Capacitative Ca²⁺Entry via Orai1 and Stromal Interacting Molecule 1 (STIM1) Regulates Adenylyl Cyclase Type 8

Abstract

Capacitative Ca 2ϩ entry (CCE), which occurs through the plasma membrane as a result of Ca 2ϩ store depletion, is mediated by stromal interacting molecule 1 (STIM1), a sensor of intracellular Ca 2ϩ 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 Ca 2ϩ entry reconstituted by coexpression of Orai1 and STIM1 has the functional properties of CCE, we used the Ca 2ϩ -calmodulin stimulated adenylyl cyclase type 8 (AC8), which responds selectively to CCE, whereas other modes of Ca 2ϩ entry, including those activated by arachidonate and the ionophore ionomycin, are ineffective. In addition, the Ca 2ϩ 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 CCEstimulated 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 Ca 2ϩ -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.

Ca 2 + signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels

“…Some Ca 2+ channels are present in multienzyme protein complexes with proteins that generate their activating ligands (1). It is intriguing to note that Ca 2+ signaling involving the Ca 2+ channel Orai1 that acts in animal immune signaling involves Orai1:adenylyl cyclase protein complexes and microdomains of cyclic nucleotide elevation (40,41). Alternatively, the receptor could act through cytosolic kinase activity to activate channels and initiate downstream CNGC-dependent Ca 2+ signaling cascades.…”

Direct demonstration of discrete Ca2+ microdomains associated with different isoforms of adenylyl cyclase

“…pharmacological disruption of STIM1 recruitment to lipid-raft regions of the PM significantly reduced CCE-dependent AC8 activity (Martin et al, 2009).…”

“…The reason for this selectivity is currently unknown. Functional studies have indirectly suggested that a close association between AC8 and CCE channels might underlie the phenomenon (Gu and Cooper, 2000;Martin et al, 2009). This is supported by the targeting of Ca 2+ -sensitive ACs and key CCE proteins to sphingolipid and cholesterolrich regions of the plasma membrane (PM), known as lipid rafts (a subset of which are the morphologically identifiable caveolae) (Fagan et al, 2000b;Smith et al, 2002).…”

“…It has been hypothesized that this property might, at least, partially, reflect the residence of these ACs in lipid-raft regions of the PM alongside sites of CCE (Fagan et al, 2000b;Smith et al, 2002). The possibility of functional coupling between the Ca 2+ -sensitive ACs and known molecular components of CCE channels (STIM1 and Orai) has recently been demonstrated and suggests a degree of intimacy, although direct protein-protein interactions were not detected (Martin et al, 2009 (Chiono et al, 1995;Fagan et al, 1998;Fagan et al, 2000b). Earlier attempts to directly address the functional disparity of the sensitivity of the Ca 2+ -inhibitable AC6 to CCE but not to Ca 2+ release from the ER employed an AC6/aequorin chimera (Nakahashi et al, 1997 -sensitive AC compared with global Ca 2+ changes, the absolute levels of Ca 2+ detected by the AC6/aequorin sensor during release and entry were actually remarkably similar, 1160±136 nM and 1225±117 nM, respectively (Nakahashi et al, 1997), and could not explain the selectivity of AC6 for CCE over Ca 2+ mobilization.…”

Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu

“…These ACs are regulated by the ubiquitous process of capacitative Ca 2+ entry (CCE) (Parekh and Putney, 2005;Cheng et al, 2011) that is triggered by Ca 2+ store depletion, and are unresponsive to other forms of Ca 2+ rise (Fagan et al, 1996;Martin et al, 2009;Willoughby and Cooper, 2007). The selective regulation of AC8 by CCE depends upon the residence of AC8 in lipid rafts Smith et al, 2002) in close proximity to store-operated Ca 2+ entry channels (Willoughby et al, 2010a).…”