PT - JOURNAL ARTICLE AU - Sabine Télémaque AU - Swapnil Sonkusare AU - Terrie Grain AU - Sung W. Rhee AU - Joseph R. Stimers AU - Nancy J. Rusch AU - James D. Marsh TI - Design of Mutant β<sub>2</sub> Subunits as Decoy Molecules to Reduce the Expression of Functional Ca<sup>2+</sup> Channels in Cardiac Cells AID - 10.1124/jpet.107.128215 DP - 2008 Apr 01 TA - Journal of Pharmacology and Experimental Therapeutics PG - 37--46 VI - 325 IP - 1 4099 - http://jpet.aspetjournals.org/content/325/1/37.short 4100 - http://jpet.aspetjournals.org/content/325/1/37.full SO - J Pharmacol Exp Ther2008 Apr 01; 325 AB - Calcium influx through long-lasting (“L-type”) Ca2+ channels (CaV) drives excitation-contraction in the normal heart. Dysregulation of this process contributes to Ca2+ overload, and interventions that reduce expression of the pore-forming α1 subunit may alleviate cytosolic Ca2+ excess. As a molecular approach to disrupt the assembly of CaV1.2 (α1C) channels at the cell membrane, we targeted the Ca2+ channel β2 subunit, an intracellular chaperone that interacts with α1C via its β interaction domain (BID) to promote CaV1.2 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 CaV1.2 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 α1C protein of CaV1.2 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, CaV1.3, but they did not reduce transient Ca2+ currents attributed to CaV3 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. The American Society for Pharmacology and Experimental Therapeutics