Inhibition by Calcium of Mammalian Adenylyl Cyclases*

Ca²⁺ regulates mammalian adenylyl cyclases in a type-specific manner. Stimulatory regulation is moderately well understood. By contrast, even the concentration range over which Ca²⁺ inhibits adenylyl cyclases AC5 and AC6 is not unambiguously defined; even less so is the mechanism of inhibition. In the present study, we compared the regulation of Ca²⁺-stimulable and Ca²⁺-inhibitable adenylyl cyclases expressed in Sf9 cells with tissues that predominantly express these activities in the mouse brain. Soluble forms of AC5 containing either intact or truncated major cytosolic domains were also examined. All adenylyl cyclases, except AC2 and the soluble forms of AC5, displayed biphasic Ca²⁺ responses, suggesting the presence of two Ca²⁺ sites of high (∼0.2 μm) and low affinity (∼0.1 mm). With a high affinity, Ca²⁺ (i) stimulated AC1 and cerebellar adenylyl cyclases, (ii) inhibited AC6 and striatal adenylyl cyclase, and (iii) was without effect on AC2. With a low affinity, Ca²⁺inhibited all adenylyl cyclases, including AC1, AC2, AC6, and both soluble forms of AC5. The mechanism of both high and low affinity inhibition was revealed to be competition for a stimulatory Mg²⁺ site(s). A remarkable selectivity for Ca²⁺was displayed by the high affinity site, with a K _i value of ∼0.2 μm, in the face of a 5000-fold excess of Mg²⁺. The present results show that high and low affinity inhibition by Ca²⁺ can be clearly distinguished and that the inhibition occurs type-specifically in discrete adenylyl cyclases. Distinction between these sites is essential, or quite spurious inferences may be drawn on the nature or location of high affinity binding sites in the Ca²⁺-inhibitable adenylyl cyclases.