The MODY-associated L114P mutation increases islet glucagon secretion and limits insulin secretion resulting in transient neonatal diabetes and glucose dyshomeostasis in adults.

The gain-of-function mutation in the TALK-1 K channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of β-cell electrical activity and glucose-stimulated insulin secretion.

TALK-1-mediated alterations of β-cell mitochondrial function and insulin secretion impair glucose homeostasis on a diabetogenic diet.

Mitochondrial Ca ([Ca]) homeostasis is critical for β-cell function and becomes disrupted during the pathogenesis of diabetes. [Ca] uptake is dependent on elevations in cytoplasmic Ca ([Ca]) and endoplasmic reticulum Ca ([Ca]) release, both of which are regulated by the two-pore domain K channel TALK-1. Here, utilizing a novel β-cell TALK-1-knockout (β-TALK-1-KO) mouse model, we found that TALK-1 limited β-cell [Ca] accumulation and ATP production.

Preparing recombinant "Split AEP" for protein labeling.

A variant originated from Oldenlandia affinis asparaginyl ligase, OaAEP1-C247A, has emerged as an ideal tool for protein labeling. However, its preparation was laborious and time-consuming. It is recombinantly produced as a zymogen, requiring acid activation and four chromatographic steps; despite these extensive steps, the catalytically active enzyme exhibited only moderate purity.

The MODY-associated L114P mutation increases islet glucagon secretion and limits insulin secretion resulting in transient neonatal diabetes and glucose dyshomeostasis in adults.

The gain-of-function mutation in the TALK-1 K channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of β-cell electrical activity and glucose-stimulated insulin secretion (GSIS).

G protein-coupled receptor inhibition of beta-cell electrical excitability and insulin secretion depends on Na/K ATPase activation.

G-coupled somatostatin or α2-adrenergic receptor activation stimulated β-cell NKA activity, resulting in islet Ca fluctuations. Furthermore, intra-islet paracrine activation of β-cell G-GPCRs and NKAs by δ-cell somatostatin secretion slowed Ca oscillations, which decreased insulin secretion. β-cell membrane potential hyperpolarization resulting from G-GPCR activation was dependent on NKA phosphorylation by Src tyrosine kinases.

Liraglutide increases islet Ca oscillation frequency and insulin secretion by activating hyperpolarization-activated cyclic nucleotide-gated channels.

Aim: To determine whether hyperpolarization-activated cyclic nucleotide-gated (HCN) channels impact glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) modulation of islet Ca handling and insulin secretion.

Methods: The impact of liraglutide (GLP-1 analogue) on islet Ca 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.

Glucose-Dependent miR-125b Is a Negative Regulator of β-Cell Function.

Impaired pancreatic β-cell function and insulin secretion are hallmarks of type 2 diabetes. miRNAs are short, noncoding RNAs that silence gene expression vital for the development and function of β cells. We have previously shown that β cell-specific deletion of the important energy sensor AMP-activated protein kinase (AMPK) results in increased miR-125b-5p levels.

TRPM7 is a crucial regulator of pancreatic endocrine development and high-fat-diet-induced β-cell proliferation.

The melastatin subfamily of the transient receptor potential channels (TRPM) are regulators of pancreatic β-cell function. TRPM7 is the most abundant islet TRPM channel; however, the role of TRPM7 in β-cell function has not been determined. Here, we used various spatiotemporal transgenic mouse models to investigate how TRPM7 knockout influences pancreatic endocrine development, proliferation and function.

A KCNK16 mutation causing TALK-1 gain of function is associated with maturity-onset diabetes of the young.

Maturity-onset diabetes of the young (MODY) is a heterogeneous group of monogenic disorders of impaired pancreatic β cell function. The mechanisms underlying MODY include β cell KATP channel dysfunction (e.g.

Sexually dimorphic roles for the type 2 diabetes-associated C2cd4b gene in murine glucose homeostasis.

Aims/hypothesis: Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B.

Methods: CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis.

Tetraspanin-7 regulation of L-type voltage-dependent calcium channels controls pancreatic β-cell insulin secretion.

Key Points: Tetraspanin (TSPAN) proteins regulate many biological processes, including intracellular calcium (Ca ) handling. TSPAN-7 is enriched in pancreatic islet cells; however, the function of islet TSPAN-7 has not been identified. Here, we characterize how β-cell TSPAN-7 regulates Ca handling and hormone secretion.

Lactate activation of α-cell K channels inhibits glucagon secretion by hyperpolarizing the membrane potential and reducing Ca entry.

