Decoy calcium channel beta subunits modulate contractile function in myocytes

To test the hypothesis that mutated beta2-subunits of the L-type calcium channel could serve as a decoy and interdict calcium channel trafficking and function, we engineered a beta2 subunit that contained the beta interaction domain for alpha1c subunit interaction, but lacked N- and C-terminal domai...

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Bibliographic Details
Published in:Molecular and cellular biochemistry 2003-01, Vol.242 (1-2), p.3-10
Main Authors: Fan, Q Ivy, Vanderpool, Kathleen M, O'Connor, Jessica, Marsh, James D
Format: Article
Language:English
Subjects:
Adenoviridae - genetics
Adenovirus
Animals
Blotting, Western
Calcium Channels - chemistry
Calcium Channels - genetics
Calcium Channels - metabolism
Cells, Cultured
Green Fluorescent Proteins
Luminescent Proteins
Male
Muscle Cells - chemistry
Muscle Cells - metabolism
Muscle Contraction
Myocardium - cytology
Myocardium - metabolism
Protein Subunits - genetics
Protein Subunits - metabolism
Proteins
Rats
Rats, Sprague-Dawley
Rodents
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Summary:To test the hypothesis that mutated beta2-subunits of the L-type calcium channel could serve as a decoy and interdict calcium channel trafficking and function, we engineered a beta2 subunit that contained the beta interaction domain for alpha1c subunit interaction, but lacked N- and C-terminal domains that might be essential for sarcolemmal localization. An adenoviral vector was constructed containing the gene for the beta-interaction domain (BID) fused to green fluorescence protein (GFP), using a vector containing only GFP as control. Freshly plated, dissociated adult rat myocytes were infected and expression and function were assessed at 60 h. Fluorescence microscopy confirmed GFP expression; immunoblot analysis confirmed dose-dependent GFP-BID expression. Mechanical properties of adult rat ventricular myocytes were evaluated using a video edge-detection system. Contractility analysis (optical/video, field stimulation) demonstrated that contracting cells decreased from 60 to 2%. Contractile amplitude (percent shortening) decreases significantly from 5.6 vs. 2.4% with no change in time to peak twitch. Recombinant adenovirus overexpressing mutated beta2 subunits in adult mammalian myocytes can markedly alter excitation-contraction coupling. This paradigm may offer new approaches to understanding and modulating EC coupling.
ISSN:0300-8177
1573-4919
DOI:10.1023/A:1021128017974