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Design of mutant beta2 subunits as decoy molecules to reduce the expression of functional Ca2+ channels in cardiac cells.

Authors :
Télémaque, Sabine
Sonkusare, Swapnil
Grain, Terrie
Rhee, Sung W
Stimers, Joseph R
Rusch, Nancy J
Marsh, James D
Source :
The Journal of Pharmacology and Experimental Therapeutics; April 2008, Vol. 325 Issue: 1 p37-46, 10p
Publication Year :
2008

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.

Details

Language :
English
ISSN :
00223565 and 15210103
Volume :
325
Issue :
1
Database :
Supplemental Index
Journal :
The Journal of Pharmacology and Experimental Therapeutics
Publication Type :
Periodical
Accession number :
ejs14396080
Full Text :
https://doi.org/10.1124/jpet.107.128215