Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC324

Poster Communications

Thapsigargin and the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol differentially regulate the association between Orai and STIM proteins in human platelets.

C. Galán1, L. Albarrán1, A. Berna-Erro1, G. M. Salido1, J. A. Rosado1

1. Physiology, University of Extremadura, Caceres, Spain.

Orai1 is a plasma membrane protein that conducts store-operated Ca2+ entry (SOCE), a mechanism regulated by the filling state of the Ca2+ stores, whose depletion can be detected by the sensor protein STIM1. Isoforms of these proteins, STIM2, Orai2 and Orai3, have been identified, although their role in intracellular Ca2+ homeostasis is less characterized (Roberts-Thomson et al., 2010). In non-excitable cells such as human platelets, physiological agonists stimulate Ca2+ entry via different mechanisms, including SOCE and second messenger-operated Ca2+ entry (SMOCE), activated, in turn, by signalling molecules such as diacylglycerol (DAG) or protein kinase C (Rosado & Sage, 2000; Salido et al., 2009). We have investigated the expression and interaction of Orai and STIM proteins in human platelets. Blood was drawn from healthy drug-free volunteers with approval of the local ethical committees and in accordance with the Declaration of Helsinki. Platelets were isolated as described (Jardin et al., 2008). Orai and STIM mRNA transcripts were detected by reverse transcription polymerase chain reaction (RT-PCR). Co-immunoprecipitation followed by Western blotting was performed as described previously (Jardin et al., 2008). In resting platelets we detected interaction between Orai1 and Orai2, Orai3, STIM1 and STIM2, as well as between STIM1 and STIM2. In cells loaded with dimethyl-BAPTA by incubation with 10 µM dimethyl BAPTA-AM for 30 min, depletion of the intracellular Ca2+ stores with the inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) thapsigargin (TG) in a Ca2+-free medium significantly enhanced the association between Orai1 and both Orai2 and Orai3 by 49 and 25%, respectively (p<0.05 Students t-test; n=4). Moreover, TG enhanced the association of STIM2 with either STIM1 or Orai1 to 300% and 219%, respectively, compared to untreated controls (p<0.05; n=4). On the other hand, in the presence of 1 mM extracellular calcium, platelet stimulation with the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol, to activate SMOCE, solely enhanced the association of Orai1 with Orai2 and STIM2 by 39% and 85%, respectively (p<0.05; n=4). In conclusion, we report the expression of different Orai and STIM isoforms in human platelets and the differential regulation of their interactions as a consequence of store depletion or DAG signalling activation.

Where applicable, experiments conform with Society ethical requirements