Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC115

Poster Communications

Regulatory Mechanism of STIM1 Translocation into Plasma Membrane in HEK293 cells

B. Zeng1, S. Xu1

1. Hull York Medical School, University of Hull, Hull, United Kingdom.


STIM1 is a Ca2+ sensing molecule mainly localized in cytosol. Once the Ca2+ stores are depleted, STIM1 translocates to the plasma membrane (PM) and activates Ca2+ influx through Orai channels. This process is a major mechanism for store-operated Ca2+ entry (SOCE). Here we investigated the regulatory mechanism of STIM1 translocation. The translocation of STIM1 from intracellular compartments to the PM was induced by thapsigargin (Tg) in HEK293 cells stably expressing YFP-tagged STIM1. The Tg-induced STIM1 puncta occurred and then persisted on the PM in both Ca2+- and Ca2+-free bath solutions. Activation of G protein-coupled receptor by trypsin significantly evoked a Ca2+ release, and accompanied by STIM1 translocation into the PM. The formation of STIM1 puncta induced by trypsin was much faster than that caused by Tg, and these puncta disappeared afterwards if extracellular Ca2+ was applied. Application of SOCE inhibitor 2-APB abolished the STIM1 membrane translocation, but resulted in STIM1 clustering in the cell. However, Gd3+ and SKF-96365 did not show any effect on STIM1 translocation. Disruption of cytoskeleton by colchicine and cytochalasin D did not prevent STIM1 translocation, suggesting the trafficking process does not rely on microtubule and actin. Rho kinase inhibitor Y-27632 and phosphoinositide 3-kinase inhibitor wortmannin did not alter the STIM1 puncta formation induced by Tg or trypsin. Cellular ATP depletion by azide showed no effect on STIM1 translocation. We also observed the involvement of ryanodine-sensitive Ca2+ store, both caffeine and 4-chloro-3-ethylphenol (4-CEP) were able to induce STIM1 translocation, but the dynamics and puncta pattern were different. Cells treated with caffeine at high concentration showed STIM1 puncta occurring slower than which induced by Tg. Application of 4-CEP led to puncta formation at first, and then changed STIM1 to a clustered pattern similar to that caused by 2-APB. We concluded that STIM translocation could be an important target for store-operated channel pharmacology. These results provide new insights for understanding the regulatory mechanism of STIM1 in SOCE.

Where applicable, experiments conform with Society ethical requirements