Skip to Content
MilliporeSigma
  • STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes.

STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes.

Proceedings of the National Academy of Sciences of the United States of America (2015-08-12)
Guiling Zhao, Tianyu Li, Didier X P Brochet, Paul B Rosenberg, W J Lederer
ABSTRACT

In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca(2+) sensor, is unclear with respect to its cellular localization, its Ca(2+)-dependent mobilization, and its action on Ca(2+) signaling. Confocal microscopy was used to measure Ca(2+) signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional SR (jSR). Depleting SR Ca(2+) using thapsigargin (2-10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca(2+) depletion. Additionally, we found no store-operated Ca(2+) entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca(2+) content and increased SR Ca(2+) leak. These changes in Ca(2+) signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca(2+) ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca(2+) leak and that these actions are independent of store-operated Ca(2+) entry, a process that is absent in normal heart cells.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Potassium hydroxide, anhydrous, ≥99.95% trace metals basis
Sigma-Aldrich
Potassium hydroxide solution, 0.1 M in ethanol
Sigma-Aldrich
Calcium chloride solution, 0.025 M
Sigma-Aldrich
Potassium hydroxide solution, 0.5 M
Sigma-Aldrich
Potassium hydroxide solution, 0.1 M
Sigma-Aldrich
Potassium hydroxide solution, 0.5 M in ethanol
Sigma-Aldrich
Calcium chloride solution, 3.2 mM
Sigma-Aldrich
Glycerol, JIS special grade, ≥99.0%
Sigma-Aldrich
Glycerol, SAJ first grade, ≥98.0%
Sigma-Aldrich
Potassium hydroxide solution, 0.02 M in ethanol
Sigma-Aldrich
Potassium hydroxide solution, 1 M
Sigma-Aldrich
Potassium hydroxide solution, 0.1 M in isopropanol
Sigma-Aldrich
Sodium hydroxide-16O solution, 20 wt. % in H216O, 99.9 atom % 16O
Sigma-Aldrich
Calcium chloride, anhydrous, powder, 99.99% trace metals basis
Sigma-Aldrich
Glycerol, FCC, FG
Sigma-Aldrich
Calcium chloride, AnhydroBeads, −10 mesh, ≥99.9% trace metals basis
Sigma-Aldrich
Sodium phosphate, 96%
Sigma-Aldrich
Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2O
Sigma-Aldrich
Magnesium chloride, AnhydroBeads, −10 mesh, 99.99% trace metals basis
Sigma-Aldrich
Magnesium chloride, AnhydroBeads, −10 mesh, 99.9% trace metals basis
Sigma-Aldrich
Glycerol, BioUltra, for molecular biology, anhydrous, ≥99.5% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 10% in H2O
Sigma-Aldrich
Calcium chloride, AnhydroBeads, −10 mesh, ≥99.99% trace metals basis
Sigma-Aldrich
Magnesium chloride, powder, <200 μm
Sigma-Aldrich
Calcium chloride solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Magnesium chloride, BioReagent, suitable for insect cell culture, ≥97.0%
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
Calcium chloride
Sigma-Aldrich
Glycerol, for molecular biology, ≥99.0%
Sigma-Aldrich
Glycerol, BioXtra, ≥99% (GC)