Abstract 5309: beta-Arrestin 1 (bArr1) and Ankyrin 2 (ANK2) Regulate Cav1.2 Trafficking through Complex G Protein Mediated Signaling
Mechanisms governing the trafficking of cardiac voltage dependent calcium channels (Cav1.2) remain largely unknown. We have previously shown that neuronal calcium channels are internalized into clathrin coated vesicles upon G protein coupled receptor (GPCR) activation. Here, we hypothesize that GPCR mediated signaling regulate Cav1.2 trafficking and surface expression through interaction with cytoskeletal and signaling molecules.
Methods: Sequential immunoprecipitation in adult rat cardiomyocytes was used to investigate the interaction between Cav1.2, cytoskeletal and signaling molecules. Live cell imaging and immunofluorescence microscopy were used to measure changes in the cellular localization of Cav1.2 and these proteins upon activation of GPCR signaling.
Results: Cav1.2 interacts with ANK2, βArr1 and spectrin. Sustained β adrenergic receptor (βAR) activation increases Cav1.2/spectrin and decreases Cav1.2/ANK2 and Cav1.2/βArr1 interactions. Sustained βAR activation induces rapid (<5 min) Cav1.2 internalization and dissociation from ANK2 as Cav1.2/ANK2 co-localization is markedly reduced (−58% p<0.01) in isoproterenol treated myocytes. A cell permeant peptide (1.4 μg/ml) that disrupts Cav1.2/ANK2 interaction decreases (−40±12%, p<0.01) Cav1.2 cell surface expression. Pretreatment with pertussis toxin prevents Cav1.2 internalization suggesting that Gi/o mediates this response. Similarly, Src kinase inhibition reduces (by 90%) Cav1.2 internalization. Differential labeling of pre-existing Cav1.2 at the cell membrane and newly inserted Cav1.2 using fluorophore conjugated dihydropyridines reveal that activation of Gi/o proteins underlies Cav1.2 internalization and insertion. In contrast, activation of the muscarinic M2 receptor (10μM carbachol) causes only insertion of new channels (n=14) and fails to alter the association of Cav1.2 with ANK2 and spectrin. Finally PI3 kinase inhibition (10nM wortmannin) prevents Cav1.2 membrane insertion.
Conclusions: Our findings reveal novel signaling mechanisms that regulate Cav1.2 trafficking, internalization and membrane insertion in a receptor subtype dependent manner. These mechanisms may be central to pathologies associated with a hyperadrenergic state.
This research has received full or partial funding support from the American Heart Association, AHA National Center.