Research ArticleIon Channels

TALK-1 channels control β cell endoplasmic reticulum Ca2+ homeostasis

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Sci. Signal.  19 Sep 2017:
Vol. 10, Issue 497, eaan2883
DOI: 10.1126/scisignal.aan2883

TALKing about K+, Ca2+, and ER stress

Insulin secretion is triggered upon depolarization of β cells, which activates voltage-gated Ca2+ channels in the plasma membrane. Ca2+ that enters the β cells is taken up by the endoplasmic reticulum (ER) and released near Ca2+-activated K+ channels in the plasma membrane that repolarize β cells. Vierra et al. showed that TALK-1 channels provided the K+ influx into the ER that counterbalanced Ca2+ release from the ER in mouse and human β cells. ER Ca2+ content is reduced in diabetic β cells, which can result in ER stress and further accentuate β cell dysfunction. The authors found that mRNA markers of ER stress were decreased in islets from TALK-1 knockout mice fed a high-fat diet and suggest that targeting TALK-1 activity may suppress ER stress and improve β cell ER Ca2+ handling in diabetes.


Ca2+ handling by the endoplasmic reticulum (ER) serves critical roles in 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. 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.


Pancreatic β cell Ca2+ influx triggers insulin secretion, and endoplasmic reticulum (ER) Ca2+ (Ca2+ER) handling plays a key role in this process (1). Ca2+ER serves many essential functions in β cells, such as controlling protein processing and metabolism, and defects in Ca2+ER homeostasis can trigger the unfolded protein response (2). The importance of precise β cell Ca2+ER handling is evident in type 1 and type 2 diabetes mellitus (T2DM), during which Ca2+ER homeostasis is disrupted, leading to β cell dysfunction and eventual destruction (18). Impaired Ca2+ER handling also causes defects in glucose-stimulated insulin secretion (GSIS), contributing to hyperglycemia (3, 9). Therefore, treatments that reduce ER stress in the context of β cell dysfunction improve glucose tolerance (1012). However, although it is known that β cell Ca2+ER concentrations are perturbed in diabetes (2, 47), the molecular determinants that set β cell Ca2+ER are poorly understood.

Maintenance of Ca2+ER homeostasis requires that Ca2+ movement across the ER membrane is balanced with a simultaneous K+ flux in the opposite direction (1315). Without this K+ countercurrent, Ca2+ release from the ER would rapidly generate a negative charge on the inside of the ER membrane, inhibiting further Ca2+ER release. To date, only a few ER K+ channels have been identified, including trimeric intracellular cation–A (TRIC-A) channels, which regulate Ca2+ER stores in myocytes (16, 17); TRIC-B channels, which control Ca2+ER homeostasis in alveolar epithelial cells and osteoblasts (18, 19); and small-conductance Ca2+-activated K+ (KCa) channels (SK channels), which modulate Ca2+ER uptake in neurons and cardiomyocytes (20). Genetic ablation or pharmacological inhibition of these channels impairs Ca2+ER handling. For example, knockout (KO) of TRIC-A or TRIC-B channels results in increased Ca2+ER stores, presumably due to the loss of a K+ countercurrent that regulates the ability of Ca2+ to exit the ER (15, 16, 18). Despite the importance of K+ countercurrents in maintaining Ca2+ER homeostasis, nothing is known about the mediators or functions of β cell ER K+ countercurrents.

ER localization has been reported for several two-pore domain K+ (K2P) channels, including TASK-1 (21), TASK-3 (22), TASK-5 (23), TWIK-2 (24, 25), and THIK-2 (24). Although the subcellular localization of TALK-1 channels has not been reported, a protein interactome study has determined that a majority (>60%) of the proteins interacting with TALK-1 are ER-resident proteins (26). Similarly, a human pancreatic islet cDNA library generated and screened in a membrane yeast two-hybrid assay to identify islet TALK-1–interacting proteins detected multiple ER-resident proteins that interact with TALK-1 (27). In accordance with these observations, TALK-1 shows substantial intracellular staining in human and mouse pancreatic β cells (28). Although these findings suggest that TALK-1 channels may serve an intracellular role, investigations of intracellular K2P channels have focused primarily on elucidating the factors that enable their functional expression on the plasma membrane, and an ER function for K2P channels has not been identified.

In β cells, TALK-1 contributes to plasma membrane potential (Vm) hyperpolarization, thereby regulating cytosolic Ca2+ (Ca2+c) influx and insulin secretion (28). TALK-1 is distributed in pancreatic islets and gastric somatostatin cells (29, 30) and is the most abundant islet K+ channel at the transcriptional level (3133). A primary physiological function of β cell TALK-1 channels is to limit glucose-induced islet electrical and Ca2+c oscillations, controlling second-phase pulsatile insulin secretion (28). Furthermore, a nonsynonymous polymorphism in TALK-1 (rs1535500) associated with T2DM (3436) causes a gain of function in TALK-1 activity (28), which may impair Ca2+c oscillations and pulsatile insulin secretion. However, the molecular mechanisms underlying TALK-1 regulation of islet Ca2+c oscillations remain unclear.

Here, we tested the hypothesis that TALK-1 channels were functional in the ER and mediated ER K+ countercurrents that support β cell Ca2+ER homeostasis. By measuring Ca2+ER, Ca2+c, and single-channel K2P currents on the ER membrane, we demonstrated that TALK-1 channels conducted ER K+ countercurrents that enhanced Ca2+ER leak in mouse and human β cells. We found that TALK-1 control of β cell Ca2+ER modulated islet Ca2+c dynamics, which has important implications for understanding the regulation of Ca2+c oscillations that underlie pulsatile insulin secretion. Moreover, we showed that other ER-localized K2P channels, such as TASK-1, could function in an identical manner. Inhibition of K+ currents through either TALK-1 or TASK-1 increased steady-state Ca2+ER concentrations, demonstrating that the K+ channel function of these proteins was essential for their effects on Ca2+ER homeostasis. Moreover, islets from mice lacking TALK-1 channels showed reduced signs of ER stress induced by chronic exposure to a high-fat diet (HFD), suggesting that defects in TALK-1 channel activity can perturb ER health and contribute to islet dysfunction in T2DM. Overall, these findings identify an intracellular function of K2P channels and reveal TALK-1 channels as a possible therapeutic target to modulate Ca2+ER homeostasis to reduce β cell ER stress in diabetes.


TALK-1 activity promotes Ca2+ER leak

TALK-1’s prominent intracellular staining pattern (28) and physical association with several ER-resident proteins (26) suggested that TALK-1 could be localized to the ER. To determine the subcellular localization of TALK-1, we performed immunofluorescence staining of mouse pancreas sections and detected colocalization of TALK-1 with the ER marker calreticulin (Fig. 1A). In addition, coexpression of a TALK-1/mCherry fusion protein and an ER-targeted indicator (37) in mouse islet cells revealed TALK-1 in the ER (fig. S1).

Fig. 1 TALK-1 channels modulate β cell Ca2+ER homeostasis.

