Abstract P131: An Akt-Phosphomimetic Sequence of the Cavb2 C-Terminal Region Protects L-Type Calcium Channels from Protein Degradation

Abstract

Alteration in the density or function of L-Type Calcium Channels (LTCCs) has been related to cardiovascular diseases such as heart failure and diabetic cardiomyopathy. It could therefore be envisioned that increasing LTCC density may improve cardiac function in heart failure. Recently, we determined that the Ser-Thr kinase Akt plays a key role in regulating cardiac inotropism through the modulation of LTCC density and function. Specifically, we found that the LTCC pore-forming channel subunit Cava1.2 contains highly evolutionary conserved PEST sequences (signals for rapid proteolytic degradation) that are responsible for direct Cava1.2 protein degradation. Phosphorylation of the C-terminal coiled coil of the Cavb2 chaperone subunit enhances LTCC protein stability by preventing PEST-mediated Cava1.2 degradation. The aim of this study was to further dissect this Akt-dependent fine-tuning mechanism regulating LTCC density, searching for potential Akt-phosphomimetic (APM) molecules that could enhance LTCC density. Using yeast two-hybrid screening, we found that APM Cavb2 sequences interact with the globular domain of Cavb2 itself in a solvent-exposed region that we named Tail Interacting Domain (TID). Biochemical and functional assays as well as site-specific mutagenesis in TID identified the minimal aminoacid sequence responsible for the TID-tail interaction. In addition, through an approach comprising western blot analyses, fluorescent-based calcium assays, calcium current (ICaL) measurements, molecular modeling and peptide arrays, we identified the minimal APMs that efficiently protects Cava1.2 from protein degradation. Based on our in vitro results, we suggest that the identified APM sequence/peptide could be used as a “therapeutic approach”; for increasing or reestablishing impaired cardiac contractility in mouse models of cardiomyopathy in which the LTCC density is altered, thus enhancing inotropism.