TALK-1 reduces delta-cell endoplasmic reticulum and cytoplasmic calcium levels limiting somatostatin secretion.

OBJECTIVE: Single-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 ß-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. METHODS: To 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. RESULTS: TALK-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. CONCLUSIONS: These 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.

Cytokine-mediated changes in K+ channel activity promotes an adaptive Ca2+ response that sustains ß-cell insulin secretion during inflammation.

Cytokines present during low-grade inflammation contribute to ß-cell dysfunction and diabetes. Cytokine signaling disrupts ß-cell glucose-stimulated Ca2+ influx (GSCI) and endoplasmic reticulum (ER) Ca2+ ([Ca2+]ER) handling, leading to diminished glucose-stimulated insulin secretion (GSIS). However, cytokine-mediated changes in ion channel activity that alter ß-cell Ca2+ handling remain unknown. Here we investigated the role of K+ currents in cytokine-mediated ß-cell dysfunction. Kslow currents, which control the termination of intracellular Ca2+ ([Ca2+]i) oscillations, were reduced following cytokine exposure. As a consequence, [Ca2+]i and electrical oscillations were accelerated. Cytokine exposure also increased basal islet [Ca2+]i and decreased GSCI. The effect of cytokines on TALK-1 K+ currents were also examined as TALK-1 mediates Kslow by facilitating [Ca2+]ER release. Cytokine exposure decreased KCNK16 transcript abundance and associated TALK-1 protein expression, increasing [Ca2+]ER storage while maintaining 2nd phase GSCI and GSIS. This adaptive Ca2+ response was absent in TALK-1 KO islets, which exhibited decreased 2nd phase GSCI and diminished GSIS. These findings suggest that Kslow and TALK-1 currents play important roles in altered ß-cell Ca2+ handling and electrical activity during low-grade inflammation. These results also reveal that a cytokine-mediated reduction in TALK-1 serves an acute protective role in ß-cells by facilitating increased Ca2+ content to maintain GSIS.

TALK-1 channels control ß cell endoplasmic reticulum Ca2+ homeostasis.

Ca2+ handling by the endoplasmic reticulum (ER) serves critical roles in controlling pancreatic ß 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. We demonstrated that K+ flux through ER-localized TALK-1 channels facilitated Ca2+ release from the ER in mouse and human ß cells. We found that ß 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 ß 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 ß cell ER Ca2+ and suggest that TALK-1 may be a therapeutic target to reduce ER Ca2+ handling defects in ß cells during the pathogenesis of diabetes.

Osteopontin activates the diabetes-associated potassium channel TALK-1 in pancreatic ß-cells.

Glucose-stimulated insulin secretion (GSIS) relies on ß-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 ß-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 ß-cells and is upregulated under pre-diabetic conditions to help maintain normal ß-cell function; however, the functional role of iOPN in ß-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 ß-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 ß-cells. Intracellular OPN was also knocked down in ß-cells to test its effect on ß-cell TALK-1 channel activity. Reducing ß-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 ß-cells. These results reveal the first protein interactions with the TALK-1 channel and found that an interaction with iOPN increased ß-cell TALK-1 K+ currents. The TALK-1/iOPN complex caused Vm hyperpolarization and reduced ß-cell glucose-stimulated Ca2+ influx, which is predicted to inhibit GSIS.