(A) Representative images of a mouse pancreas section stained for TALK-1 and calreticulin. Scale bar, 10 μm. Images are representative of results obtained from three mice. (B) β cell Ca2+ER measurements made with the genetically encoded Ca2+ER indicator D4ER. Cells were perfused with solutions containing indicated glucose concentrations and 50 μM CPA (n = 3 mice per genotype). (C) Area under the curve (AUC) analysis of Ca2+ER under low (2 mM) and high (11 mM) glucose conditions from (B). AU, arbitrary units. (D) CPA-induced reduction in Ca2+ER, presented as percent of maximum Ca2+ER of wild-type (WT) β cells from (B). (E) WT and TALK-1 KO β cells were perfused with the indicated solutions; 11 mM glucose (G) and 125 μM diazoxide (Dz) were present throughout the experiment. (F) Fold increase in Ca2+ in response to the indicated treatments. (G) Ca2+ AUC for the period after addition of 2.5 mM Ca2+ to the extracellular buffer (t = 1000 to 1750 s) [n = 5 mice per genotype for (E) to (G)]. Statistical significance was determined by Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.005.

Although TALK-1 conducts K+ currents on the plasma membrane in β cells (28), it has not yet been determined whether K2P channels in the ER, such as TALK-1, are functional. To test whether TALK-1 channel function could affect Ca2+ER homeostasis, we first directly measured β cell Ca2+ER from control (wild-type) and TALK-1 KO islets (28) with the ER-targeted, genetically encoded Ca2+ indicator D4ER (Fig. 1B) (38). Under both low and high glucose conditions, TALK-1 KO β cells had significantly higher Ca2+ER concentrations (Fig. 1, B and C). Inhibition of sarco-endoplasmic Ca2+ adenosine triphosphatases (SERCAs) with cyclopiazonic acid (CPA) produced a greater decrease in Ca2+ER in KO β cells compared with controls (Fig. 1D). Absolute Ca2+ER concentrations in KO β cells remained above wild-type β cells after application of CPA. These findings suggest that inhibition of SERCAs was insufficient to completely empty β cell Ca2+ER stores, as observed in neurons (39), and imply that TALK-1 channels were a critical determinant of β cell steady-state Ca2+ER concentrations. Because a slight reduction in Ca2+ER stimulates β cell proliferation (40), we tested whether TALK-1 activity affected β cell number or proliferation. The absence of TALK-1 did not alter islet cellular composition nor did it impair adaptive proliferation [as determined by 5-bromo-2′-deoxyuridine (BrdU) incorporation] in response to a short-term (1-week) HFD stimulus (41) (fig. S2, A to F), suggesting that inhibition of TALK-1 did not influence islet cell composition.

To further confirm that TALK-1 modulates Ca2+ER, we quantified Ca2+ER indirectly in wild-type and KO β cells by measuring Ca2+c in response to multiple stimuli. Treating β cells with the Ca2+ ionophore ionomycin in the absence of extracellular Ca2+ resulted in more Ca2+ release from KO than wild-type β cells (fig. S3, A and B), suggesting increased intracellular Ca2+ stores. We next perfused isolated wild-type and TALK-1 KO β cells with Ca2+-free buffer containing diazoxide to selectively monitor Ca2+c independently of Ca2+ entry through plasma membrane channels (Fig. 1E). Under these conditions, TALK-1 KO β cells exhibited lower basal Ca2+c, and the addition of CPA produced a larger increase in Ca2+c (Fig. 1, E and F), suggesting reduced Ca2+ER leak and increased Ca2+ER stores. After washout of CPA, addition of Ca2+ to the extracellular buffer led to a similar amount of Ca2+c influx in wild-type and TALK-1 KO cells, showing that activation of store-operated Ca2+ entry (SOCE) was not impaired in TALK-1 KO β cells (Fig. 1, E and F). However, the reduced basal Ca2+c observed without external Ca2+ was maintained in the presence of extracellular Ca2+ in TALK-1 KO β cells (Fig. 1, E and G).

Because TALK-1 is also detected in human β cells (28), we next examined whether TALK-1 was present in the ER of human β cells. Immunofluorescent staining of human pancreas sections revealed colocalization of TALK-1 with the ER marker calreticulin (Fig. 2A). To assess TALK-1–mediated regulation of human β cell Ca2+ER, we measured CPA-induced Ca2+ER release in β cells expressing a dominant-negative TALK-1 (TALK-1 DN) construct (Fig. 2B). The TALK-1 DN contains a pore mutation, which inhibits K+ conductance when it interacts with endogenous TALK-1 (28). The TALK-1 DN construct also contains a P2A element between the sequences encoding TALK-1 and an mCherry reporter, allowing us to detect cells expressing the TALK-1 DN by mCherry fluorescence; β cells were identified by poststaining for insulin (28). Inhibition of TALK-1 with the TALK-1 DN caused a significant increase in CPA-induced Ca2+ER release (Fig. 2C), demonstrating that TALK-1 modulates Ca2+ER homeostasis in human β cells.

Fig. 2 TALK-1 channels modulate human β cell Ca2+ER homeostasis.

(A) Representative image of a human pancreas section stained for TALK-1 and calreticulin. Scale bar, 10 μm. (B) Representative recordings of intracellular Ca2+ in human β cells transfected with either TALK-1 DN or mCherry control. Present throughout were 11 mM glucose, 0 mM Ca2+, 125 μM diazoxide, and 1 mM EGTA. (C) Quantification of the fold change in the Ca2+ AUC in response to treatment with CPA in human β cells. The number of β cells per donor is indicated on the graph. Statistical significance was determined by Student’s t test and one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test; *P < 0.05, ***P < 0.005. Avg., average.

To test whether TALK-1 activity reduced Ca2+ER storage, we examined the effects of TALK-1 expression on Ca2+ER in human embryonic kidney–293 (HEK293) cells. Two channel-forming isoforms of TALK-1 (1b and 1a) (42) colocalized with an ER-targeted yellow fluorescent protein (YFP) marker in these cells (Fig. 3A). We next assessed the consequences of TALK-1 expression on Ca2+ER homeostasis in these cells. To minimize potential deleterious effects of protein overexpression, we used the TALK-1 DN mutant as a control, which permitted dissociation of the effects of TALK-1–mediated K+ conductance and protein-protein interactions on Ca2+ER homeostasis. Overexpression of K+-conducting wild-type TALK-1 yielded a substantial increase in basal Ca2+c and a concomitant reduction in total Ca2+ released from the ER during CPA-induced inhibition of SERCAs when compared to the non–K+-conducting TALK-1 DN channels (Fig. 3, B to D). These observations suggest that TALK-1 channel activity promotes Ca2+ER leak. Addition of Ca2+ to the extracellular buffer produced a larger increase in Ca2+c in wild-type TALK-1 compared to TALK-1 DN–expressing cells, and similar to CPA-induced Ca2+ER release, stimulation of inositol 1,4,5-trisphosphate (IP3)–triggered Ca2+ER release with carbachol elicited a greater response in cells expressing TALK-1 DN (Fig. 3, B and E). Thus, the K+-channel function of TALK-1 is sufficient to alter Ca2+ER homeostasis.

Fig. 3 The K+ channel function of TALK-1 contributes to its regulation of Ca2+ER homeostasis.

(A) TALK-1b and TALK-1a colocalize with the ER marker ER-YFP. Images are representative of three independent experiments. Scale bars, 10 μm. (B) Representative recordings of HEK293 cells expressing either WT TALK-1 or TALK-1 DN and perfused with the indicated solutions; 10 mM glucose was present throughout the experiment. (C) Normalized Ca2+ AUC for the period during treatment with CPA (t = 250 to 600 s). (D) Ca2+ AUC for the period after addition of 2.5 mM Ca2+ to the extracellular buffer (t = 1000 to 1750 s). (E) Fold increase in Ca2+ in response to treatment with the muscarinic receptor agonist carbachol [n = 3 independent experiments for (B) to (D)]. Statistical significance was determined by Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.005.

