The subunits KCNQ1 and KCNE1 generate the slowly activating, delayed rectifier potassium current, I_Ks, that responds to sympathetic stimulation and is critical for human cardiac repolarization. The A-kinase anchoring protein Yotiao facilitates macromolecular complex formation between I_Ks and protein kinase A (PKA) to regulate phosphorylation of KCNQ1 and I_Ks currents following beta-adrenergic stimulation. We have previously shown that adenylyl cyclase Type 9 (AC9) is associated with a KCNQ1-Yotiao-PKA complex and facilitates isoproterenol-stimulated phosphorylation of KCNQ1 in an immortalized cell line. However, requirement for AC9 in sympathetic control of I_Ks in the heart was unknown. Using a transgenic mouse strain expressing the KCNQ1-KCNE1 subunits of I_Ks, we show that AC9 is the only adenylyl cyclase (AC) isoform associated with the KCNQ1-KCNE1-Yotiao complex in the heart. Deletion of AC9 resulted in the loss of isoproterenol-stimulated KCNQ1 phosphorylation in vivo, even though AC9 represents less than 3% of total cardiac AC activity. Importantly, a significant reduction of isoproterenol-stimulated I_Ks currents was also observed in adult cardiomyocytes from I_Ks-expressing AC9KO mice. AC9 and Yotiao co-localize with N-cadherin, a marker of intercalated disks and cell–cell junctions, in neonatal and adult cardiomyocytes, respectively. In conclusion, AC9 is necessary for sympathetic regulation of PKA phosphorylation of KCNQ1 in vivo and for functional regulation of I_Ks in adult cardiomyocytes.

The establishment of macromolecular complexes by scaffolding proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for cardiac functions. We identify a novel AC scaffold, the Popeye domain‐containing (POPDC) protein. The POPDC family of proteins is important for cardiac pacemaking and conduction, due in part to their cAMP‐dependent binding and regulation of TREK‐1 potassium channels. We show that TREK‐1 binds the AC9:POPDC1 complex and copurifies in a POPDC1‐dependent manner with AC9 activity in heart. Although the AC9:POPDC1 interaction is cAMP‐independent, TREK‐1 association with AC9 and POPDC1 is reduced upon stimulation of the β‐adrenergic receptor (βAR). AC9 activity is required for βAR reduction of TREK‐1 complex formation with AC9:POPDC1 and in reversing POPDC1 enhancement of TREK‐1 currents. Finally, deletion of the gene‐encoding AC9 ( Adcy9) gives rise to bradycardia at rest and stress‐induced heart rate variability, a milder phenotype than the loss of Popdc1 but similar to the loss of Kcnk2 (TREK‐1). Thus, POPDC1 represents a novel adaptor for AC9 interactions with TREK‐1 to regulate heart rate control.

Highly homologous members of the Gα_i family, Gα_i1-3, have distinct tissue distributions and physiological functions, yet their biochemical and functional properties are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gα_i1 effector that is poorly activated by Gα_i2. In a proteomic proximity labeling screen we observed a strong preference for Gα_i1 relative to Gα_i2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gα_i1 into Gα_i2 or substitution of a single amino acid, A230 in Gα_i2 with the corresponding D in Gα_i1, largely rescues PRG activation and interactions with other potential Gα_i targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, separation at the HD-Ras-like domain (RLD) interface is more pronounced in Gα_i2 than Gα_i1. Mutation of A230 to D in Gα_i2 stabilizes HD-RLD interactions via ionic interactions with R145 in the HD which in turn modify the conformation of Switch III. These data support a model where D229 in Gα_i1 interacts with R144 and stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gα_i2 is unable to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gα_i1 or Gα_i2 by G protein-coupled receptors.

CAMP influences multiple aspects of cardiac biology, including the regulation of contraction, relaxation, and overall stress responses. The functional outcomes of cAMP are driven by the spatial arrangement of enzymes that produce and degrade cAMP (adenylyl cyclase [AC] and phosphodiesterase, respectively), together with the downstream targets of cAMP. We performed proximity-dependent biotin identification (BioID) of near-neighbor interactions proteomics in cardiomyocytes to generate proximity maps for three cardiac AC isoforms. Processing and trafficking functions were the most common gene ontology terms for ACs, with AC5 and AC6 generating unique proximal protein sets compared to AC9. AC5/6 proximal proteins showed significant localization at the endo/sarcoplasmic reticulum with roles in calcium handling, whereas those near AC9 were more abundant at the plasma membrane. Upon treatment with the hypertrophic stimulus norepinephrine, only a few calcium handling proteins were differentially labeled for AC5/6, but not AC9. Endogenous AC activity copurified with vesicle transport, signaling, and muscle contraction proteins that were identified by BioID and/or pulldown of FLAG-tagged AC (FLAG-mass spectrometry) in cardiomyocytes, including ryanodine receptor 2, ATP2A2 (sarco/endoplasmic reticulum Ca²⁺-ATPase 2), epidermal growth factor receptor, syntrophin alpha 1, and vesicle-associated membrane protein 2. Finally, overlapping BioID and FLAG-mass spectrometry datasets suggested heterodimerization of AC5 and AC6, while super-resolution electron microscopy spatial mapping validated homodimerization and heterodimerization of these AC isoforms on the plasma membrane. Overall, our comprehensive network analysis has identified new binding partners and shed light on the spatial and functional significance of AC-containing macromolecular complexes in heart.

Subcellular targeting of signaling enzymes influences where and when various modes of intracellular communication operate. Macromolecular complexes of signal transduction and signal termination elements favor reversible control of repetitive processes. This includes adrenergic stimulation of excitation-contraction coupling in the heart. Long isoforms of A-kinase anchoring protein 18 (AKAP18γ and δ) modulate this process via regulation of calcium uptake into the sarcoplasmic reticulum through the Ca²⁺ATPase 2a (SERCA2a). AKAP18 proximity-proteomic screening in cardiomyocytes identifies networks for protein kinase A (PKA) and ubiquitin-specific proteinases (USPs). A 2'phosphoesterase domain on AKAP18 interfaces with the USP4 isoform at the Z bands of sarcomeres. PKA stimulates USP4 activity in the presence of the anchoring protein. AKAP18 anchored PKA phosphorylates serine 829 on USP4, a conserved residue near the active site of this deubiquitinase. Antibodies against the pSer⁸²⁹ motif show that adrenergic stimulation enhances phosphorylation of USP4 in mouse adult cardiomyocytes. In related studies, elevated USP4 phosphorylation at Ser⁸²⁹ is detected in human post myocardial infraction tissue as compared to healthy tissue. Thus, phosphorylation of sarcoplasmic USP4 may be a cardioprotective response. Pharmacological inhibition of PKA or deletion of the AKAP7/18 gene in mice decreases calcium flux through the exchanger. This suggests that loss of the anchoring protein impacts SERCA2 action. Thus, AKAP18/PKA/USP4 complexes are well positioned to influence the rate and magnitude of calcium reuptake during the cardiac cycle.

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. ONE-SENTENCE SUMMARY: Adenylyl cyclase type 9 binds in an isoproterenol-dependent manner to the POPDC1:TREK-1 complex regulating heart rate.