RESUMEN
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.
Asunto(s)
Canales de Calcio/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Canales de Calcio/análisis , Humanos , Secreción de Insulina , Insulinoma , Fosforilación , Células Tumorales CultivadasRESUMEN
Heterologous expression studies have shown that the activity of voltage-gated Ca2+ channels is regulated by their beta subunits in a beta subunit isoform-specific manner. In this study we therefore investigated if one or several beta subunit isoforms associate with L-type Ca2+ channels in different regions of mammalian brain. All four beta subunit isoforms (beta1b, beta2, beta3, and beta4) are expressed in cerebral cortex as shown in immunoblots. Immunoprecipitation of (+)-[3H]isradipine-labeled L-type channels revealed that the majority of beta subunit-associated L-type channels was associated with beta3 (42 +/- 8%) and beta4 (42 +/- 7%) subunits, whereas beta1b and beta2 were present in a smaller fraction of channel complexes. beta3 and beta4 were also the major L-type channel beta subunits in hippocampus. In cerebellum beta1b, beta2, and beta3 but not beta4 subunits were expressed at lower levels than in cortex. Accordingly, beta4 was the most prominent beta subunit in cerebellar L-type channels. This beta subunit composition was very similar to the one determined for 125I-omega-conotoxin-GVIA-labeled N-type and 125I-omega-conotoxin-MVIIC-labeled P/Q-type channel complexes in cerebral cortex and cerebellum. Our data show that all four beta subunit isoforms associate with L-type Ca2+ channels in mammalian brain. This beta subunit heterogeneity may play an important role for the fine tuning of L-type channel function and modulation in neurons.
Asunto(s)
Canales de Calcio/química , Proteínas Musculares/química , Proteínas del Tejido Nervioso/química , Neuronas/química , Receptores Colinérgicos/química , Animales , Química Encefálica , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio/metabolismo , Canales de Calcio Tipo L , Electroforesis en Gel de Poliacrilamida , Cobayas , Isradipino/metabolismo , Venenos de Moluscos/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Proteínas del Tejido Nervioso/metabolismo , Péptidos/metabolismo , Conformación Proteica , Conejos , Receptores Colinérgicos/metabolismo , omega-Conotoxina GVIARESUMEN
Monoclonal antibodies that recognize skeletal muscle dihydropyridine-sensitive calcium channel subunits were used to identify similar proteins in neuronal and small cell carcinoma cell lines. alpha 1-related proteins were detected by FACS analysis on the surface of human neuroblastoma (IMR 32) and small cell carcinoma (DMS 273 and DMS 114) cell lines. alpha 1-like polypeptides from these cells were isolated and partially characterized. The polypeptides exhibit an M(r) similar to that of the L-type channel alpha 1 subunit and are recognized by two distinct anti-alpha 1 mAbs. The data provide biochemical evidence for structural similarities between the alpha 1 subunit of small cell carcinoma and neuronal cell lines. Similarly, an alpha 2-like protein was characterized from these cells. Because alpha 2 is a subunit shared by many subtypes of calcium channels, these data suggest that subunits other than the pore-forming alpha 1 subunit may play an important role in the etiology of Lambert-Eaton syndrome. We demonstrate directly that small cell carcinoma and a cell line derived from peripheral neurons share L-type calcium channel-related proteins and a protein common to many voltage-gated calcium channel subtypes. These data support a model that proposes that cross-reactivity of anti-tumor cell antibodies with presynaptic elements, possibly calcium channels, plays a role in the development of Lambert-Eaton syndrome.