To confirm the specificity of the effect of TALK-1 on Ca2+ER, we compared Ca2+ER storage in cells stably and inducibly expressing different K2P channels (Fig. 4A and fig. S4, A and B) (43). Similar to transfected cells, TALK-1 induction reduced Ca2+ER stores (Fig. 4A). We also found that expression of TASK-1 (Fig. 4A) and TASK-3 (fig. S5, A and B) channels (21, 22) also caused a reduction in Ca2+ER. We confirmed the Ca2+ER reduction in TALK-1– and TASK-1–expressing cells by directly measuring Ca2+ER using the genetically encoded Ca2+ER indicator T1ER (Fig. 4B) (44). However, not all K2P channels influenced Ca2+ER, as demonstrated by the absence of a Ca2+ER phenotype after induction of TREK-2 or TREK-1 channels (Fig. 4A and fig. S5, C to F).

Fig. 4 Pharmacological manipulation of K2P channel activity can alter steady-state Ca2+ER concentrations.

(A) Representative recordings of CPA-induced Ca2+ER release in cell lines with tetracycline-inducible expression of the indicated K2P channels. Ca2+ AUC in response to CPA is shown to the right (representative of n = 3 independent experiments; NI, not induced). (B) Direct quantification of Ca2+ER concentration in HEK293 cells with inducible expression of TALK-1, TASK-1, TREK-2, TREK-1, and the Ca2+ER indicator T1ER (n = 3 independent experiments). (C and D) Treatment of TASK-1–expressing cells with ML365 restores Ca2+ER to prechannel expression concentrations (C); AUC quantification (D) (n = 3 independent experiments). (E) Mouse α cells were treated with ML365 in the presence of 11 mM glucose and 125 μM diazoxide (representative of n = 3 independent experiments). The response to CPA is quantified in (F) (n = 3 independent experiments). Statistical significance was determined by Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.005.

This finding prompted us to examine whether pharmacological modulation of K2P channels could be used to manipulate Ca2+ER storage. Because specific pharmacology for TALK-1 channels does not presently exist, we tested whether selective TASK-1 inhibition with ML365 [a small molecule antagonist of TASK-1 (45) and a partial TASK-3 inhibitor] could influence Ca2+ER. Inhibition of TASK-1 channel activity with ML365 treatment significantly restored Ca2+ER loss caused by TASK-1 channel induction (Fig. 4, C and D). Because ML365 also partially blocks TASK-3, treatment of TASK-3–expressing cells with this compound caused a modest increase in Ca2+ER (fig. S5, A and B). However, ML365 did not influence Ca2+ER in cells expressing TREK-1 or TREK-2 (fig. S5, C to F). These data further support the hypothesis that K+ flux through ER K2P channels enhances Ca2+ER leak.

Although these observations suggested that pharmacological modulation of K2P channels could be used to regulate Ca2+ER, immortalized cell lines expressing TASK-1 may not recapitulate Ca2+ER handling of primary tissues. Thus, we tested whether pharmacological blockade of TASK-1 could alter Ca2+ER in primary islet α cells, where they regulate glucagon secretion (46). ML365 treatment increased α cell Ca2+ER stores (Fig. 4, E and F). These data demonstrate that TASK-1 affects α cell Ca2+ER homeostasis and that pharmacological inhibition of Ca2+ER-modulating K2P channels could be used to control Ca2+ER in primary islet cells.

TASK-1 mutations have been implicated in pulmonary arterial hypertension (PAH), one of which (G203D) is a dominant-negative mutation that directly impairs K+ conductance through TASK-1 (47). Expression of TASK-1 G203D produced a significantly greater increase in Ca2+ER stores compared to that of control TASK-1 channels (fig. S5, G and H), an effect similar to that of expressing the TALK-1 DN mutant, which increased Ca2+ER stores compared to cells expressing wild-type TALK-1. These observations imply that defects in TASK-1 K+ conductance may inappropriately increase Ca2+ER or impair physiologically important Ca2+ER fluxes.

TALK-1 and TASK-1 form functional channels across the ER membrane

During Ca2+ER release, K+ moves across the ER membrane to maintain ER electroneutrality and sustain the driving force for Ca2+ER release (14, 16, 48, 49). To directly assess whether TALK-1 functions as an ER K+ channel, we used nuclear patch clamp electrophysiology (50) to measure channel activity on the outer nuclear membrane, which is continuous with the ER (Fig. 5, A and B). In nuclei from cells expressing TREK-2, which does not affect Ca2+ER, we did not detect TREK-2 channel activity (Fig. 5C). However, nuclei from cells expressing TALK-1 (Fig. 5D) or TASK-1 (Fig. 5E) exhibited single-channel openings consistent with their respective biophysical profiles, suggesting that TALK-1 and TASK-1 form functional channels on the ER membrane.

Fig. 5 Functional TALK-1 and TASK-1 channels are present in the ER membrane.

(A) Nuclear patch clamp of the outer nuclear membrane (ONM) permits detection of ER ion channels. (B) Representative image of isolated mouse islet nuclei with patch pipette positioned on nucleus. (C) Recordings obtained from the nucleus of a TREK-2–expressing HEK293 cell (representative of five nuclei). (D) Current trace obtained from the nucleus of a TALK-1–expressing HEK293 cell. Right: Representative current amplitude histograms (representative of eight nuclei). (E) As in (D) but recorded from the nucleus of a TASK-1–expressing cell (representative of seven nuclei). (F) Representative current traces obtained from WT mouse nuclei; patches held at −50 mV (representative of 42 nuclei). (G) Single-channel current-voltage relationships from nucleus recordings obtained from TALK-1–expressing (n = 8) and TASK-1–expressing (n = 7) HEK293 cells and WT islet cells (n = 42). (H) Percent of nuclei with K2P-channel–like channel activity detected in WT and TALK-1 KO β cells (n = 42 nuclei; four mice per genotype). Statistical significance was determined by Student’s t test; *P < 0.05.

These results implied that TALK-1 and TASK-1 regulate Ca2+ER homeostasis by allowing K+ flux across the ER membrane. We further tested whether TALK-1 modulation of Ca2+ER release depended on K+ flux by manipulating the cytosolic K+ concentration. Using digitonin-permeabilized HEK293 cells expressing wild-type or TALK-1 DN and the genetically encoded Ca2+ER indicator G-CEPIA1er (37), we examined Ca2+ER leak in response to SERCA inhibition with CPA. In the presence of K+, Ca2+ER leak was faster in cells expressing wild-type TALK-1 compared to TALK-1 DN (fig. S6, A to D). Therefore, K+ flux through TALK-1 supports the movement of Ca2+ across the ER membrane. We also examined whether TALK-1 functions as an ER K+ channel in primary cells by performing nuclear patch clamp recordings on nuclei isolated from wild-type and TALK-1 KO islets. We detected single-channel openings (Fig. 5F) with a current amplitude comparable to cloned TALK-1 in 55.6 ± 6.3% of wild-type islet cell nuclei (Fig. 5G). However, only 31.2 ± 2.7% of nuclei from TALK-1 KO islets displayed K2P-like channel openings (Fig. 5H). Together, our findings suggest that TALK-1 and TASK-1 form functional channels on the ER membrane, allowing for a K+ countercurrent that supports Ca2+ER leak and helps to set Ca2+ER.

