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CARDIOVASCULAR

Design of Mutant β₂ Subunits as Decoy Molecules to Reduce the Expression of Functional Ca²⁺ Channels in Cardiac Cells

Departments of Internal Medicine (S.T., T.G., J.D.M.) and Pharmacology and Toxicology (S.S., S.W.R., J.R.S., N.J.R.), College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas

Calcium influx through long-lasting ("L-type") Ca²⁺ channels (Ca_V) drives excitation-contraction in the normal heart. Dysregulation of this process contributes to Ca²⁺ overload, and interventions that reduce expression of the pore-forming ₁ subunit may alleviate cytosolic Ca²⁺ excess. As a molecular approach to disrupt the assembly of Ca_V1.2 (_1C) channels at the cell membrane, we targeted the Ca²⁺ channel β₂ subunit, an intracellular chaperone that interacts with _1C via its β interaction domain (BID) to promote Ca_V1.2 channel expression. Recombinant adenovirus expressing either the full β₂ subunit (Full-β₂) or truncated β₂ subunit constructs lacking either the C terminus, N terminus, or both (N-BID, C-BID, and BID, respectively) fused to green fluorescent protein were developed as potential decoys and overexpressed in HL-1 cells. Fluorescence microscopy revealed that the localization of Full-β₂ at the surface membrane was associated with increased Ca²⁺ current mainly attributed to Ca_V1.2 channels. In contrast, truncated N-BID and C-BID constructs showed punctate intracellular expression, and BID showed a diffuse cytosolic distribution. Total expression of the _1C protein of Ca_V1.2 channels was similar between groups, but HL-1 cells overexpressing C-BID and BID exhibited reduced Ca²⁺ current. C-BID and BID also attenuated Ca²⁺ current associated with another L-type Ca²⁺ channel, Ca_V1.3, but they did not reduce transient Ca²⁺ currents attributed to Ca_V3 channels. These results suggest that β₂ subunit mutants lacking the N terminus may preferentially disrupt the proper localization of L-type Ca²⁺ channels in the cell membrane. Cardiac-specific delivery of these decoy molecules in vivo may represent a gene-based treatment for pathologies involving Ca²⁺ overload.