RESUMEN
Facilitation of calcium current by depolarizing prepulses has been observed in many cells including cardiac muscle. The mechanism underlying prepulse facilitation is controversial with respect to the requirements of channel subunits and cAMP kinase. We found that coexpression of the cardiac alpha1C-a subunit with the cardiac beta2a subunit significantly promotes the facilitation of I(Ba) by strong depolarizing prepulses. The magnitude of I(Ba) facilitation depended on the voltage potential of the prepulse and the interval duration between prepulse and test pulse. Prepulse facilitation was not affected by coexpression of AKAP79 and conditions favoring cAMP-dependent phosphorylation. Prepulse facilitation was also observed in cells expressing an alpha1C-a subunit which was truncated at residue 1733 removing the cAMP kinase site at Ser-1928. Facilitation was abolished by coexpression of the alpha2delta-1 or alpha2delta-3 subunit. We conclude that the expressed alpha1C-a beta2a complex is sufficient to support prepulse facilitation. Facilitation is prevented by coexpression of the alpha2delta subunit.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Canales de Calcio/química , Canales de Calcio/metabolismo , Proteínas Portadoras , Miocardio/metabolismo , Proteínas de Anclaje a la Quinasa A , Animales , Canales de Calcio/genética , Canales de Calcio Tipo L , Línea Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Potenciales de la Membrana , Ratones , Conformación Proteica , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Stable cell lines are potentially excellent tools for large-scale screening of new compounds. Two carboxyterminal-deleted constructs of the two splice variants a and b of the calcium channel class C alpha 1 subunit were expressed stably in HEK 293 cells. Each cell line produced regular L-type calcium currents. The opening and closing of the calcium channel elicited by potassium depolarization was followed by Fura-2 transients. These transients were blocked by the calcium channel blocker mibefradil with a concentration for 50% inhibition of 1.7 microM. The cell lines expressing the truncated cardiac alpha 1C-a or smooth muscle alpha 1C-b calcium channel were both blocked by nisoldipine under patch clamp conditions. Nisoldipine interacted with higher affinity with the alpha 1C-b channel than with the alpha 1C-a channel. These results indicate that the two cell lines retain the differential dihydropyridine sensitivity of smooth muscle and cardiac calcium channels and may be potential tools for the screening of L-type calcium channel blockers.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Bencimidazoles/farmacología , Línea Celular , Dihidropiridinas/metabolismo , Dihidropiridinas/farmacología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Fura-2/química , Corazón/efectos de los fármacos , Mibefradil , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocardio/metabolismo , Nisoldipino/farmacología , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Potasio/metabolismo , Relación Estructura-Actividad , Tetrahidronaftalenos/farmacología , TransfecciónRESUMEN
Nuclear factor I (NFI) is a member of a family of dimeric DNA-binding proteins that are involved both in the initiation of adenovirus DNA replication and in the stimulation of transcriptional activation. We have used fluorescence in situ hybridisation (FISH) to map one of four known genes encoding an NFI protein, the human NFI/X gene, to chromosome 19p1.3. Secondary sites of hybridisation observed at 5p1.5, 1q4.2-4.4, 1p2.1-2.2, and 20p1.3 most likely are attributable to partial sequence homologies with related NFI genes.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Cromosomas Humanos Par 19 , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Humanos , Hibridación Fluorescente in Situ , Factores de Transcripción NFI , Proteínas Nucleares , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Proteína 1 de Unión a la Caja YRESUMEN
The L-type alpha(1C) (Ca(v)1.2) calcium channel is the major calcium entry pathway in cardiac and smooth muscle. We inactivated the Ca(v)1.2 gene in two independent mouse lines that had indistinguishable phenotypes. Homozygous knockout embryos (Ca(v)1. 2-/-) died before day 14.5 postcoitum (p.c.). At day 12.5 p.c., the embryonic heart contracted with identical frequency in wild type (+/+), heterozygous (+/-), and homozygous (-/-) Ca(v)1.2 embryos. Beating of isolated embryonic cardiomyocytes depended on extracellular calcium and was blocked by 1 microm nisoldipine. In (+/+), (+/-), and (-/-) cardiomyocytes, an L-type Ba(2+) inward current (I(Ba)) was present that was stimulated by Bay K 8644 in all genotypes. At a holding potential of -80 mV, nisoldipine blocked I(Ba) of day 12.5 p.c. (+/+) and (+/-) cells with two IC(50) values of approximately 0.1 and approximately 1 microm. Inhibition of I(Ba) of (-/-) cardiomyocytes was monophasic with an IC(50) of approximately 1 microm. The low affinity I(Ba) was also present in cardiomyocytes of homozygous alpha(1D) (Ca(v)1.3) knockout embryos at day 12.5 p.c. These results indicate that, up to day 14 p.c., contraction of murine embryonic hearts requires an unidentified, low affinity L-type like calcium channel.