During physiological cell stimulation, many processes are engaged that might eventually lead to Ca²⁺ entry. One can roughly separate the different Ca²⁺ entries as 1) activated by a second messenger produced by an agonist (receptor-activated Ca²⁺ entry, RACE) and 2) linked to the depletion of the ER Ca²⁺ store, the SOCE (store-operated Ca²⁺ entry). The latter is well characterized and involved the proteins STIM1 (the ER Ca²⁺ sensor) and Orai1 (the Ca²⁺ entry channel). Regarding the RACE pathway, it appears that a diversity of activation mechanisms as well as ionic channels is involved. As agonist stimulation produced both second messengers and a depletion of the ER, it is difficult to separate both types of influx. In this study, our aim was to differentiate between RACE and SOCE pathways during agonist-induced cell stimulation, using electrophysiological and imaging approaches on a human endothelial cell line (EA.hy926).

In Ca²⁺ containing medium, histamine induced an ER Ca²⁺ depletion of about 15% of what was achieved by thapsigargin (TG), while in the absence of Ca²⁺, histamine fully depleted the ER. This pointed to a modest involvement of the SOCE upon histamine stimulation. In whole-cell perforated patch, histamine and TG activated both an inwardly rectified current, when 10 mM Ba²⁺ (and 2 mM Ca²⁺) was present in the bath. The current shared characteristics with the I_CRAC, like the inward rectification, the block by La³⁺ and the behavior in divalent free medium. Surprisingly, in presence of 10 mM Ca²⁺ in the bath (without Ba²⁺) the currents activated by TG and histamine were clearly different. In particular, histamine activated an outwardly rectified current, blocked by Ba²⁺, while TG activated a similar, but smaller current as in Ba²⁺ containing medium. We also showed that during voltage clamp recordings, the ER gets more depleted in 10 mM Ba²⁺ compared to 10 mM Ca²⁺ medium, explaining the activation of a CRAC-like current (due to store depletion) upon histamine in Ba²⁺ medium. In Ca²⁺ medium, the outward current activated by histamine was increased upon Na⁺ removal and inhibited by Ni²⁺ and KB-R7943, arguing in favor of the Na⁺/Ca²⁺ exchanger (NCX). Ni²⁺ and KB-R7943 also inhibited histamine-, while not TG-induced Ca²⁺ entry, measured by fura-2. These data led us conclude that under physiological conditions, part of the RACE was due to the NCX, working in the reverse mode, that accomplishes sufficient ER refilling to largely prevent the activation of SOCE.