Design of Mutant β2 Subunits as Decoy Molecules to Reduce the Expression of Functional Ca2+ Channels in Cardiac Cells
AbstractAbstract Calcium influx through long-lasting (“L-type”) Ca 2+ channels (Ca V ) drives excitation-contraction in the normal heart. Dysregulation of this process contributes to Ca 2+ overload, and interventions that reduce expression of the pore-forming α 1 subunit may alleviate cytosolic Ca 2+ excess. As a molecular approach to disrupt the assembly of Ca V 1.2 (α 1C ) channels at the cell membrane, we targeted the Ca 2+ channel β 2 subunit, an intracellular chaperone that interacts with α 1C via its β interaction domain (BID) to promote Ca V 1.2 channel expression. Recombinant adenovirus expressing either the full β 2 subunit (Full-β 2 ) or truncated β 2 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-β 2 at the surface membrane was associated with increased Ca 2+ current mainly attributed to Ca V 1.2 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 α 1C protein of Ca V 1.2 channels was similar between groups, but HL-1 cells overexpressing C-BID and BID exhibited reduced Ca 2+ current. C-BID and BID also attenuated Ca 2+ current associated with another L-type Ca 2+ channel, Ca V 1.3, but they did not reduce transient Ca 2+ currents attributed to Ca V 3 channels. These results suggest that β 2 subunit mutants lacking the N terminus may preferentially disrupt the proper localization of L-type Ca 2+ channels in the cell membrane. Cardiac-specific delivery of these decoy molecules in vivo may represent a gene-based treatment for pathologies involving Ca 2+ overload.