Marek K Korzeniowski, Barbara Baird, David Holowka
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引用次数: 8
Abstract
Oligomerization of the Ca2+ sensor, STIM1, in the endoplasmic reticulum (ER) membrane, caused by depletion of ER Ca2+ stores, results in STIM1 coupling to the plasma membrane Ca2+ channel protein, Orai1, to activate Ca2+ influx in a process known as store-operated Ca2+ entry. We use fluorimetry-based fluorescence resonance energy transfer (FRET) to monitor changes in STIM1 oligomerization in COS7 cells transfected with STIM1 constructs containing selected truncations, deletions, and point mutations, and labeled with donor and acceptor fluorescent proteins at either the luminal (N-terminal) or the cytoplasmic (C-terminal) ends. Our results with sequential truncations of STIM1 from the C-terminus support previous evidence that the CRAC activation domain (CAD/SOAR, human sequence 342-448) is an oligomer-promoting segment of STIM1, and they show that truncation just after CAD/SOAR (1-448) causes significantly elevated basal cytoplasmic Ca2+ and spontaneous STIM1 clustering. We find that a 14 amino acid sequence just C-terminal of CAD/SOAR (449-462) prevents spontaneous clustering and activation of STIM1 in COS7 cells. In response to store depletion, C-terminally labeled STIM1 without CAD/SOAR clusters together with CAD/SOAR-containing STIM1 constructs. However, these donor-acceptor pairs do not undergo a stimulated increase in FRET, exhibiting instead a decrease in FRET consistent with a stimulated conformational extension in full length STIM1. We find that the 14 amino acid sequence plays a regulatory role in this process. Overall, our FRET results provide evidence in live cells that Ca2+ store depletion stimulates a conformational extension in the cytoplasmic segment of STIM1 that accompanies its oligomerization.
期刊介绍:
AIMS Biophysics is an international Open Access journal devoted to publishing peer-reviewed, high quality, original papers in the field of biophysics. We publish the following article types: original research articles, reviews, editorials, letters, and conference reports. AIMS Biophysics welcomes, but not limited to, the papers from the following topics: · Structural biology · Biophysical technology · Bioenergetics · Membrane biophysics · Cellular Biophysics · Electrophysiology · Neuro-Biophysics · Biomechanics · Systems biology