Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC92

Poster Communications

Modulation of the dynamic interaction between STIM1, Orai1 and TRPC1 by the cytoskeleton in HEK-293 cells

N. Dionisio1, C. Galán1, G. M. Salido1, J. A. Rosado1

1. Department of Physiology, University of Extremadura, 10003-C

Store-operated Ca2+ entry (SOCE) is one of the most relevant pathways for Ca2+ influx in non-electrically excitable cells. A conformational coupling mechanism between the endoplasmic reticulum Ca2+ sensor STIM1 and Ca2+ permeable channels in the plasma membrane has been recently reported to account for the activation of SOCE upon depletion of the intracellular Ca2+ stores (Hogan et al., 2010). In addition, TRPC proteins have been shown to associate with Orai1 and STIM1 in a dynamic ternary complex regulated by the occupation of membrane receptors, which might play an important role in the activation of Ca2+ entry (Salido et al., 2011). A number of studies have reported a role for the cytoskeleton modulating the activation of SOCE; therefore, here we have investigated whether the interaction between STIM1 and the Ca2+ permeable channels is modulated by the actin or microtubular network. Human embryonic kidney 293 (HEK293) cells were obtained from the American Type Culture Collection. Cytosolic free Ca2+ concentration measurement, immunoprecipitation and Western blotting were performed as previously described (Ben-Amor et al., 2006; Woodard et al., 2010). Cell treatment for 30 min with the microtubular disrupter colchicine (30 µM) significantly enhanced the activation of SOCE by 23% (p<0.05 Student’s t-test; n=6), as well as increased thapsigargin (TG)-evoked association between STIM1 and Orai1 or TRPC1 by 75 and 44%, respectively (p<0.05; n=6). Conversely, stabilization of the microtubules by treatment with 10 µM paclitaxel for 30 min attenuated SOCE by 60% and the interaction between STIM1 and the Ca2+ channels Orai1 and TRPC1 by 50 and 60%, respectively (p<0.05; n=6), altogether suggesting that the microtubules act as a negative regulator of SOCE. Stabilization of the cortical actin filament layer by treatment for 30 min with 10 µM jasplakinolide abolished TG-evoked association between STIM1, Orai1 and TRPC1 and reduced SOCE by 60% (p<0.05; n=6). Interestingly, disruption of the actin filament network by treatment with 10 µM cytochalasin D for 40 min did not significantly modify TG-evoked association between STIM1 and Orai1 or TRPC1 but enhanced TG-stimulated SOCE by 70% (p<0.05; n=6). Finally, inhibition of calmodulin by treatment for 10 min with 1 µM calmidazolium enhances SOCE by 63%. In addition, disruption of the actin cytoskeleton results in inhibition of TG-evoked association of calmodulin with Orai1 and TRPC1. In summary, we demonstrate that the cytoskeleton plays an essential role in the regulation of SOCE through the modulation of the interaction between STIM1 and the Ca2+ permeable channels Orai1 and TRPC1.

Where applicable, experiments conform with Society ethical requirements