Calmidazolium and arachidonate activate a calcium entry pathway that is distinct from store-operated calcium influx in HeLa cells

Agonists that deplete intracellular Ca²⁺ stores also activate Ca²⁺ entry, although the mechanism by which store release and Ca²⁺ influx are linked is unclear. A potential mechanism involves ‘store-operated channels’ that respond to depletion of the intracellular Ca²⁺ pool. Although SOCE (store-operated Ca²⁺ entry) has been considered to be the principal route for Ca²⁺ entry during hormonal stimulation of non-electrically excitable cells, recent evidence has suggested that alternative pathways activated by metabolites such as arachidonic acid are responsible for physiological Ca²⁺ influx. It is not clear whether such messenger-activated pathways exist in all cells, whether they are truly distinct from SOCE and which metabolites are involved. In the present study, we demonstrate that HeLa cells express two pharmacologically and mechanistically distinct Ca²⁺ entry pathways. One is the ubiquitous SOCE route and the other is an arachidonate-sensitive non-SOCE. We show that both these Ca²⁺ entry pathways can provide long-lasting Ca²⁺ elevations, but that the channels are not the same, based on their differential sensitivity to 2-aminoethoxydiphenyl borate, LOE-908 {(R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate} and gadolinium. In addition, non-SOCE and not SOCE was permeable to strontium. Furthermore, unlike SOCE, the non-SOCE pathway did not require store depletion and was not sensitive to displacement of the endoplasmic reticulum from the plasma membrane using jasplakinolide or ionomycin pretreatment. These pathways did not conduct Ca²⁺ simultaneously due to the dominant effect of arachidonate, which rapidly curtails SOCE and promotes Ca²⁺ influx via non-SOCE. Although non-SOCE could be activated by exogenous application of arachidonate, the most robust method for stimulation of this pathway was application of the widely used calmodulin antagonist calmidazolium, due to its ability to activate phospholipase A₂.