STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes.
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.
Laboratory of Molecular Cardiology, Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MD 21201; Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201;
Guiling
G
Zhao
Department of Medicine, Ion Channel Research Group, Sarah Steadman Nutrition and Metabolism Center, Duke University School of Medicine, Durham, NC 27710.
Tianyu
T
Li
Laboratory of Molecular Cardiology, Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MD 21201; Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201;
Didier X P
DX
Brochet
Department of Medicine, Ion Channel Research Group, Sarah Steadman Nutrition and Metabolism Center, Duke University School of Medicine, Durham, NC 27710.
Paul B
PB
Rosenberg
Laboratory of Molecular Cardiology, Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MD 21201; Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201; jlederer@umaryland.edu.
W J
WJ
Lederer
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D006352:Heart Ventricles / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000502:physiology*; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D000071737:Stromal Interaction Molecule 1; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D000071737:Stromal Interaction Molecule 1; D000099211:Phospholamban; C518857:Stim1 protein, rat
STIM1; diffusion coefficient; heart; phospholamban; store-operated calcium entry