TALK-1 regulation of β cell Ca2+ER handling modulates islet Ca2+ oscillations

To dissect the role of TALK-1 modulation of Ca2+ER during β cell Ca2+ influx, we controlled β cell Ca2+c influx with K+-induced depolarization of diazoxide-treated cells in the presence or absence of the SERCA inhibitor thapsigargin (Fig. 6A) (51). Under these conditions, the role of plasma membrane TALK-1 channels is effectively dissociated from its intracellular functions: Diazoxide circumvents the depolarizing effects of glucose by activating KATP channels, and K+ depolarization activates voltage-dependent Ca2+ channels (VDCCs) independently of K+ channel activity (52). Subtracting the control trace from the thapsigargin-treated trace revealed the Ca2+ER contribution to the Ca2+c signal (Fig. 6B). During Ca2+ influx, Ca2+ER uptake was observed (Fig. 6B, downward deflection), whereas Ca2+ER release occurred after the depolarizing K+ pulse (Fig. 6B, upward component). We found reduced Ca2+ER release in KO β cells (Fig. 6, B and C), in accordance with our finding that TALK-1 channel activity promotes Ca2+ER release.

Fig. 6 TALK-1 regulates Ca2+ER handling during plasma membrane Ca2+ influx in β cells.

(A) Intracellular Ca2+ oscillations in response to pulses of 45 mM K+ (K45) for 40 s in the presence or absence of thapsigargin (1.25 μM). Recordings were performed in the presence of 11 mM glucose, 2.5 mM Ca2+, and 125 μM diazoxide. (B) Subtraction of the thapsigargin-treated trace from the control trace in (A) reveals the kinetics of Ca2+ER uptake and release. (C) Quantification of average Ca2+ER uptake and release in WT and TALK-1 KO β cells (n = 3 mice per genotype). (D) Effect of CPA on glucose-stimulated Ca2+ influx in WT and KO islets. (E) AUC analysis of glucose-stimulated Ca2+ influx for periods corresponding to low glucose (2G), high glucose (11G), and CPA (11G + CPA) (n = 49 WT and 53 TALK-1 KO islets). Statistical significance was determined by Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.005.

β cell Ca2+ER release has been implicated in the activation of hyperpolarizing Ca2+-activated K+ currents (5355). When stimulated with glucose, KO islets show accelerated Ca2+ oscillations (28), which may be due to changes in Ca2+ER control of the Vm and VDCC activity. We tested this by depleting Ca2+ER using CPA in wild-type and TALK-1 KO islets undergoing glucose-stimulated Ca2+ oscillations (Fig. 6D). Under low-glucose conditions, basal Ca2+c concentrations were modestly lower in KO islets (Fig. 6E). Upon stimulation with high glucose, Ca2+ influx was significantly greater in KO islets (Fig. 6E). However, Ca2+ER depletion with CPA normalized Ca2+c concentrations in KO islets to similar to those in wild-type islets (Fig. 6E). Because depletion of Ca2+ER removes the contribution of the ER from the glucose-stimulated Ca2+c signal, this finding suggested that TALK-1 influences β cell Ca2+c by modulating Ca2+ER handling, which in turn regulates plasma membrane K+ currents and Ca2+ influx. Therefore, we proceeded to test the relationship between TALK-1 regulation of Ca2+ER release and β cell Ca2+-activated K+ currents.

The termination of each electrical oscillation is triggered by a slowly activating Ca2+-dependent K+ current termed Kslow, which is mediated by intermediate-conductance KCa channels, apamin-insensitive SK KCa channels, and KATP channels (5557). The β cell ER can release Ca2+ close to the plasma membrane (58), and Kslow activity is sensitive to Ca2+ER release (5355). In KO islets, Vm repolarization is reduced by about 50% at the termination of each electrical oscillation (28), suggesting that Kslow may be impaired in KO islets. We tested this notion by measuring Kslow in wild-type and KO β cells. Kslow amplitude (Fig. 7A, inset) was reduced in KO β cells by 48 ± 17% relative to wild-type β cells (Fig. 7, B and C). TALK-1 is not activated by Ca2+c in oocytes (59), and we also found that TALK-1 activity in HEK293 (Fig. 7D) or β cells (Fig. 7E) was insensitive to Ca2+c, making it unlikely that TALK-1 is a constituent channel of Kslow. These findings suggest that TALK-1 may modulate β cell Kslow indirectly through control of Ca2+ER homeostasis. To assess whether modulation of K2P channels activity affects depolarization-induced Ca2+ER uptake and release, we inhibited TASK-1 in α cells with ML365. We found that TASK-1 channel inhibition reduced Ca2+ER release induced by Vm depolarization (Fig. 7, G) (51), suggesting that TASK-1 facilitates α cell Ca2+ER release.

Fig. 7 Reduced Kslow currents are associated with altered Ca2+ER dynamics.

(A and B) Representative Kslow currents recorded from WT (A) and TALK-1 KO (B) β cells. The peak of the Kslow tail current is indicated by the arrow. (C) Quantification of Kslow currents recorded from WT and TALK-1 KO β cells (n = 26 cells; 4 mice per genotype). (D and E) Average whole-cell currents recorded in HEK293 cells expressing TALK-1 with intracellular buffer containing low Ca2+ (50 nM, black line) or high Ca2+ (5 μM, green line) in HEK293 [(D); n = 11 cells per condition] and mouse β cells [(E); n = 15 (50 nM Ca2+) and 13 cells (5 μM)]. (F) Depolarization-induced Ca2+ influx in mouse α cells treated with vehicle or ML365. (G) AUC analysis of rising (rise) and decaying (fall) phase of Ca2+ influx in α cells suggests reduced Ca2+-induced Ca2+ER release in ML365-treated α cells (n = 11 cells per condition). Statistical significance was determined by Student’s t test; *P < 0.05, ***P < 0.005. n.s., not significant.

TALK-1 channel activity exacerbates islet ER stress

Reduced β cell Ca2+ER content is associated with ER stress and islet dysfunction in diabetes (4, 6063). Our results indicated that TALK-1 channels were a determinant of β cell Ca2+ER concentrations and suggested that TALK-1 channel activity could exacerbate Ca2+ER depletion, which leads to ER stress. Therefore, we determined whether the absence of functional TALK-1 channels affected islet responses to the metabolic stress of a HFD. After 1 week of HFD feeding, the expression of genes involved in ER stress signaling did not differ between wild-type and TALK-1 KO islets (Fig. 8A). SERCA abundance is reported to change as a function of Ca2+ER content (64, 65); however, we did not detect differences in the expression of mRNAs encoding SERCA2b and SERCA3 in wild-type and TALK-1 KO islets. However, after prolonged (20 weeks) exposure to an HFD, TALK-1 KO islets exhibited decreased expression of multiple ER stress genes, as well as significantly decreased expression of mRNA encoding SERCA2b and SERCA3 (Fig. 8B). The decreased SERCA expression may represent a compensatory mechanism to reduce Ca2+ER overloading and maintain β cell Ca2+ER concentrations in an optimal range.

Fig. 8 TALK-1 channel activity exacerbates ER stress.

