Abstract

Capacitative Ca²⁺ entry (CCE), which occurs through the plasma membrane as a result of Ca²⁺ store depletion, is mediated by stromal interacting molecule 1 (STIM1), a sensor of intracellular Ca²⁺ store content, and the pore-forming component Orai1. However, additional factors, such as C-type transient receptor potential (TRPC) channels, may also participate in the CCE apparatus. To explore whether the store-dependent Ca²⁺ entry reconstituted by coexpression of Orai1 and STIM1 has the functional properties of CCE, we used the Ca²⁺-calmodulin stimulated adenylyl cyclase type 8 (AC8), which responds selectively to CCE, whereas other modes of Ca²⁺ entry, including those activated by arachidonate and the ionophore ionomycin, are ineffective. In addition, the Ca²⁺ entry mediated by previous CCE candidates, diacylglycerol-activated TRPC channels, does not activate AC8. Here, we expressed Orai1 and STIM1 in HEK293 cells and saw a robust increment in CCE, and a proportional increase in CCE-stimulated AC8 activity. Inhibitors of the CCE assembly process ablated the effects on cyclase activity in both AC8-overexpressing HEK293 cells and insulin-secreting MIN6 cells endogenously expressing Ca²⁺-sensitive AC isoforms. AC8 is believed to be closely associated with the source of CCE; indeed, not only were AC8, Orai1, and STIM1 colocalized at the plasma membrane but also all three proteins occurred in lipid rafts. Together, our data indicate that Orai1 and STIM1 can be integral components of the cAMP and CCE microdomain associated with adenylyl cyclase type 8.