how they contribute to the pathogenesis of diabetes, and 2) my future as an independent investigator. My training will be facilitated by the detailed research plan, the exceptionally qualified mentors, and the outstanding training resources and facilities available through Vanderbilt University.
Diabetes affects 422 million people worldwide, including almost 10% of the American population, and is caused in-part by pancreatic ?-cell failure. The proposed study will determine the mechanisms by which TALK-1, the most highly expressed K+ channel in ?-cells, contributes to ?-cell ER stress, mitochondrial dysfunction, and therefore ?-cell failure during the pathogenesis of diabetes. The proposed study is expected to significantly contribute to our understanding of how ?-cells become impaired during diabetes and illuminate a new therapeutic target for the treatment of metabolic diseases.
