Ca2+ handling by the endoplasmic reticulum (ER) serves critical roles controlling pancreatic β-cell function and becomes perturbed during the pathogenesis of diabetes. ER Ca2+ homeostasis is determined by ion movements across the ER membrane, including K+ flux through K+ channels. Here, we demonstrated that K+ flux through ER-localized TALK-1 channels facilitated Ca2+ release from the ER in mouse and human β-cells. We found that β-cells from mice lacking TALK-1 exhibited reduced basal cytosolic Ca2+ and increased ER Ca2+ concentrations, suggesting reduced ER Ca2+ leak. These changes in Ca2+ homeostasis were presumably due to TALK-1-mediated ER K+ flux, because we recorded K+ currents mediated by functional TALK-1 channels on the nuclear membrane, which is continuous with the ER. Moreover, overexpression of K+-impermeable TALK-1 channels in HEK293 cells did not reduce ER Ca2+ stores. Reduced ER Ca2+ content in β-cells is associated with ER stress and islet dysfunction in diabetes, and islets from TALK-1-deficient mice fed a high-fat diet showed reduced signs of ER stress, suggesting that TALK-1 activity exacerbated ER stress. Our data establish TALK-1 channels as key regulators of β-cell ER Ca2+, and suggest that TALK-1 may be a therapeutic target to reduce ER Ca2+ handling defects in β-cells during the pathogenesis of diabetes.

ObjectiveSingle-cell RNA sequencing studies have revealed that the type-2 diabetes associated two-pore domain K+ (K2P) channel TALK-1 is abundantly expressed in somatostatin-secreting δ-cells. However, a physiological role for TALK-1 in δ-cells remains unknown. We previously determined that in β-cells, K+ flux through endoplasmic reticulum (ER)-localized TALK-1 channels enhances ER Ca2+ leak, modulating Ca2+ handling and insulin secretion. As glucose amplification of islet somatostatin release relies on Ca2+-induced Ca2+ release (CICR) from the δ-cell ER, we investigated whether TALK-1 modulates δ-cell Ca2+ handling and somatostatin secretion.MethodsTo define the functions of islet δ-cell TALK-1 channels, we generated control and TALK-1 channel-deficient (TALK-1 KO) mice expressing fluorescent reporters specifically in δ- and α-cells to facilitate cell type identification. Using immunofluorescence, patch clamp electrophysiology, Ca2+ imaging, and hormone secretion assays, we assessed how TALK-1 channel activity impacts δ- and α-cell function.ResultsTALK-1 channels are expressed in both mouse and human δ-cells, where they modulate glucose-stimulated changes in cytosolic Ca2+ and somatostatin secretion. Measurement of cytosolic Ca2+ levels in response to membrane potential depolarization revealed enhanced CICR in TALK-1 KO δ-cells that could be abolished by depleting ER Ca2+ with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors. Consistent with elevated somatostatin inhibitory tone, we observed significantly reduced glucagon secretion and α-cell Ca2+ oscillations in TALK-1 KO islets, and found that blockade of α-cell somatostatin signaling with a somatostatin receptor 2 (SSTR2) antagonist restored glucagon secretion in TALK-1 KO islets.ConclusionsThese data indicate that TALK-1 reduces δ-cell cytosolic Ca2+ elevations and somatostatin release by limiting δ-cell CICR, modulating the intraislet paracrine signaling mechanisms that control glucagon secretion.

