Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC114

Poster Communications

Homer1 has a role in Store Operated Calcium Entry in human platelets.

I. Jardin1, G. M. Salido1, J. A. Rosado1

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

Store-operated calcium entry (SOCE) is a process controlled by the filling state of intracellular calcium stores and mediated by a complex of signalling proteins, including the endoplasmic reticulum calcium sensor STIM1 and the calcium permeable channels Orai1 and TRPCs (Salido et al., 2011). Homer1 is a scaffold protein that participates in targeting and localization of ryanodine receptors, IP3 receptors, STIM1 and TRPs via its N-terminal EVH1 domain that interacts with the proline-rich sequences of the PPXXF motif which are found in many target and ligand proteins (Worley et al., 2007; Gasperini et al., 2009; Shalygin et al., 2010). Here we have investigated the role of Homer1 in SOCE and its association and subsequent modulation of calcium signal proteins in human platelets. Platelets were obtained from blood drawn from healthy drug-free volunteers with approval of the local ethical committees and in accordance with the Declaration of Helsinki. Intracellular free calcium concentration ([Ca2+]c) was determined by spectrofluorimetry and protein-protein interactions by Western blotting. In a Ca2+-free medium thapsigargin evoked a prolonged increase in [Ca2+]c and increased the association between Homer1 and IP3R, hTRPC1, STIM1 and Orai1. The subsequent addition of Ca2+ (300 µM) to the external medium induces a sustained increase in [Ca2+]c indicative of SOCE. Electrotransjection of PPKKFR, a specific peptide that emulates the proline-rich sequences of the PPXXF motif that interacts with Homer1 impairing its interaction with others proteins, resulted in an attenuation of thapsigargin-stimulated interaction between STIM1 and Orai1 by 20% and significantly reduced SOCE by 34% (p<0.05 Student’s t-test; n=4). This effect was not found when we used the negative control peptide PPKKRR. Summarizing, Homer1 acts as a scaffolding protein for the SOCE signalling complex in human platelets.

Where applicable, experiments conform with Society ethical requirements