Heterologous expression studies have shown that the activity of voltage-gated Ca 2؉ channels is regulated by their ␤ subunits in a ␤ subunit isoform-specific manner. In this study we therefore investigated if one or several ␤ subunit isoforms associate with L-type Ca 2؉ channels in different regions of mammalian brain.All four ␤ subunit isoforms (␤1b, ␤2, ␤3, and ␤4) are expressed in cerebral cortex as shown in immunoblots. Immunoprecipitation of (؉)-[3 H]isradipine-labeled Ltype channels revealed that the majority of ␤ subunitassociated L-type channels was associated with ␤3 (42 ؎ 8%) and ␤4 (42 ؎ 7%) subunits, whereas ␤1b and ␤2 were present in a smaller fraction of channel complexes. ␤3 and ␤4 were also the major L-type channel ␤ subunits in hippocampus. In cerebellum ␤1b, ␤2, and ␤3 but not ␤4 subunits were expressed at lower levels than in cortex. Accordingly, ␤4 was the most prominent ␤ subunit in cerebellar L-type channels. This ␤ subunit composition was very similar to the one determined for 125 I--conotoxin-GVIA-labeled N-type and 125 I--conotoxin-MVIIClabeled P/Q-type channel complexes in cerebral cortex and cerebellum.Our data show that all four ␤ subunit isoforms associate with L-type Ca 2؉ channels in mammalian brain. This ␤ subunit heterogeneity may play an important role for the fine tuning of L-type channel function and modulation in neurons.Voltage-gated Ca 2ϩ channels control the depolarization-induced influx of extracellular Ca 2ϩ into neurons and other electrically excitable cells. They exist as hetero-oligomeric complexes of different subunits (␣1, ␣2-␦, and ␤). Different types of neuronal Ca 2ϩ channels (termed L-, N-, P-, Q-, and R-type; 1) are discriminated by biophysical and pharmacological criteria (for reviews see Refs. 2-5). N-and P/Q-type channels are blocked by peptide toxins (-CTx

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.

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.