title: Design of mutant beta2 subunits as decoy molecules to reduce the expression of functional Ca2+ channels in cardiac cells.
abstract: 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 alpha(1) subunit may alleviate cytosolic Ca(2+) excess. As a molecular approach to disrupt the assembly of Ca(V)1.2 (alpha(1C)) channels at the cell membrane, we targeted the Ca(2+) channel beta(2) subunit, an intracellular chaperone that interacts with alpha(1C) via its beta interaction domain (BID) to promote Ca(V)1.2 channel expression. Recombinant adenovirus expressing either the full beta(2) subunit (Full-beta(2)) or truncated beta(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-beta(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 alpha(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 beta(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.
authors.affiliation: Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham, #832, Little Rock, AR 72205, USA. stelemaque@uams.edu
authors.forename: Sabine
authors.initials: S
authors.lastname: Télémaque
authors.forename: Swapnil
authors.initials: S
authors.lastname: Sonkusare
authors.forename: Terrie
authors.initials: T
authors.lastname: Grain
authors.forename: Sung W
authors.initials: SW
authors.lastname: Rhee
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Stimers
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Rusch
authors.forename: James D
authors.initials: JD
authors.lastname: Marsh
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000187:drug effects* / Q000235:genetics; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D016503:Drug Delivery Systems / Q000379:methods; D015195:Drug Design; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D018716:Molecular Mimicry; D009154:Mutation; D009206:Myocardium / Q000737:chemistry* / Q000166:cytology; D015202:Protein Engineering / Q000379:methods; D021122:Protein Subunits / Q000235:genetics / Q000627:therapeutic use; D051381:Rats
chemical_list: D020746:Calcium Channels, L-Type; D018076:DNA, Complementary; D021122:Protein Subunits; D002118:Calcium