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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
  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/497/eaan2883/DC1

    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.

  • Supplementary Materials for:

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

    Nicholas C. Vierra, Prasanna K. Dadi, Sarah C. Milian, Matthew T. Dickerson, Kelli L. Jordan, Patrick Gilon, David A. Jacobson*

    *Corresponding author. Email: david.a.jacobson{at}vanderbilt.edu

    This PDF file includes:

    • 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.

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    Citation: N. C. Vierra, P. K. Dadi, S. C. Milian, M. T. Dickerson, K. L. Jordan, P. Gilon, D. A. Jacobson, TALK-1 channels control β cell endoplasmic reticulum Ca2+ homeostasis. Sci. Signal. 10, eaan2883 (2017).

    © 2017 American Association for the Advancement of Science

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