Design of mutant beta2 subunits as decoy molecules to reduce the expression of functional Ca2+ channels in cardiac cells.
J Pharmacol Exp Ther
; 325(1): 37-46, 2008 Apr.
Article
em En
| MEDLINE
| ID: mdl-18184831
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Desenho de Fármacos
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Terapia Genética
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Mimetismo Molecular
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Canais de Cálcio Tipo L
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Mutação
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Miocárdio
Tipo de estudo:
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
J Pharmacol Exp Ther
Ano de publicação:
2008
Tipo de documento:
Article
País de afiliação:
Estados Unidos