Inhibition by calcium of mammalian adenylyl cyclases

Ca2+ regulates mammalian adenylyl cyclases in a type-specific manner. Stimu latory regulation is moderately well understood, By contrast, even the conc entration range over which Ca2+ inhibits adenylyl cyclases AC5 and AC6 is n ot unambiguously defined; even less so is the mechanism of inhibition. In t he present study, we compared the regulation of Ca2+-stimulable and Ca2+-in hibitable adenylyl cyclases expressed in Sf9 cells with tissues that predom inantly express these activities in the mouse brain. Soluble forms of AC5 c ontaining either intact or truncated major cytosolic domains were also exam ined. All adenylyl cyclases, except AC2 and the soluble forms of AC5, displ ayed biphasic Ca2+ responses, suggesting the presence of two Ca2+ sites of high (similar to 0.2 mu M) and low affinity (similar to 0.1 mM). With a hig h affinity, Ca2+ (i) stimulated AC1 and cerebellar adenylyl cyclases, (ii) inhibited AC6 and striatal adenylyl cyclase, and (iii) was without effect o n 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 a nd 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 similar to 0.2 mu M, in the face of a 50 00-fold excess of Mg2+. The present results show that high and low affinity inhibition by Ca2+ can be clearly distinguished and that the inhibition oc curs type-specifically in discrete adenylyl cyclases, Distinction between t hese sites is essential, or quite spurious inferences may be drawn on the n ature or location of high affinity binding sites in the Ca2+-inhibitable ad enylyl cyclases.