Received for publication July 23, 2007.
Revised January 8, 2008.
Accepted for publication January 8, 2008.

Design of Mutant ₂ Subunits as Decoy Molecules to Reduce the Expression of Functional Ca ²⁺ Channels in Cardiac Cells

Abstract

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 ₂ subunit, an intracellular chaperone that interacts with _1C via its beta interaction domain (BID) to promote Ca_V1.2 channel expression. Recombinant adenovirus expressing either the full ₂ subunit (Full-₂) or truncated ₂ subunit constructs lacking either the C-, N-terminus or both (N-BID, C-BID, BID, respectively) fused to GFP were developed as potential decoys and overexpressed in HL-1 cells. Fluorescence microscopy revealed that the localization of Full-₂ 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 did not reduce transient ("T-type") Ca²⁺ currents attributed to Ca_V3 channels. These results suggest that ₂ 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.

Key words: Adenovirus, Calcium currents, Cardiomyocytes, Gene therapy, L-type calcium channels, b2 subunits