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, 9, 84-97

TALK-1 Reduces Delta-Cell Endoplasmic Reticulum and Cytoplasmic Calcium Levels Limiting Somatostatin Secretion

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TALK-1 Reduces Delta-Cell Endoplasmic Reticulum and Cytoplasmic Calcium Levels Limiting Somatostatin Secretion

Nicholas C Vierra et al. Mol Metab.

Abstract

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 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.

Keywords: Endoplasmic reticulum; Hormone secretion; Islet; KCNK16; Paracrine; Two-pore domain K(+) channel.

Figures

Figure 1
Figure 1
TALK-1 channels are expressed in mouse and human δ-cells. (A) Mouse pancreas section stained for TALK-1 (green) and somatostatin (red) (representative of N = 3 mice). (B) Mouse pancreas section stained for TALK-1 (green), ER (GRP94, red), and SST (cyan). (C) Human pancreas section stained for TALK-1 (green) and somatostatin (red) (representative of N = 3 pancreata). (D) Human pancreas section stained for TALK-1 (green), ER (GRP94, red), and somatostatin (E) K2P currents recorded from WT and TALK-1 KO δ-cells (N = 3 mice per genotype). (F) K2P currents recorded from human δ-cells expressing TALK-1 DN or control mCherry. (N = 3 islet preparations); *P < 0.05, **P < 0.005.
Figure 2
Figure 2
Glucose synchronizes WT and TALK-1 KO δ-cellCa2+cwith β-cells. (A) Recording of Ca2+c from a WT islet expressing GCaMP6s in δ-cells and loaded with the red Ca2+ dye Cal590-AM (black: representative WT islet Cal590-AM signal excluding GCaMP6s-positive δ-cells, green: representative WT δ-cell GCaMP6s signals, purple: average WT δ-cell GCaMP6s signal; lines above indicate glucose concentrations; colored regions delineate periods used for cross-correlation analysis). (B) Recording of Ca2+c from a TALK-1 KO islet expressing GCaMP6s in δ-cells and loaded with the red Ca2+ dye Cal590-AM (black: representative TALK-1 KO islet Cal590-AM signal excluding GCaMP6s-positive δ-cells, green: representative TALK-1 KO δ-cell GCaMP6s signals, and purple: average TALK-1 KO δ-cell GCaMP6s signal). (C) Average correlation between WT β- and δ-cells at 1 mM glucose (N = 13 islets). (D) Average correlation between WT β- and δ-cells at 11 mM glucose (N = 13 islets). (E) Average correlation between WT β- and δ-cells at 11 mM glucose + 50 μM CPA (N = 13 islets). (F) Average correlation between TALK-1 KO β- and δ-cells at 1 mM glucose (N = 12 islets). (G) Average correlation between TALK-1 KO β- and δ-cells at 11 mM glucose (N = 12 islets). (H) Average correlation between TALK-1 KO β- and δ-cells at 11 mM glucose + 50 μM CPA (N = 12 islets). (I). Average δ-cell Ca2+ oscillation frequency in WT and TALK-1 KO δ-cells measured under the indicated conditions (N = 3 mice per genotype). (J) Average maximum GCaMP6s fluorescence amplitude in WT and TALK-1 KO δ-cells measured under the indicated conditions (N = 3 mice per genotype). (K) Comparison of percent of δ-cells exhibiting Ca2+ oscillations under indicated glucose conditions (N = 3 mice per genotype); *P < 0.05; **P < 0.005.
Figure 3
Figure 3
TALK-1 limits changes in δ-cellCa2+cand somatostatin secretion. (A) Ca2+c recorded in single Fura-2-loaded WT δ-cells perfused with the indicated treatments (representative of N = 3 mice per genotype). (B) Ca2+c recorded in single Fura-2-loaded TALK-1 KO δ-cells perfused with the indicated treatments (representative of N = 3 mice per genotype). (C) Increase in Ca2+c (area under the curve, AUC) in WT and TALK-1 KO δ-cells (AUC with 11 mM glucose was quantified between 0 and 800 s and AUC with EGTA and no extracellular Ca2+ was quantified between 1300 and 1500 s; N = 3 mice per genotype). (D) Maximum Ca2+c oscillation amplitude (F/Fmin) measured in WT and TALK-1 KO δ-cells (N = 6 mice per genotype). (E) Somatostatin secretion from WT and TALK-1 KO islets at 1 and 11 mM glucose (N = 4–7 islet preparations per genotype). (F) Representative Ca2+c transients in WT, TALK-1 KO, and human δ-cells subjected to depolarizing pulses in the presence of 11 mM glucose or 5 mM 2-deoxyglucose and 5 μM rotenone. The fold increase in δ-cell Ca2+ in response to increasingly is quantified in (G) (N = 12 cells (WT); 12 cells (TALK-1 KO); 7 cells (human)). *P < 0.05, **P < 0.005, ***P < 0.0005; ANOVA followed by Tukey's multiple comparison test.
