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

Abstract

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 Ki value, whereas the Ki 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.