Stim1 and Orai1 Mediate CRAC Currents and Store-Operated Calcium Entry Necessary for Endothelial Cell Proliferation

Abstract

Recent breakthroughs in the store-operated calcium (SOC) entry pathway have identified Stim1 as the endoplasmic reticulum (ER) calcium sensor and Orai1 as the pore forming subunit of the highly calcium selective CRAC channel. Previous studies have suggested that endothelial cell (EC) SOC is encoded by members of the Canonical Transient Receptor Potential (TRPC) channel family, either TRPC1 or TRPC4. Here we show that passive store depletion or receptor activation by thrombin or VEGF activates SOC entry pathway in primary EC with classical SOC pharmacological features. EC possess the archetypical store-depletion activated CRAC current. By amplifying currents in divalent free bath solutions, we show that EC CRAC has similar characteristics to that recorded from RBL cells, namely a similar time course of activation, sensitivity to 2-APB and low concentrations of lanthanides, the same inwardly rectifying I/V relationship, very positive reversal potential, and large sodium currents displaying the typical phenomenon of depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOC entry and CRAC currents in EC which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, complete knockdown of either TRPC1 or TRPC4 proteins had no effect on SOC entry in EC. Smaller CRAC current densities in EC compared to those recorded in RBL cells were due to lower expression of Stim1. Ectopic expression of Stim1 in EC increased their CRAC currents to a size comparable to those in RBL cells. Knockdown of either Stim1, Stim2 or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim1. These results are first to establish the requirement of Stim1/Orai1 in the endothelial SOC pathway necessary for proliferation.