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.

Pancreatic α-cells exhibit oscillations in cytosolic Ca2+ (Ca2+c), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca2+c oscillations have not been elucidated. As β-cell Ca2+c oscillations are regulated in part by Ca2+-activated K+ (Kslow) currents, this work investigated the role of Kslow in α-cell Ca2+ handling and GCG secretion. α-Cells displayed Kslow currents that were dependent on Ca2+ influx through L- and P/Q-type voltage-dependent Ca2+ channels (VDCCs) as well as Ca2+ released from endoplasmic reticulum stores. α-Cell Kslow was decreased by small-conductance Ca2+-activated K+ (SK) channel inhibitors apamin and UCL 1684, large-conductance Ca2+-activated K+ (BK) channel inhibitor iberiotoxin (IbTx), and intermediate-conductance Ca2+-activated K+ (IK) channel inhibitor TRAM 34. Moreover, partial inhibition of α-cell Kslow with apamin depolarized membrane potential ( Vm) (3.8 ± 0.7 mV) and reduced action potential (AP) amplitude (10.4 ± 1.9 mV). Although apamin transiently increased Ca2+ influx into α-cells at low glucose (42.9 ± 10.6%), sustained SK (38.5 ± 10.4%) or BK channel inhibition (31.0 ± 11.7%) decreased α-cell Ca2+ influx. Total α-cell Ca2+c was similarly reduced (28.3 ± 11.1%) following prolonged treatment with high glucose, but it was not decreased further by SK or BK channel inhibition. Consistent with reduced α-cell Ca2+c following prolonged Kslow inhibition, apamin decreased GCG secretion from mouse (20.4 ± 4.2%) and human (27.7 ± 13.1%) islets at low glucose. These data demonstrate that Kslow activation provides a hyperpolarizing influence on α-cell Vm that sustains Ca2+ entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when α-cell Ca2+c is elevated during secretagogue stimulation, Kslow activation helps to preserve GCG secretion.

Aim To determine whether hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels impact glucagon‐like peptide‐1 (GLP‐1) receptor (GLP‐1R) modulation of islet Ca2+ handling and insulin secretion. Methods The impact of liraglutide (GLP‐1 analogue) on islet Ca2+ handling, HCN currents and insulin secretion was monitored with fluorescence microscopy, electrophysiology and enzyme immunoassays, respectively. Furthermore, liraglutide‐mediated β‐to‐δ‐cell cross‐communication was assessed following selective ablation of either mouse islet δ or β cells. Results Liraglutide increased β‐cell Ca2+ oscillation frequency in mouse and human islets under stimulatory glucose conditions. This was dependent in part on liraglutide activation of HCN channels, which also enhanced insulin secretion. Similarly, liraglutide activation of HCN channels also increased β‐cell Ca2+ oscillation frequency in islets from rodents exposed to a diabetogenic diet. Interestingly, liraglutide accelerated Ca2+ oscillations in a majority of islet δ cells, which showed synchronized Ca2+ oscillations equivalent to β cells; therefore, we assessed if either cell type was driving this liraglutide‐mediated islet Ca2+ response. Although δ‐cell loss did not impact liraglutide‐mediated increase in β‐cell Ca2+ oscillation frequency, β‐cell ablation attenuated liraglutide‐facilitated acceleration of δ‐cell Ca2+ oscillations. Conclusion The data presented here show that liraglutide‐induced stimulation of islet HCN channels augments Ca2+ oscillation frequency. As insulin secretion oscillates with β‐cell Ca2+, these findings have important implications for pulsatile insulin secretion that is probably enhanced by liraglutide activation of HCN channels and therapeutics that target GLP‐1Rs for treating diabetes. Furthermore, these studies suggest that liraglutide as well as GLP‐1‐based therapies enhance δ‐cell Ca2+ oscillation frequency and somatostatin secretion kinetics in a β‐cell‐dependent manner.