Investigating the role of TALK-1 in modulating ER stress and β-cell dysfunction
β-cell dysfunction during the pathogenesis of Type 2 Diabetes Mellitus (T2DM) results in inadequate insulin secretion and hyperglycemia. Endoplasmic reticulum (ER) calcium ([Ca2+]ER) handling plays an important role in regulating insulin secretion, and is modulated by ER resident K+ channels. The two-pore domain potassium channel (K2P), TALK-1, is highly expressed in the pancreatic β-cell where it serves to control [Ca2+]ER release by providing a K+ countercurrent. Recently, a mutation (R13E) in TALK-1 has been identified that causes neonatal diabetes. We found that this mutation results in a loss of plasma membrane TALK-1 currents, whereas its intracellular function at the ER is enhanced, leading to [Ca2+]ER release. We hypothesize that the TALK-1(R13E) results in increased ER membrane TALK-1, thereby enhancing [Ca2+]ER depletion and ER-stress. To test this, we are using a genetically encoded [Ca2+]ER indicator (D4ER) to investigate the effect of TALK-1 on [Ca2+]ER with and without the R13E mutation. We predict that TALK-1 expression will cause [Ca2+]ER depletion, which will be further enhanced with the R13E mutation. Furthermore, we will also investigate the effect of TALK-1 and TALK-1 R13E on the ER-stress response by using a luciferase-based assay to monitor ER-stress in response to tunicamycin, palmitate, thapsigargin, and H2O2. Finally, we will investigate whether TALK-1 is differentially localized on the ER and plasma membranes under stressful conditions, and whether this is altered with the TALK-1(R13E). This research is significant, as it will elucidate how TALK-1 contributes to ER-stress, and how subtle changes in this pathway lead to β-cell dysfunction.