Objective: Elevations in pancreatic α-cell intracellular Ca ([Ca]) lead to glucagon (GCG) secretion. Although glucose inhibits GCG secretion, how lactate and pyruvate control α-cell Ca handling is unknown. Lactate enters cells through monocarboxylate transporters (MCTs) and is also produced during glycolysis by lactate dehydrogenase A (LDHA), an enzyme expressed in α-cells.

The pore-forming subunit MCU of the mitochondrial Ca uniporter is required for normal glucose-stimulated insulin secretion in vitro and in vivo in mice.

Aims/hypothesis: Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca importer (MCU) complex is thought to represent the main route for Ca transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo.

Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction.

The activation of neuronal plasma membrane Ca channels stimulates many intracellular responses. Scaffolding proteins can preferentially couple specific Ca channels to distinct downstream outputs, such as increased gene expression, but the molecular mechanisms that underlie the exquisite specificity of these signaling pathways are incompletely understood. Here, we show that complexes containing CaMKII and Shank3, a postsynaptic scaffolding protein known to interact with L-type calcium channels (LTCCs), can be specifically coimmunoprecipitated from mouse forebrain extracts.

Glucose-mediated inhibition of calcium-activated potassium channels limits α-cell calcium influx and glucagon secretion.

Pancreatic α-cells exhibit oscillations in cytosolic Ca (Ca), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca oscillations have not been elucidated. As β-cell Ca oscillations are regulated in part by Ca-activated K (K) currents, this work investigated the role of K in α-cell Ca handling and GCG secretion.

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 β-cells, K flux through endoplasmic reticulum (ER)-localized TALK-1 channels enhances ER Ca leak, modulating Ca handling and insulin secretion.

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

Cytokines present during low-grade inflammation contribute to β-cell dysfunction and diabetes. Cytokine signaling disrupts β-cell glucose-stimulated Ca influx (GSCI) and endoplasmic reticulum (ER) Ca ([Ca]) handling, leading to diminished glucose-stimulated insulin secretion (GSIS). However, cytokine-mediated changes in ion channel activity that alter β-cell Ca handling remain unknown.

Simultaneous Real-Time Measurement of the β-Cell Membrane Potential and Ca Influx to Assess the Role of Potassium Channels on β-Cell Function.

Stimulus-secretion coupling in pancreatic β-cells requires Ca influx through voltage-dependent Ca channels, whose activity is controlled by the plasma membrane potential (V ). Here, we present a method of measuring fluctuations in the β-cell V and Ca influx simultaneously, which provides valuable information about the ionic signaling mechanisms that underlie insulin secretion. This chapter describes the use of perforated patch clamp electrophysiology on cells loaded with a fluorescent intracellular Ca indicator, which permits the stable recording conditions needed to monitor the V and Ca influx in β-cells.

TALK-1 channels control β cell endoplasmic reticulum Ca homeostasis.

Ca handling by the endoplasmic reticulum (ER) serves critical roles in controlling pancreatic β cell function and becomes perturbed during the pathogenesis of diabetes. ER Ca 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 Ca release from the ER in mouse and human β cells.

Chronic β-Cell Depolarization Impairs β-Cell Identity by Disrupting a Network of Ca-Regulated Genes.

We used mice lacking , a key component of the β-cell K-channel, to analyze the effects of a sustained elevation in the intracellular Ca concentration ([Ca]) on β-cell identity and gene expression. Lineage tracing analysis revealed the conversion of β-cells lacking into pancreatic polypeptide cells but not to α- or δ-cells. RNA-sequencing analysis of FACS-purified β-cells confirmed an increase in gene expression and revealed altered expression of more than 4,200 genes, many of which are involved in Ca signaling, the maintenance of β-cell identity, and cell adhesion.

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

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.

Selective Small Molecule Activators of TREK-2 Channels Stimulate Dorsal Root Ganglion c-Fiber Nociceptor Two-Pore-Domain Potassium Channel Currents and Limit Calcium Influx.

The two-pore-domain potassium (K2P) channel TREK-2 serves to modulate plasma membrane potential in dorsal root ganglia c-fiber nociceptors, which tunes electrical excitability and nociception. Thus, TREK-2 channels are considered a potential therapeutic target for treating pain; however, there are currently no selective pharmacological tools for TREK-2 channels. Here we report the identification of the first TREK-2 selective activators using a high-throughput fluorescence-based thallium (Tl) flux screen (HTS).

Fibronectin binding to the Treponema pallidum adhesin protein fragment rTp0483 on functionalized self-assembled monolayers.

Past work has shown that Treponema pallidum, the causative agent of syphilis, binds host fibronectin (FN). FN and other host proteins are believed to bind to rare outer membrane proteins (OMPs) of T. pallidum, and it is postulated that this interaction may facilitate cell attachment and mask antigenic targets on the surface.