L-type calcium channels in insulin-secreting cells: biochemical characterization and phosphorylation in RINm5F cells.
Mol Endocrinol
; 11(5): 619-29, 1997 May.
Article
em En
| MEDLINE
| ID: mdl-9139805
ABSTRACT
Opening of dihydropyridine-sensitive voltage-dependent L-type Ca2+-channels (LTCCs) represents the final common pathway for insulin secretion in pancreatic beta-cells and related cell lines. In insulin-secreting cells their exact subunit composition is unknown. We therefore investigated the subunit structure of (+)-[3H]isradipine-labeled LTCCs in insulin-secreting RINm5F cells. Using subunit-specific antibodies we demonstrate that alpha1C subunits (199 kDa, short form) contribute only a minor portion of the total alpha1 immunoreactivity in membranes and partially purified Ca2+-channel preparations. However, alpha1C forms a major constituent of (+)-[3H]isradipine-labeled LTCCs as 54% of solubilized (+)-[3H]isradipine-binding activity was specifically immunoprecipitated by alpha1C antibodies. Phosphorylation of immunopurified alpha1C with cAMP-dependent protein kinase revealed the existence of an additional 240-kDa species (long form), that remained undetected in Western blots. Fifty seven percent of labeled LTCCs were immunoprecipitated by an anti-beta-antibody directed against all known beta-subunits. Isoform-specific antibodies revealed that these mainly corresponded to beta1b- and beta3-subunits. We found beta2- and beta4-subunits to be major constituents of cardiac and brain L-type channels, respectively, but not part of L-type channels in RINm5F cells. We conclude that alpha1C is a major constituent of dihydropyridine-labeled LTCCs in RINm5F cells, its long form serving as a substrate for cAMP-dependent protein kinase. beta1b- and beta3-Subunits were also found to associate with L-type channels in these cells. These isoforms may therefore represent biochemical targets for the modulation of LTCC activity in RINm5F cells.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Canais de Cálcio
/
Ilhotas Pancreáticas
/
Insulina
Limite:
Humans
Idioma:
En
Revista:
Mol Endocrinol
Assunto da revista:
BIOLOGIA MOLECULAR
/
ENDOCRINOLOGIA
Ano de publicação:
1997
Tipo de documento:
Article
País de afiliação:
Alemanha