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Research Article
Endoplasmic reticulum potassium–hydrogen exchanger and small conductance calcium-activated potassium channel activities are essential for ER calcium uptake in neurons and cardiomyocytes
Malle Kuum, Vladimir Veksler, Joanna Liiv, Renee Ventura-Clapier, Allen Kaasik
Journal of Cell Science 2012 125: 625-633; doi: 10.1242/jcs.090126
Malle Kuum
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Vladimir Veksler
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Joanna Liiv
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Renee Ventura-Clapier
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Allen Kaasik
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  • For correspondence: allen.kaasik@ut.ee
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  • Fig. 1.
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    Fig. 1.

    Replacement of K+ ions with Na+ or TEA+ inhibits in situ ER Ca2+ uptake. (A–D) Time course of luminal Ca2+-dependent fluorescence after Ca2+ (100 nM) addition (left panels) and mean values of maximal fluorescence (right panels) in GT1-7 cells (A,C) or in primary cortical neurons (B,D). Physiological K+ (ctrl) was replaced with equimolar Na+ (A,B) or TEA+ (C,D). (E) Tension transients elicited by 5 mM caffeine in ventricular permeabilized fibers after 5 minutes of SR Ca2+ loading in the presence of K+ or TEA+ (left panel) and mean values of tension–time integrals (right panel). Here and in subsequent figures *P<0.05, **P<0.01, ***P<0.001 versus control conditions (taken as 1).

  • Fig. 2.
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    Fig. 2.

    Inhibitors of KATP channels as well as KATP channel openers have no effect on ER Ca2+ uptake in GT1-7 neurons. Time course of luminal Ca2+-dependent fluorescence after Ca2+ addition (left panels) and mean values of maximal fluorescence (right panels) in the presence of KATP channel inhibitors 5-HD (100 μM; A) and glimepiride (glim, 50 μM; B), and KATP channel openers pinacidil (pin, 200 μM; C) and diazoxide (diaz, 150 μM; D).

  • Fig. 3.
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    Fig. 3.

    Inhibitors of SKCa channels, UCL 1684 and dequalinium inhibit ER Ca2+ uptake. (A,B,D,E) Time course of luminal Ca2+-dependent fluorescence after Ca2+ addition (left panels) and mean values of maximal fluorescence (right panels) in GT1-7 neurons (A,D) or in primary cortical neurons (B,E) in the presence of UCL 1684 (50 μM; A,B) or dequalinium (deq, 100 μM; D,E) as compared with control conditions (ctrl). (C,F) Force transients in ventricular permeabilized fibers elicited by 5 mM caffeine after 5 minutes of SR Ca2+ loading in the presence of UCL 1684 (C) or dequalinium (F; left panels) and mean values of tension–time integrals (right panels).

  • Fig. 4.
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    Fig. 4.

    Effects of KHE and NHE inhibitors on ER Ca2+ uptake in neurons, and force transients in ventricular permeabilized fibers elicited by caffeine after SR Ca2+ loading. (A–C) Propranolol (prop, 500 μM) inhibited ER Ca2+ uptake in GT1-7 cells (A), primary cortical neurons (B) and cardiac fibers (C). (D) Inhibitory effects of propranolol on Ca2+ uptake in cardiac fibers was opposed by 30 μM nigericin. (E,F) Quinine (quin, 500 μM; E) but not cariporide (carip, 50 μM; F) had an inhibitory effect on ER Ca2+ uptake in GT1-7 neurons.

  • Fig. 5.
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    Fig. 5.

    SKCa, KATP channels and LETM1 colocalize with ER in cortical neurons. Antibody staining of SKCa (A), KATP staining (glibenclamide-BODIPY FL, B) and antibody staining of LETM1 (C) partially overlap with the ER marker pDsRed2-ER. Left panels show dispersed pattern of signals. Higher magnification images (right panels) of the boxed regions show that signals from the channels and LETM1 clearly colocalize with the ER marker. Arrows indicate finger-like invaginations of the ER–nuclear membrane.

  • Fig. 6.
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    Fig. 6.

    SKCa and KATP channels and LETM1 visualization in cortical neurons expressing a fluorescent mitochondrial marker (DsRed2-mito). Staining of SKCa and LETM1 with antibodies and KATP with glibenclamide-BODIPY FL shows that these proteins are not restricted to mitochondria.

  • Fig. 7.
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    Fig. 7.

    Imaging of KATP channels, LETM1 and SKCa in cardiomyocytes. (A,B) Mitochondria visualized with Mitotracker Green (higher magnification in B). (C) T-tubules visualized with DiOC16. (D,E) SR visualized with anti-SERCA2 (higher magnification in E). (F,G) KATP signal (glibenclamide-BODIPY FL) is mostly mitochondrial (higher magnification in G). (H,I) Antibody staining of LETM1 (higher magnification in I) is the same as that of the SR. (J,K) Antibody staining of SKCa (higher magnification in K) is predominantly similar to that of the SR although some dispersed signal could be coming from mitochondria.

  • Fig. 8.
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    Fig. 8.

    Western blot analysis of the ER-enriched fraction from brain. This fraction contains SKCa (lane 1) and LETM1 (lane 2). Note that this ER fraction is rich in SERCA2 (lane 3) but is free of mitochondrial contamination of cytochrome c oxidase (lane 4). This mitochondrial protein is abundantly present in the mitochondria-enriched fraction (lane 5, positive control).

  • Fig. 9.
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    Fig. 9.

    Proposed model of counterion movement through the ER membrane during Ca2+ uptake. Ca2+ ions are pumped into the ER lumen by SERCA. This is accompanied by countertransport of protons (two to three protons per two Ca2+ ions). To compensate for the remaining positive charge, Cl− ions enter through CIC channels that are associated with extrusion of protons (one proton per two Cl− ions). To avoid matrix alkalization and concomitant inhibition of SERCA, protons should re-enter through KHE (or LETM1). To compensate for the loss of K+ ions from the lumen, K+ might re-enter through SKCa channels open only during the uptake phase.

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Research Article
Endoplasmic reticulum potassium–hydrogen exchanger and small conductance calcium-activated potassium channel activities are essential for ER calcium uptake in neurons and cardiomyocytes
Malle Kuum, Vladimir Veksler, Joanna Liiv, Renee Ventura-Clapier, Allen Kaasik
Journal of Cell Science 2012 125: 625-633; doi: 10.1242/jcs.090126
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Research Article
Endoplasmic reticulum potassium–hydrogen exchanger and small conductance calcium-activated potassium channel activities are essential for ER calcium uptake in neurons and cardiomyocytes
Malle Kuum, Vladimir Veksler, Joanna Liiv, Renee Ventura-Clapier, Allen Kaasik
Journal of Cell Science 2012 125: 625-633; doi: 10.1242/jcs.090126

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