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 Ca(2+). This property provides a sensitive feedback mechanism of the Ca(2+) 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 Ca(2+) inhibits AC5. Because previous studies had showed that Ca(2+) antagonized the activation of adenylyl cyclase brought about by Mg(2+), we have now evaluated the Mg(2+)-binding domain in the catalytic site as the potential site of the interaction, using a number of mutations of AC5 with impaired Mg(2+) activation. Mg(2+) activation exerted contrasting effects on the high and low affinity Ca(2+) inhibition. In both wild type and mutants, activation by Mg(2+) 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 Mg(2+). Parallel changes were noted in the efficacies of Ca(2+), Sr(2+), and Ba(2+) in the mutant species, suggesting a simple mutation in a shared domain. Strikingly, forskolin, which activates by a mechanism different from Mg(2+), did not modify inhibition by Ca(2+). 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 Ca(2+) are exerted on different conformations of the Mg(2+)-binding sites in the catalytic domain of AC5.
Hu
Biao
B
Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Nakata
Hiroko
H
Gu
Chen
C
De Beer
Tonny
T
Cooper
Dermot M F
DM
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001464:Barium / Q000737:chemistry; D002118:Calcium / Q000494:pharmacology*; D020134:Catalytic Domain; D002460:Cell Line; D005576:Colforsin / Q000737:chemistry; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017353:Gene Deletion; D006801:Humans; D007313:Insecta; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013324:Strontium / Q000737:chemistry
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D005576:Colforsin; D001464:Barium; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; C100622:adenylyl cyclase 2; C419510:adenylyl cyclase type V; D008274:Magnesium; D002118:Calcium; D013324:Strontium