(A) Reverse-transcribed RNA from islets isolated from WT and TALK-1 KO mice fed a HFD for 1 week was subjected to real-time quantitative polymerase chain reaction (qPCR) to measure total Xbp1, spliced Xbp1, CHOP, BiP, Atp2a2 (SERCA2b), and Atp2a3 (SERCA3) expression (n = 4 to 5 mice per genotype). (B) Reverse-transcribed RNA from islets isolated from WT and TALK-1 KO mice fed a HFD for 20 weeks was subjected to real-time qPCR to measure total Xbp1, spliced Xbp1, CHOP, BiP, Dnajc3, Hsp90, Atp2a2 (SERCA2b), and Atp2a3 (SERCA3) expression (n = 3 to 4 mice per genotype). (C) INS-1 cells cotransfected with TALK-1 DN mutant, WT TALK-1, or TALK-1 A277E and an ATF6-promoter luciferase reporter (p5xATF6-GL3) were treated with vehicle (VHL) [dimethyl sulfoxide (DMSO); 0.0125%, v/v] or tunicamycin (Tm) (0.25 μg/ml) for 16 to 20 hours before cell lysis and luciferase assay (n = 4 independent experiments). (D) INS-1 cells were cotransfected with TALK-1 WT or TALK-1 A277E and pCMV-D4ER to measure basal Ca2+ER concentrations in 11 mM glucose (n = 4 independent experiments). Statistical significance was determined by Student’s t test; *P < 0.05, **P < 0.01.

We also assessed whether the T2DM-linked gain-of-function polymorphism (rs1535500) encoding TALK-1 A277E (28) interfered with ER function. Activating transcription factor 6 (ATF6) transcriptional activation occurs in response to Ca2+ER depletion and protein misfolding (65, 66), which we measured in INS-1 cells [with a luciferase reporter containing five tandem repeats of ATF6-binding sites (67, 68)] after application of tunicamycin, which inhibits protein glycosylation and causes protein misfolding and ER stress. INS-1 cells expressing wild-type TALK-1 or TALK-1 A277E were significantly more susceptible to tunicamycin-induced ATF6 activation than cells expressing the nonconducting TALK-1 DN. Furthermore, TALK-1 A277E activated ATF6 to a significantly greater extent than wild-type TALK-1 (Fig. 8C). However, coexpression of TALK-1 A277E with the TALK-1 DN reduced ATF6 activation to amounts comparable to that induced by expression of TALK-1 DN alone (Fig. 8C). INS-1 cells expressing TALK-1 A277E showed reduced Ca2+ER concentrations compared to cells expressing wild-type TALK-1 (Fig. 8D). Together, our findings indicate that TALK-1 channels control Ca2+ER fluxes in the islet, which may regulate plasma membrane ion channel activity, electrical excitability, and Ca2+ER concentrations important for protein processing (fig. S7).


Tight regulation of β cell Ca2+ER is required to sustain insulin synthesis, metabolism, and intracellular Ca2+ signaling, and perturbations in Ca2+ER handling contribute to diabetes pathogenesis. β cell Ca2+ER is controlled by several proteins including SERCAs (38, 62), IP3 receptors (69), ryanodine receptors (2, 69, 70), and the translocon (71). However, the ubiquitous distribution of most Ca2+ER handling proteins precludes their clinical use in treating diabetes. Here, we demonstrated that pharmacological manipulation of K2P channels can be used to control primary cell Ca2+ER. Moreover, our data indicated that inhibiting TALK-1 channel activity could protect islets from ER stress induced by prolonged exposure to a HFD. These observations suggest the exciting potential of using K2P channels such as TALK-1 as a therapeutic target to reduce β cell ER dysfunction under diabetic conditions.

Ca2+ER is determined by a balance of SERCA activity, Ca2+ER release, and Ca2+ER buffering. Ca2+ER release shifts the ER membrane potential [Vm(ER)] toward the Ca2+ER reversal potential [ECa2+(ER)], where net Ca2+ER efflux would stop. However, the K+ countercurrent across the ER membrane maintains Vm(ER) positive of ECa2+(ER), facilitating Ca2+ER release (16, 20, 49, 72). Our data indicate that ER TALK-1 K+ currents support the electrochemical driving force for Ca2+ER release, consistent with our observation that TALK-1 overexpression decreased Ca2+ER storage by increasing Ca2+ER leak. Conversely, inhibition of TALK-1 should move the Vm(ER) closer to ECa2+(ER), resulting in reduced Ca2+ER leak and increased Ca2+ER (15), a prediction in accordance with the phenotype of TALK-1 KO β cells.

Although a diminished contribution of Ca2+ER leak to bulk Ca2+c might be predicted to impair GSIS, TALK-1 KO islets exhibit increased glucose-stimulated Ca2+ influx and insulin secretion (28). These observations suggest that β cell Ca2+ influx through VDCCs is increased after loss of TALK-1 channels. Ca2+ER can play important roles in controlling plasma membrane channels that tune VDCC activity. For example, β cell Ca2+ER release influences insulin secretion through modulation of currents that hyperpolarize the Vm, such as Kslow, thereby indirectly controlling VDCC activity. In TALK-1 KO β cells, reduced Ca2+ER release presumably results in less Kslow activation, leading to Vm depolarization and more persistent electrical activity, culminating in a net increase in Ca2+c and enhanced GSIS. Ca2+ER release serves an important role in regulating second-phase Ca2+c influx and insulin secretion, which are increased when SERCAs are inhibited pharmacologically and is also observed in islets lacking SERCA3 (38, 73, 74). Similarly, we find that both second-phase Ca2+c and insulin secretion are increased in the absence of functional TALK-1 channels (28). These observations suggest that TALK-1 modulation of the Ca2+ER handling, which controls second-phase insulin secretion, is a key physiological function of β cell TALK-1 channels.

As mentioned above, TALK-1 KO islets exhibit increased insulin secretion, a higher frequency of Ca2+c oscillations, and increased plateau fraction (the fraction of time spent in electrically excitable periods) (28). The present study helps resolve the molecular mechanisms underlying these phenotypes. A rationale for the increased plateau fraction and oscillation frequency in TALK-1 KO islets is that ER TALK-1 channels sustain Ca2+ER release, which results in greater Kslow activity and Vm hyperpolarization. Because this Ca2+ER release is reduced in TALK-1 KO β cells, Kslow activation is diminished, resulting in an increased plateau fraction. This is in accordance with observations that depletion of Ca2+ER inhibits Kslow and accelerates Ca2+c oscillations (53, 75). Ca2+ER concentrations also determine the activation of depolarizing store-operated currents, as demonstrated by the acceleration of Vm and Ca2+c oscillations after treatment of islets with SERCA inhibitors (7577), presumably due to inhibition of Kslow and activation of SOCE. These observations highlight that SOCE serves an important role in shaping islet Ca2+c oscillations, and future studies are required to dissect the relationship between TALK-1 modulation of Ca2+ER stores, SOCE, Vm and Ca2+c oscillations, and insulin secretion.

ER K+ channels are also important for Ca2+ER uptake, as demonstrated by the influence of ER-localized SK channels in regulating neuronal and cardiomyocyte ER and SR (sarcoplasmic reticulum) Ca2+ uptake (20). SK channels are predicted to preserve ER pH homeostasis through activation of an ER K+/H+ antiporter that promotes ER H+ entry to balance SERCA-mediated H+ loss during Ca2+ uptake (20). Although we cannot exclude a role for K2P channels in modulating SERCA function, we found reduced basal Ca2+ER when TALK-1 was heterologously expressed and conversely found increased Ca2+ER in TALK-1 KO β cells. These findings suggest that if TALK-1 controls Ca2+ER uptake, it would presumably do so by inhibiting SERCA function, in contrast to SK channels, which enhance SERCA function. Any effects of TALK-1 on SERCA activity could be through indirect mechanisms modulated by Ca2+ER, such as mitochondrial ATP production, which energizes the β cell SERCA pump (78).

