A critical interplay between Ca2+ inhibition and activation by Mg2+ of AC5 revealed by mutants and chimeric constructs

Adenylyl cyclase type 5 (AC5) is sensitive to both high and low affinity inhibition by Ca2+. This property provides a sensitive feedback mechanism of the Ca2+ entry that is potentiated by cAMP in sources where AC5 is commonly expressed (e.g. myocardium). Remarkably little is known about the molecular mechanism whereby Ca2+ inhibits AC5. Because previous studies had showed that Ca2+ antagonized the activation of adenylyl cyclase brought about by Mg2+, we have now evaluated the Mg2+-binding domain in the catalytic site as the potential site of the interaction, using a number of mutations of AC5 with impaired Mg2+ activation. Mg2+ activation exerted contrasting effects on the high and low affinity Ca2+ inhibition. In both wild type and mutants, activation by Mg2+ decreased the absolute amount of high affinity inhibition without affecting the K-i value, whereas the K-i value for low affinity inhibition was decreased. These effects were directly proportional to the sensitivity of the mutants to Mg2+. Parallel changes were noted in the efficacies of Ca2+, Sr2+, and Ba2+ in the mutant species, suggesting a simple mutation in a shared domain. Strikingly, forskolin, which activates by a mechanism different from Mg2+, did not modify inhibition by Ca2+. Deletion of the N terminus and the C1b domain of AC5 and a chimera formed with AC2 confirmed that the catalytic domain alone was responsible for high affinity inhibition. We therefore conclude that both low and high affinity inhibition by Ca2+ are exerted on different conformations of the Mg2+-binding sites in the catalytic domain of AC5.