Figure 4
Figure 4
Electrical activity and VDCC currents in WT and TALK-1 KO δ-cells. (A) Vm recording from a WT δ-cell treated with indicated glucose concentrations (representative of recordings obtained from N = 12 cells/4 mice per genotype). (B) Vm recording from a TALK-1 KO δ-cell treated with indicated glucose concentrations (representative of recordings obtained from N = 12 cells/4 mice per genotype). (C) Quantification of plateau fraction in WT and TALK-1 KO δ-cells (N = 12 cells/4 mice per genotype). (D and E) Average Vm in WT and TALK-1 δ-cells at 1 and 11 mM glucose. (N = 12 cells/4 mice per genotype). (F) VDCC currents recorded from WT (representative of N = 9 cells/3 mice per genotype) and TALK-1 KO (representative of N = 8 cells/3 mice per genotype) δ-cells. VDCC current densities are quantified in (G); *P < 0.05.
Figure 5
Figure 5
CICR is increased in TALK-1 KO δ-cells. (A, B) Average increase in Ca2+c following high K+ (45 mM)-induced depolarization in WT and TALK-1 KO δ-cells, treated with vehicle or with 5 μM thapsigargin (Tg). SERCAs were energized with 11 mM glucose and KATP was activated with 125 μM diazoxide. Subtraction of the signal obtained from Tg treated cells from vehicle treated cells reveals the contribution of the ER to the Ca2+c signal (C) (N = 3 mice per genotype). (D) Average change in Ca2+c in response to treatment with diazoxide (125 μM) and depolarization with 45 mM K+ in WT and TALK-1 KO δ-cells (N = 3 mice per genotype); *P < 0.05. (E) Representative Ca2+c transients in WT and TALK-1 KO δ-cells subjected to short depolarizing pulses. The fold increase in δ-cell Ca2+ in response to increasingly long depolarizations in the presence or absence of CPA (25 μM) is quantified in (F) (N = 9 cells (WT); 7 cells (TALK-1 KO); 3 mice per genotype). (G) Ca2+c in WT and TALK-1 KO δ-cells was assessed before and after treatment with CPA. (N = 3 mice per genotype); *P < 0.05, **P < 0.005.
Figure 6
Figure 6
Reduced glucagon secretion from TALK-1 KO islets. (A) Isolated islets from WT and TALK-1 KO mice were perifused with the indicated glucose concentrations (N = 4 mice per genotype). (B) Glucagon AUC for the period corresponding to glucagon secretion in 1 mM glucose (0–18 min). (C) Glucagon AUC for the period corresponding to glucagon secretion in 11 mM glucose (18–45 min). (D) K2P current density in WT and TALK-1 KO α-cells (N = 3 mice per genotype). (E) Human pancreas sections stained for TALK-1 using two different antibodies and glucagon. (F) K2P current density in human α-cells expressing either TALK-1 DN mutant or mCherry control. Cells were post-stained for glucagon; only glucagon-positive cells were analyzed (N = 10 α-cells per condition/five donors); *P < 0.05; **P < 0.005.
Figure 7
Figure 7
TALK-1 KO α-cells exhibit altered Ca2+dynamics only in intact islets. (A) Recordings of intracellular Ca2+ responses in WT α-cells, in islets expressing the genetically encoded Ca2+ indicator GCaMP3 specifically in α-cells (representative of islet α-cells from N = 3 mice). (B) Recordings of intracellular Ca2+ responses in TALK-1 KO α-cells, in islets expressing the genetically encoded Ca2+ indicator GCaMP3 specifically in α-cells (representative of islet α-cells from N = 3 mice). (C) Recordings of intracellular Ca2+ responses in single WT α-cells expressing the genetically encoded Ca2+ indicator GCaMP3 (representative of α-cells from N = 3 mice). (D) Recordings of intracellular Ca2+ responses in single TALK-1 KO α-cells expressing the genetically encoded Ca2+ indicator GCaMP3 (representative of α-cells from N = 3 mice). (E and F) Comparison of percent oscillating α-cells and Ca2+ AUC determined from WT and TALK-1 KO α-cells under the indicated conditions (N = 3 mice per genotype). (G) Islets were incubated for 1 h with the indicated treatments (SSTR2 antagonist: 500 nM CYN154806); N = 10 mice per genotype (glucose only); 3 mice per genotype (+CYN154806); *P < 0.05.

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