Abstract. To identify and localize the protein products of genes encoding distinct L-type calcium channels in central neurons, anti-peptide antibodies specific for the class C and class D otl subunits were produced. Anti-CNC1 directed against class C immunoprecipitated 75 % of the L-type channels solubilized from rat cerebral cortex and hippocampus. Anti-CND1 directed against class D immunoprecipitated only 20% of the L-type calcium channels. Immunoblotting revealed two size forms of the class C L-type al subunit, L¢~ and Lc2, and two size forms of the class D L-type t~l subunit, Lo~ and Lm. The larger isoforms had apparent molecular masses of •200-210 kD while the smaller isoforms were 180-190 kD, as estimated from electrophoresis in gels polymerized from 5 % acrylamide.Imrnunocytochemical studies using CNC1 and CND1 antibodies revealed that the o~1 subunits of both L-type calcium channel subtypes are localized mainly in neuronal cell bodies and proximal dendrites. Relatively dense labeling was observed at the base of major dendrites in many neurons. Staining in more distal dendritic regions was faint or undetectable with CND1, while a more significant level of staining of distal dendrites was observed with CNC1, particularly in the dentate gyrus and the CA2 and CA3 areas of the hippocampus. Class C calcium channels were concentrated in clusters, while class D calcium channels were generally distributed in the cell surface membrane of cell bodies and proximal dendrites. Our results demonstrate multiple size forms and differential localization of two subtypes of L-type calcium channels in the cell bodies and proximal dendrites of central neurons. The differential localization and multiple size forms may allow these two channel subtypes to participate in distinct aspects of electrical signal integration and intracellular calcium signaling in neuronal cell bodies. The preferential localization of these calcium channels in cell bodies and proximal dendrites implies their involvement in regulation of calciumdependent functions occurring in those cellular compartments such as protein phosphorylation, enzyme activity, and gene expression.

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Embryos homozygous for mutations in the unc-52, pat-2, pat-3, and unc-112 genes of C. elegans exhibit a similar Pat phenotype. Myosin and actin are not organized into sarcomeres in the body wall muscle cells of these mutants, and dense body and M-line components fail to assemble. The unc-52 (perlecan), pat-2 (α-integrin), and pat-3 (β-integrin) genes encode ECM or transmembrane proteins found at the cell–matrix adhesion sites of both dense bodies and M-lines. This study describes the identification of the unc-112 gene product, a novel, membrane-associated, intracellular protein that colocalizes with integrin at cell–matrix adhesion complexes. The 720–amino acid UNC-112 protein is homologous to Mig-2, a human protein of unknown function. These two proteins share a region of homology with talin and members of the FERM superfamily of proteins.We have determined that a functional UNC-112::GFP fusion protein colocalizes with PAT-3/β-integrin in both adult and embryonic body wall muscle. We also have determined that UNC-112 is required to organize PAT-3/β-integrin after it is integrated into the basal cell membrane, but is not required to organize UNC-52/perlecan in the basement membrane, nor for DEB-1/vinculin to localize with PAT-3/β-integrin. Furthermore, UNC-112 requires the presence of UNC-52/perlecan and PAT-3/β-integrin, but not DEB-1/vinculin to become localized to the muscle cell membrane.

We describe the isolation and characterization of several rat brain cDNAs that are homologous to the a, subunit of heart and skeletal muscle dihydropyridine-sensitive Ca channels. Northern blot analysis of 32 cDNAs shows that they can be grouped into four distinct classes (A, B, C, and D), each corresponding to a distinct hybridization pattern of brain mRNAs. Southern blot and DNA sequencing suggest that each class of cDNA represents a distinct gene or gene family. In the regions sequenced, the rat brain class C and D gene products share =75% amino acid identity with the rabbit skeletal muscle Ca channel. In addition, the class C polypeptide is almost identical to the rabbit cardiac Ca channel (97% identity The exact subunit composition of neuronal Ca channels has not been determined. Biochemical analysis of the dihydropyridine (DHP) receptor/Ca channel of skeletal muscle shows that it is composed offive distinct subunits (8, 9). DNA cloning has revealed the primary structure of the skeletal muscle a,, a2, 8, and y subunits (10-12, 30). More recently, the structure of the cardiac a1 subunit has also been reported (13). Both the skeletal muscle and cardiac a1-subunit genes encode large polypeptides (212 and 242 kDa, respectively) and are structurally similar to the a subunit of Na channels (14,15,31). Both the Na-channel a subunit and the Cachannel a1 subunit possess four internal repeats, each containing six putative transmembrane regions. In one of these transmembrane domains, every third residue is positively charged; this transmembrane helix is thought to contain the voltage sensor of the molecule (16).Cardiac ventricular myocytes express mainly DHP-sensitive Ca channels with the slow inactivating properties classified as L-type, and Xenopus oocytes injected with cardiac mRNA express the same kind of channels. In agreement with this, microinjected synthetic RNA derived from the cloned cardiac a1-subunit cDNA directs the synthesis of similar channels in oocytes (13). Furthermore, expression of the skeletal muscle a,-subunit cDNA in myotubes from mdg mice and in mouse L cells results in functional DHP-sensitive Ca currents (17, 18). Thus, the a1 subunit acts both as a DHP receptor and as a voltage-sensitive ion channel. The functional role of the other Ca-channel subunits is not clear, although they may be involved in regulation of the a1 subunit (13).Here we show that rat brain expresses a family of genes that are related to the a1 subunit of skeletal muscle and of cardiac Ca channels. That the isolated cDNAs encode Ca channels is supported by DNA sequence data and by hybrid depletion experiments using Xenopus oocytes injected with rat brain RNA. Antisense oligomers for three of the four classes of cDNA partially (and in one case almost fully) inhibit the expression of rat brain Ca channels synthesized in oocytes. These same oligomers do not affect the expression ofbrain K or Na channels. These results strongly suggest that at least a portion ofobserved Ca-channel diversity in neurons is the result of ...