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Design of mutant beta2 subunits as decoy molecules to reduce the expression of functional Ca2+ channels in cardiac cells.
- Source :
-
The Journal of pharmacology and experimental therapeutics [J Pharmacol Exp Ther] 2008 Apr; Vol. 325 (1), pp. 37-46. Date of Electronic Publication: 2008 Jan 09. - 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.
- Subjects :
- Animals
Calcium metabolism
Calcium Channels, L-Type genetics
Cell Line
DNA, Complementary genetics
Down-Regulation drug effects
Drug Delivery Systems methods
Humans
Myocardium cytology
Protein Engineering methods
Protein Subunits genetics
Protein Subunits therapeutic use
Rats
Calcium Channels, L-Type drug effects
Drug Design
Genetic Therapy methods
Molecular Mimicry
Mutation
Myocardium chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1521-0103
- Volume :
- 325
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- The Journal of pharmacology and experimental therapeutics
- Publication Type :
- Academic Journal
- Accession number :
- 18184831
- Full Text :
- https://doi.org/10.1124/jpet.107.128215