Ca2+, Sr2+, and Ba2+ identify distinct regulatory sites on adenylyl cyclase (AC) types VI and VIII and consolidate the apposition of capacitative cation entry channels and Ca2+-sensitive ACs

Ca2+-sensitive adenylyl cyclases may act as early integrators of the two major second messenger-signaling pathways mediated by Ca2+ and cBMP, Ca2+ stimulation of adenylyl cyclase type I (ACI) and adenylyl cyclase type VIII (ACVIII) is mediated by calmodulin and the site on these adenylyl cyclases that interacts with calmodulin has been defined. By contrast, the mechanism whereby Ca2+ inhibits adenylyl cyclase type V (ACV) and adenylyl cyclase type VI (ACVI) is unknown. In this study, Ca2+, Sr2+, and Ba2+ were compared to probe the involvement of E-F hand-like domains in both Ca2+ stimulation and inhibition of ACVIII and ACVI, respectively. HEK 293 cells transfected with ACVIII cDNA and C6-2B glioma cells (where the endogenous adenylyl cyclases is predominantly ACM) were used to compare the effects of these three cations in in vitro and in vivo measurements. The in vitro data identified two Ca2+ regulatory sites for both ACVIII and ACVI, Striking;ly different potency series for these cations at mediating high affinity stimulation and inhibition of ACVIII and ACVI, respectively, effectively rule out the possibility that calmodulin or proteins utilizing similar Ca2+-binding motifs mediate inhibition of ACVI. On the other hand, the low affinity inhibition that is common to both ACVIII and ACVI showed virtually identical potency profiles for the IIa cation series, indicating a common site of action. Remarkably, whereas Sr2+ was rather ineffective at regulating these was rather ineffective at regulating these cyclases (particularly ACVI) in vitro, adequate concentrations accumulated in the vicinity of these enzymes as a consequence of capacitative cation entry to partially regulate both of these activities in vivo. This latter finding consolidates earlier observations that Ca2+-sensitive adenylyl cyclases detect and respond to capacitative cation entry rather than global cytosolic cation concentrations.