Our observations suggested that TALK-1 activity controls Ca2+ER, which affects many aspects of β cell function in health and disease. In addition to controlling Ca2+c signals, another essential function of β cell Ca2+ER handling is to maintain insulin production and processing, which is impaired under conditions of β cell stress induced by insulin resistance or decreased β cell mass. A hallmark of ER stress is increased Ca2+ER leak (2), which can be caused in β cells by reductions in the abundance of proteins that affect Ca2+ER such as SERCA2b (60, 62) or sorcin (68). Our data indicate that TALK-1 activity aggravated islet ER stress under conditions of increased systemic insulin demand. Moreover, our finding that T2DM-associated TALK-1 A277E channels exacerbated Ca2+ER leak and ER stress responses suggests that defects in TALK-1 channel activity could contribute to islet ER dysfunction in diabetes. TALK-1 transcript abundance is reduced under conditions that cause ER stress in diabetes [such as palmitate or inflammatory cytokine treatment (79)], which may be a protective mechanism to preserve β cell Ca2+ER homeostasis by reducing Ca2+ER leak. It will be important to determine how TALK-1 participates in the cellular response to other diabetes-associated ER stressors.

Mutations in other K2P channels have also been associated with various disorders that may be associated with defects in Ca2+ER handling. For example, dominant-negative mutations in TASK-1 or TASK-3 result in PAH or Birk-Barel syndrome, respectively (47). We found that a PAH-linked mutation in TASK-1 (G203D) enhanced Ca2+ER stores relative to wild-type TASK-1. Thus, in patients with the TASK-1 G203D mutation, disruptions in ER/SR Ca2+ handling may contribute to PAH (80). In pulmonary arterial smooth muscle cells, impaired Ca2+ER transfer to mitochondria leads to pulmonary vascular remodeling, a defect that can be targeted with clinically used chemical chaperones to ameliorate PAH (80, 81). Inhibition of TASK-1 using the specific inhibitor A293 causes pulmonary vascular remodeling and PAH in rats, and pharmacological activation of TASK-1 protects from the development of PAH (82). TASK-3 also controls Ca2+ER, and a mutation in KCNK9 (which encodes TASK-3, G236R) causes Birk-Barel syndrome, which is characterized by intellectual disability, hypotonia, and facial dysmorphism. TASK-3 is also implicated in mitochondrial function (83), highlighting the importance of determining the relationship between TASK-3 modulation of Ca2+ER handling and mitochondrial function. Because we found that pharmacological regulation of TASK-1 could control primary cell Ca2+ER, K2P channels (such as TASK-1, TASK-3, or TALK-1) could be targeted for cell-selective therapies to reduce ER dysfunction.

Not all K2P channels regulate Ca2+ER, as demonstrated by our finding that neither TREK-1 channels nor TREK-2 channels affected Ca2+ER homeostasis. These findings could be due to localization of these channels; TREK-1 channels are found primarily on the plasma membrane (84), whereas the subcellular localization of TREK-2 channels has not been determined. However, all K+ channels are assembled in the ER before their delivery to the plasma membrane (85). It remains to be determined how certain K2P channels (specifically, TALK-1, TASK-1, and TASK-3) regulate Ca2+ER, whereas others (specifically, TREK-1 and TREK-2) do not. Because many K+ channels, such as KATP, require a physical interaction with the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to conduct K+, K2P channels that are dependent on PIP2 for activity may not function in the ER membrane. This property could be due to intrinsically low PIP2 concentrations on the ER membrane, which limits K+ channel activity until they are transported to the plasma membrane (86). TREK-1 is highly sensitive to PIP2, whereas both TASK-1 and TASK-3 channels, both of which affect Ca2+ER, are insensitive to PIP2 (87). Future studies are needed to better understand the regulatory mechanisms underlying K2P channel activity in the ER and how these affect Ca2+ER homeostasis.

In conclusion, we demonstrate that TALK-1 in the ER regulates Ca2+ER handling, thus controlling Kslow activity, Ca2+ influx, and insulin secretion. These findings highlight a physiological function of K2P channels in the regulation of Ca2+ER. K2P channels may provide cell-selective targets to modulate Ca2+ER to treat the many diseases characterized by dysfunctional Ca2+ER handling.



Unless otherwise specified, all reagents were obtained from Sigma-Aldrich.

Mouse models

The mice used in this study were 8- to 12-week-old males on a C57Bl6/J background. The generation of Kcnk16−/− (TALK-1 KO) mice has been previously described (28). For experiments using mouse α cells, transgenic mice expressing tandem-dimer red fluorescent protein specifically in α cells were used (46). All mice used in this study were handled in compliance with protocols reviewed and approved by the Vanderbilt University Institutional Animal Care and Use Committee, according to guidelines set forth by the National Institutes of Health.

Islet isolation and culture

Mouse islets were isolated using collagenase P (Roche) digestion of the pancreas and density gradient centrifugation (5). Human islets from adult nondiabetic donors (donor information is provided in table S1) were obtained through isolation centers organized by the Integrated Islet Distribution Program. In experiments using D4ER, cells were transduced with Ad-D4ER (38) 48 hours before imaging. In human β cell experiments, cells were transfected with TALK-1 DN– or mCherry-expressing plasmids (28). Islets and dispersed cells were cultured for 24 to 48 hours before experimentation (28).

Cell culture and luciferase assays

The development of T-REx-293 cells with inducible expression of K2P channels has been previously described (43). To measure TALK-1 and TASK-1 expression in induced cells, we ran lysates from TALK-1– or TASK-1–T-REx-293 cells treated for 24 hours with or without tetracycline (1 μg/ml) induction on 4 to 12% bis-tris polyacrylamide gels (Invitrogen). The protein was then transferred to a nitrocellulose blotting membrane (Bio-Rad), which was probed with TALK-1 (#NBP1-83071, Novus Biologicals) or TASK-1 (#49433, Abcam) antibodies. Equal loading of wells was assessed by stripping and reprobing membranes with a β-actin antibody (#4970, Cell Signaling Technologies). Representative blots are shown in fig. S4. For experiments comparing the effects of wild-type TALK-1 and TALK-1 DN on Ca2+ER handling, HEK293 cells were transfected with pcDNA3.1 plasmids encoding these channels using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions and imaged 48 hours after transfection.

INS-1 (832/13) cells were cultured in RPMI 1640 supplemented with 15% fetal bovine serum and penicillin-streptomycin. INS-1 cells were transfected with p5xATF6-GL3 (#11976, Addgene) and plasmids encoding wild-type TALK-1, TALK-1 A277E, or TALK-1 DN (28). Cells were incubated overnight with vehicle (DMSO) or tunicamycin (0.25 μg/ml) for 16 to 20 hours before performing a luciferase assay using the Steady-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions.

