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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The molecular properties of two size forms of the al subunit of purified skeletal muscle calcium channels were analyzed. The minor, full-length, form, a1212, was found to have an apparent molecular mass of 214 kDa by Ferguson plot analysis, while the major, truncated, form, now designated al1o, had an apparent molecular mass of 193 kDa. Antibody mapping of the C-terminal region of allg0 with 10 anti-peptide antibodies placed the C terminus between residues 1685 and 1699. Three consensus sites for cAMP-dependent protein phosphorylation are present in the C-terminal region of a1212 but not in al1g, and they may be important for the regulation of the ion conductance activity of the calcium channel.

Site-directed antibodies corresponding to conserved putative extracellular segments of sodium channels, coupled with binding studies of radiolabeled insect-selective scorpion neurotoxins, were employed to clarify the relationship between the toxins' receptor sites and the insect sodium channel. (1) The depressant insect toxin LqhIT2 was shown to possess two noninteracting binding sites in locust neuronal membranes: a high-affinity (KD1 = 0.9 +/- 0.6 nM) and low-capacity (Bmax1 = 0.1 +/- 0.07 pmol/mg) binding site as well as a low-affinity (KD2 = 185 +/- 13 nM) and high-capacity (Bmax2 = 10.0 +/- 0.6 pmol/mg) binding site. (2) The high-affinity site serves as a target for binding competition by the excitatory insect toxin AaIT. (3) The binding of LqhIT2 was significantly inhibited in a dose-dependent manner by each of four site-directed antibodies. The binding inhibition resulted from reduction in the number of binding sites. (4) The antibody-mediated inhibition of [125I]AaIT binding differs from that of LqhIT2: three out of the four antibodies which inhibited LqhIT2 binding only partially affected AaIT binding. Two antibodies, one corresponding to extracellular and one to intracellular segments of the channel, did not affect the binding of either toxin. These data suggest that the receptors to the depressant and excitatory insect toxins (a) comprise an integral part of the insect sodium channel, (b) are formed by segments of external loops in domains I, III, and IV of the sodium channel, and (c) are localized in close proximity but are not identical in spite of the competitive interaction between these toxins.

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