Cytokines present during low-grade inflammation contribute to β-cell dysfunction and diabetes. Cytokine signaling disrupts β-cell glucose-stimulated Ca 2+ influx (GSCI) and endoplasmic reticulum (ER)Caphase GSCI and GSIS. This adaptive Ca 2+ response was absent in TALK-1 KO islets, which exhibited decreased 2 nd phase GSCI and diminished GSIS. These findings suggest that K slow and TALK-1 currents play important roles in altered β-cell Ca 2+ handling and electrical activity during lowgrade inflammation. These results also reveal that a cytokine-mediated reduction in TALK-1 serves an acute protective role in β-cells by facilitating increased Ca 2+ content to maintain GSIS.Failure of β-cells to secrete sufficient insulin precedes the onset of type 2 diabetes mellitus (T2DM) 1 . As the incidence of T2DM is rapidly increasing, it is important to identify better therapeutic options for reducing β-cell failure during the pathogenesis of the disease. Low-grade inflammation is a key contributor to β-cell dysfunction in T2DM 1-8 . Conditions of over-nutrition and inactivity result in low-grade systemic inflammation during which pro-inflammatory cytokine concentrations (e.g. tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interferon-γ (IFN-γ)) increase several fold over basal levels [1][2][3][4][8][9][10] . For example, in a rat model of T2DM pancreatic cytokine levels were all elevated above nontreated controls (e.g. TNF-α increased from 24.3 ± 3.6 pg/mg protein to 47.9 ± 3.5 pg/mg protein (P < 0.05), IL-1β increased from 25.5 ± 2.7 pg/mg protein to 29.2 ± 1.7 pg/mg protein (P < 0.05), and IFN-γ increased from 49.4 ± 4.2 pg/mg protein to 65.1 ± 6.7 pg/mg protein (P < 0.05)) 11 . The presence of these cytokines contributes to insulin resistance and diminished β-cell function 5 . Under stressful conditions (e.g. glucolipotoxicity) β-cells are also capable of secreting pro-inflammatory cytokines, which damage islet function 4,12 . Cytokine-mediated islet dysfunction correlates with increased basal intracellular Ca 2+ ([Ca 2+ ] i ), reduced glucose-stimulated Ca 2+ influx (GSCI), increased [Ca 2+ ] i oscillation frequency, altered endoplasmic reticulum (ER) Ca 2+ ([Ca 2+ ] ER ) storage, and increased apoptotic signaling [5][6][7]13 . While chronic low-grade inflammation leads to β-cell dysfunction in T2DM, the mechanisms responsible remain unresolved. Understanding how cytokines disrupt islet Ca 2+ handling may illuminate therapeutic targets for preventing β-cell failure during T2DM.

Glucose-stimulated insulin secretion (GSIS) relies on β-cell Ca2+ influx, which is modulated by the two-pore-domain K+ (K2P) channel, TALK-1. A gain-of-function polymorphism in KCNK16, the gene encoding TALK-1, increases risk for developing type-2 diabetes. While TALK-1 serves an important role in modulating GSIS, the regulatory mechanism(s) that control β-cell TALK-1 channels are unknown. Therefore, we employed a membrane-specific yeast two-hybrid (MYTH) assay to identify TALK-1-interacting proteins in human islets, which will assist in determining signaling modalities that modulate TALK-1 function. Twenty-one proteins from a human islet cDNA library interacted with TALK-1. Some of these interactions increased TALK-1 activity, including intracellular osteopontin (iOPN). Intracellular OPN is highly expressed in β-cells and is upregulated under pre-diabetic conditions to help maintain normal β-cell function; however, the functional role of iOPN in β-cells is poorly understood. We found that iOPN colocalized with TALK-1 in pancreatic sections and coimmunoprecipitated with human islet TALK-1 channels. As human β-cells express two K+ channel-forming variants of TALK-1, regulation of these TALK-1 variants by iOPN was assessed. At physiological voltages iOPN activated TALK-1 transcript variant 3 channels but not TALK-1 transcript variant 2 channels. Activation of TALK-1 channels by iOPN also hyperpolarized resting membrane potential (Vm) in HEK293 cells and in primary mouse β-cells. Intracellular OPN was also knocked down in β-cells to test its effect on β-cell TALK-1 channel activity. Reducing β-cell iOPN significantly decreased TALK-1 K+ currents and increased glucose-stimulated Ca2+ influx. Importantly, iOPN did not affect the function of other K2P channels or alter Ca2+ influx into TALK-1 deficient β-cells. These results reveal the first protein interactions with the TALK-1 channel and found that an interaction with iOPN increased β-cell TALK-1 K+ currents. The TALK-1/iOPN complex caused Vm hyperpolarization and reduced β-cell glucose-stimulated Ca2+ influx, which is predicted to inhibit GSIS.

Conformational changes in proteins are essential to their biological functions, from allosteric regulation to signal propagation, which often involve interconversions between open/close and bound/unbound pairs as observed by X-ray crystallography. However, the transient nature of the intermediates