Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

The establishment of macromolecular complexes by scaffolding proteins such as A-kinase anchoring proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for many cardiac functions. We have identified herein a novel AC scaffold, the Popeye domain-containing (POPDC) protein. Unlike other AC scaffolding proteins, POPDC1 binds cAMP with high affinity. The POPDC family of proteins are important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. TREK-1 binds the AC9:POPDC1 complex and co-purifies in a POPDC1-dependent manner with AC9-associated activity in heart. Although the interaction of AC9 and POPDC1 is cAMP independent, TREK-1 association with AC9 and POPDC1 is reduced in an isoproterenol-dependent manner, requiring an intact cAMP binding Popeye domain and AC activity within the complex. We show that deletion of Adcy9 (AC9) gives rise to bradycardia at rest and stress-induced heart rate variability. The phenotype for deletion of Adcy9 is milder than previously observed upon loss of Popdc1, but similar to loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel scaffolding protein for AC9 to regulate heart rate control.

Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases

Mycobacterium tuberculosis adenylyl cyclase (AC) Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signaling is well established, the function of their transmembrane (TM) regions remains unknown. Here we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 Å resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.One-Sentence SummaryCryo-EM structure of M. tuberculosis adenylyl cyclase Cya provides clues to the role of its transmembrane domain

Imaging Promoter Assay of Adenylyl Cyclase A Gene in Dictyostelium discoideum during Fruiting Body Formation by Dual-Color Bioluminescence Microscopy

Cyclic adenosine monophosphate (cAMP), which is derived from adenosine triphosphate through adenylyl cyclase A (acaA), acts as an intracellular secondary messenger and an extracellular chemotactic substance in important biological processes. In the social amoebae Dictyostelium discoideum, cAMP mediates cell aggregation, development, and differentiation to spore and stalk cells during fruiting body formation. The acaA gene is transcribed under the control of three different alternative promoters. This study aimed to develop a promoter assay for acaA in D. discoideum using bioluminescence microscopy. Here, we inserted green- and red-emitting luciferase genes into downstream of promoter regions 1 and 3, respectively. Promoter activities were visualized by bioluminescence microscopy. We confirmed the differential expression of acaA under the control of promoters 1 and 3 at the different stages of D. discoideum development. We also demonstrated the application of dual-color bioluminescence imaging in the development of an imaging promoter assay.