Patch clamp electrophysiology

An Axopatch 200B amplifier (Molecular Devices) was used to measure whole-cell K+ channel currents in the voltage-clamp mode; currents were digitized using a Digidata 1440, low-pass–filtered at 1 kHz and sampled at 10 kHz. For Kslow recordings, pipettes were filled with an intracellular solution (57) containing 28.4 mM K2SO4, 63.7 mM KCl, 11.8 mM NaCl, 1 mM MgCl2, 20.8 mM Hepes, 0.5 mM EGTA (pH 7.22 with KOH), and amphotericin B (~0.05 mg ml−1). Nuclear patch clamp experiments were performed using the approach described by Mak and colleagues (50). Nuclei were patched in a solution containing 150 mM KCl, 10 mM Hepes, 0.5 mM EGTA, and 0.36 mM CaCl2 (pH 7.3 with KOH). Patch electrodes were pulled to a resistance of 8 to 10 megaohms, loaded with recording solution, and coated with Sigmacote. Single-channel currents were low-pass–filtered at 1 kHz and sampled at 50 kHz. When intracellular [Ca2+] was clamped, cells were recorded using the whole-cell configuration using electrodes filled with a solution containing 140 mM KCl, 5 mM Hepes, 4 mM Mg·ATP, 1 mM EGTA, and 137 μM (for 50 nM Ca2+ final) or 946 μM (for 5 μM Ca2+ final) CaCl2 (pH 7.22 with KOH). [Ca2+] was determined using MAXCHELATOR software. The extracellular buffer used for islet cells [a modified Krebs-Ringer buffer (KRB)] contained 119 mM NaCl, 2.5 mM CaCl2, 4.7 mM KCl, 25 mM Hepes, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 11 mM glucose (pH 7.35 with NaOH). The extracellular buffer used for HEK293 cells (HEK buffer) contained 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2.5 mM CaCl2, 10 mM Hepes, and 10 mM glucose (pH 7.35 with NaOH). When assessing the Ca2+ sensitivity of TALK-1 in β cells, the extracellular buffer was supplemented with a cocktail of K+ channel inhibitors including 200 μM tolbutamide (MP Biomedicals), 10 mM tetraethylammonium chloride (Acros Organics), 100 nM apamin (Alomone Labs), 100 nM iberiotoxin (Alomone Labs), 100 nM TRAM-34 (Alomone Labs), and 10 μM nifedipine to inhibit VDCCs. Cells were recorded with a voltage-clamp protocol used to assess K2P channel currents (28). Recordings were analyzed using Clampfit 10 (Molecular Devices) and Microsoft Excel software.

Calcium imaging

Mouse and human β cells were loaded with 2 μM Fura-2 AM (Molecular Probes) and imaged as previously described (88). CPA (Alomone Labs) was used at a concentration of 50 μM; ionomycin was used at a concentration of 5 μM (Alomone Labs). Human β cells were poststained for insulin (88). In all experiments, cells were perfused with a flow of 2 ml min−1 at 37°C. For analysis of mouse β cell Ca2+ER uptake and release (51), Fura-2–loaded cells were incubated for 10 min in KRB supplemented with 11 mM glucose, 125 μM diazoxide (Enzo), and 1.25 μM thapsigargin (Alomone Labs) or vehicle. In high-[K+] stimulus buffer, NaCl was reduced accordingly to maintain osmolarity. For experiments using D4ER, cells were incubated for 20 min in KRB containing 2 mM glucose before imaging.

For assays comparing the effects of expression of K2P channels in stably transduced T-REx-293 cells, 30,000 cells per well were seeded to 384-well black-wall, clear-bottom, amine-coated plates (BD Biosciences). Channel expression was induced with tetracycline (1 μg ml−1) in culture medium, and the cells were cultured overnight in a 5% CO2 incubator at 37°C. The cells were washed using an ELx405CW plate washer (Bio-Tek Instruments Inc.) and loaded with 4 μM Fluo-4 AM (Molecular Probes) for 45 min in a 5% CO2 incubator at 37°C. The cells were then washed with buffer supplemented with 1 mM EGTA and incubated in a 5% CO2 incubator at 37°C for 8 min before imaging. Plates were then loaded into a whole-plate kinetic-imaging Functional Drug Screening System (FDSS 6000, Hamamatsu) and imaged at 37°C as previously described (43).

When assessing the effects of TASK-1 or TASK-3 channel blockade, cells were loaded 3 hours before imaging with 500 nM ML365 (Tocris) or DMSO vehicle in culture medium, and in α cells, the culture medium also contained 125 μM diazoxide. ML365 was present throughout the experiment. For high-speed imaging of α cell Ca2+ influx, cells were loaded with 5 μM Fluo-4 AM for 25 min, followed by washing with KRB (11 mM glucose). α Cells were then patched according to the perforated patch clamp protocol described above on a Nikon Eclipse TE2000-U microscope equipped with an X-Cite 120Q widefield fluorescence light source (Excelitas Technologies) and a D-104 microscope photometer (Photon Technologies Inc.). Upon obtaining a low-leak, gigaohm seal, the fluorescence light source was activated, and plasma membrane currents were recorded using the Kslow voltage-clamp protocol (57) simultaneously with Fluo-4 fluorescence. Currents and photometer signal were digitized and sampled at 10 kHz. For analysis of the effects of TALK-1 on the CPA-induced Ca2+ER leak rate, T-REx-293 cells transfected with wild-type TALK-1 or TALK-1 DN and CEPIA1-ER (#58215, Addgene) (37) were permeabilized for 4 min in a 5% CO2 incubator at 37°C in an intracellular buffer containing 140 mM potassium gluconate or 140 mM tris base (K+-free), 10 mM Hepes, 1 mM EGTA, 0.432 mM CaCl2, and 3 mM Mg·ATP, with sucrose added as needed to match osmolarity (pH 7.24), and supplemented with 50 μg ml−1 of digitonin (Santa Cruz Biotechnology). Cells were then washed for an additional 5 min in the appropriate buffer without digitonin before the start of imaging. To determine the rate constant of CPA-induced Ca2+ leak, we fitted the normalized data to a one-phase exponential decay model using GraphPad Prism 7 software. Data were analyzed using Nikon Elements, Microsoft Excel, Clampfit 10, and GraphPad Prism 7 software.

Site-directed mutagenesis

The TASK-1 G203D point mutation was generated using a previously described approach (28). The sequences of oligonucleotide primers (Integrated DNA Technologies) used to create the TASK-1 G203D mutant were 5′-ACCACCATCGGCTTCGACGACTACGTGGCGCTGCAGA-3′ (forward) and 5′-TCTGCAGCGCCACGTAGTCGTCGAAGCCGATGGTGGT-3′ (reverse). PCRs were performed in 50 μl using Q5 High-Fidelity DNA Polymerase (New England Biolabs) with 100 ng of pcDNA3.1-KCNK3 plasmid. DNA was then incubated with 1 μl of DpnI for 2 hours at 37°C. Clones were sequenced to confirm mutagenesis.

Real-time quantitative polymerase chain reaction

qPCR of cDNA obtained from mouse islets was performed according to the approach described by Tong and colleagues (62). A list of primers can be found in table S3.


Processing and staining of paraffin-embedded mouse and human pancreas sections was performed as previously described (human donor information is provided in table S2) (28). Sections were stained using primary antibodies against TALK-1 (1:175; #NBP1-83071, Novus Biologicals) and calreticulin (1:125; #N-19, Santa Cruz Biotechnology); secondary antibodies used were Alexa Fluor 488–conjugated donkey anti-rabbit (1:300; #711-546-152, Jackson ImmunoResearch) and DyLight 650–conjugated donkey anti-goat (1:250; #SA5-10089, Thermo Fisher Scientific). HEK293 cells cotransfected with TALK-1a or TALK-1b (28) and ER-targeted enhanced yellow fluorescent protein (#56589, Addgene) were washed twice with cold phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 30 min at 4°C. Cells were then incubated in PBS supplemented with 0.2% bovine serum albumin (BSA), 2% normal donkey serum (NDS; Jackson ImmunoResearch), and 0.05% Triton X-100 for 1 hour, followed by incubation in PBS containing primary antibodies against TALK-1 (1:175) and green fluorescent protein (1:300; NB600-597, Novus Biologicals), 0.2% BSA, 1% NDS, and 0.1% Triton X-100, overnight at 4°C. After removal of the primary antibody solution, the cells were subjected to two 10-min PBS washes and then incubated in the dark for 1 hour at room temperature in PBS containing 1% NDS and secondary antibodies: Alexa Fluor 488–conjugated donkey anti-rabbit (1:300) and Alexa Fluor 647–conjugated goat anti-mouse (1:300; A21237, Thermo Fisher Scientific). The secondary antibody solution was removed, and the cells were subjected to three 8-min PBS washes before imaging. All images were obtained using a Zeiss LSM 710 or Zeiss LSM 780 confocal laser scanning microscope. Images were analyzed using ImageJ software.

