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La clonación molecular y la expresión funcional del intercambiador cardíaco sarcolemal Na(+) -Ca2+
D A Nicoll1, S Longoni, K D Philipson
1Department of Medicine, UCLA School of Medicine 90024-1760.
Science (New York, N.Y.)
|October 26, 1990
Resumen
Los investigadores identificaron un clon de ADN complementario (ADNc) para el intercambiador cardíaco sarcolemal de sodio y calcio (NCX1). Este clon permite la expresión funcional del intercambiador Na(+) - Ca2+, crucial para el acoplamiento de excitación-contracción cardíaca.
Área de la Ciencia:
- Fisiología cardiovascular fisiología cardiovascular.
- Biología Molecular Biología Molecular
- La bioquímica es la bioquímica.
Sus antecedentes:
- El intercambiador Na(+) - Ca2+ del sarcolema cardíaco es vital para regular el Ca2+ intracelular durante el acoplamiento excitación-contracción.
- Comprender la base molecular de este transportador es esencial para estudiar la función cardíaca.
Objetivo del estudio:
- Identificar y caracterizar el ADN complementario (ADNc) que codifica la proteína del intercambiador cardíaco Na(+) - Ca2+.
- Para permitir estudios moleculares del transportador Na(+) -Ca2+.
Principales métodos:
- Se utilizaron anticuerpos policlonales para examinar una biblioteca de ADNc.
- Se realizó un análisis de la mancha del norte para evaluar la hibridación del ARN.
- Se realizó un análisis de hidropatía y comparaciones de similitud de secuencias.
- Péptidos sintéticos generados para la producción de anticuerpos y la inmunoblotación.
- Se inyecta ARN sintetizado en ovocitos de Xenopus para estudios de expresión funcional.
Principales resultados:
- Se identificó un clon de ADNc que codifica la proteína intercambiadora Na(+) -Ca2+.
- El ADNc se hibrida a un ARN de 7 kilobases y contiene un marco de lectura abierta de 970 aminoácidos.
- El análisis de la hidropatía sugiere múltiples hélices transmembrana, con similitud de secuencia con la ATPasa dependiente de Na(+) y K(+).
- Los anticuerpos reaccionaron con las proteínas sarcolemmales de 70, 120 y 160 kDa.
- El ARN sintetizado a partir del ADNc indujo la actividad de intercambio Na(+) - Ca2+ en los ovocitos de Xenopus.
Conclusiones:
- El clon de ADNc identificado representa el intercambiador cardíaco Na(+) -Ca2+.
- Esta herramienta molecular permite la expresión funcional y una mayor investigación del papel del transportador en la fisiología cardíaca.
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