For analysis of islet cell numbers, paraffin-embedded pancreata were processed as described above and stained using primary antibodies against insulin (1:500; #A0564, Dako), somatostatin (1:250; sc-7819, Santa Cruz Biotechnology), and glucagon (1:500; #15954-I-AP, Proteintech); secondary antibodies used were Alexa Fluor 488–conjugated donkey anti-rabbit (1:500; #711-546-152, Jackson ImmunoResearch), DyLight 650–conjugated donkey anti-goat (1:250; #SA5-10089, Thermo Fisher Scientific), and Cy3-conjugated donkey anti–guinea pig (1:500; #706-165-148, Jackson ImmunoResearch).

For analysis of HFD-induced islet-cell proliferation, age-matched wild-type and TALK-1 KO mice were placed on a HFD (60% kcal per fat; #D12492, Research Diets) for 10 days. Four days before sacrifice, mice were provided with drinking water containing BrdU (0.8 mg ml−1) supplemented with Splenda artificial sweetener (20 mg ml−1). Paraffin-embedded pancreata were processed as described above and were subjected to antigen retrieval performed in 1× NaCitrate (pH 6.0), for 14 min in a microwave at high power, followed by cooling at room temperature in 1× NaCitrate solution for 25 min. After antigen retrieval, slides were washed for 10 min in double-distilled H2O (ddH2O), followed by two 2-min washes in PBS. Sections were the stained using primary antibodies against insulin (1:500; #A0564, Dako), somatostatin (1:250; sc-7819, Santa Cruz Biotechnology), glucagon (1:500; #15954-I-AP, Proteintech), and BrdU (1:50; #G3G4, Developmental Studies Hybridoma Bank). Secondary antibodies used were Alexa Flour 647–conjugated goat anti-mouse (1:250; #A21237, Life Technologies), DyLight 488–conjugated donkey anti-mouse (1:300; #SA5-10166, Thermo Fishers Scientific), and Alexa Flour 594–conjugated donkey anti-guinea pig (1:400; #706-586-148, Jackson ImmunoResearch), DyLight 650–conjugated donkey anti-goat (1:250; #SA5-10089, Thermo Fisher Scientific), and Alexa Fluor 488–conjugated donkey anti-rabbit (1:500; #711-546-152, Jackson ImmunoResearch). Blocking was performed in a dark humidity chamber for 1 hour using Dako blocking solution (reference no. X0909). Primary antibodies were diluted to above concentrations in Dako antibody diluent solution (reference no. S3002) and incubated on the sections overnight at 4°C. After primary antibody incubation, slides were washed for 5 min in PBS twice. Secondary antibodies were diluted to the above concentrations in PBS supplemented with 5% NDS and incubated on slides in the dark for 2 hours at room temperature. Sections were then washed twice for 5 min in PBS, and 4′,6-diamidino-2-phenylindole (DAPI) was added (1:1000 for 2 min). After DAPI staining, sections were washed for 5 min in ddH2O and then mounted with a coverslip.

All sections were imaged with an Aperio ImageScope and analyzed using an algorithm developed with CytoNuclear FLv1.2 software (Aperio/Indica Labs). The algorithm is designed to take into account factors, such as nuclear staining, cytoplasm radius, nucelar size, nuclear roundness, and dye fluorescence wavelength (Cy2, Cy3, or Cy5), to identify, differentiate, and count β, δ, and α cells. The algorithm was also used to count the number of β, δ, and α cells on the slides labeled with BrdU, which was further analyzed using ImageJ software and Microsoft Excel.


The data were presented as recordings that are averaged or representative of results obtained from at least three independent cultures. All values presented are means ± SEM. Statistical differences between means were assessed using Student’s t test or one-way ANOVA, as appropriate. The significance of all experimental findings presented as fold changes was assessed by performing statistical tests on log-transformed data. P < 0.05 was considered as significant.


Fig. S1. TALK-1 exhibits ER localization.

Fig. S2. Islet cell number and proliferation are not modulated by TALK-1 activity.

Fig. S3. Intracellular Ca2+ stores are increased in TALK-1 KO islet cells.

Fig. S4. Tetracycline-inducible expression of TALK-1 and TASK-1.

Fig. S5. TASK-3 and TASK-1 K2P channel activity alter Ca2+ER concentrations.

Fig. S6. Ca2+ER leak is accelerated by TALK-1 channels.

Fig. S7. Hypothetical model depicting potential molecular mechanisms of TALK-1 channel modulation of β cell Ca2+ER handling and Ca2+C oscillations.

Table S1. Human islet donor characteristics.

Table S2. Human pancreas donor characteristics.

Table S3. Primers used for real-time qPCR.


Acknowledgments: We are grateful for the helpful discussions and input from the laboratory of A. Powers. We thank M. Merrins, University of Wisconsin–Madison, for sharing reagents used for Ca2+ER imaging experiments and for critical review of the manuscript; T. Pozzan, University of Padua, for providing pCMV-D4ER plasmid; and S. Omer for contributions to Ca2+ imaging experiments performed during this study. Human islets were procured through the Integrated Islet Distribution Program organized by National Institute of Diabetes and Digestive and Kidney Diseases. We acknowledge the assistance of the Vanderbilt Translational Pathology Shared Resource in the preparation and processing of paraffinembedded pancreata (2P30 CA068485-14; 5U24DK059637-13). Imaging and analysis of stained pancreas sections were performed with the assistance of the Vanderbilt Islet Procurement and Analysis Core (DK020593). Funding: This project was funded by NIH grants K01DK081666 and R01DK097392, Vanderbilt Diabetes Research and Training Center Pilot and Feasibility grant P60DK20593, and American Diabetes Association grant 1-17-IBS-024 (D.A.J.); Vanderbilt Molecular Endocrinology Training Program (METP) grant 5T32DK07563 and NIH grant 1F31DK109625 (N.C.V.); Vanderbilt METP grant 5T32DK007563-28 (S.C.M.); and Vanderbilt Integrated Training in Engineering and Diabetes grant T32DK101003 (M.T.D.). P.G. is the research director of the Fonds de la Recherche Scientifique, Brussels, Belgium. Confocal microscopy was performed using the Vanderbilt Cell Imaging Shared Resource (DK020593). Author contributions: N.C.V. and D.A.J. designed the project. N.C.V., P.G., and D.A.J developed the methodology. N.C.V., P.K.D., S.C.M., M.T.D., K.L.J., and D.A.J. performed experiments and analyzed the data. N.C.V. and D.A.J. wrote and edited the manuscript. Competing interests: The authors declare that they have no competing interests.
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