Abstract
Biochemical properties and subcellular distribution of the class A calcium channel alpha 1 subunits (alpha 1A) from rat and rabbit brain were examined using site-directed anti-peptide antibodies specific for rat rbA (anti-CNA3) and for rabbit BI (anti-NBI-1 and anti-NBI-2) isoforms of alpha 1A. In immunoblotting experiments, anti-CNA3 specifically identifies multiple alpha 1A polypeptides with apparent molecular masses of 210, 190, and 160 kD, and anti-NBI-1 and anti-NBI-2 specifically recognize 190-kD alpha 1A polypeptides in rat brain membrane. In rabbit brain, anti-NBI-1 or anti-NBI-2 specifically detect alpha 1A polypeptides with apparent molecular masses of 220, 200, and 190 kD, while anti-CNA3 specifically recognizes 190-kD alpha 1A polypeptides. These polypeptides evidently represent multiple isoforms of alpha 1A present in both rat and rabbit brain. Anti-CNA3 specifically immunoprecipitates high affinity receptor sites for omega-conotoxin MVIIC (Kd approximately 100 pM), whereas anti-NBI-2 immunoprecipitates two distinct affinity receptor sites for omega-conotoxin MVIIC (Kd approximately 100 pM and approximately 1 microM). Coimmunoprecipitation experiments indicate that alpha 1A subunits recognized by anti-CNA3 and anti-NBI-2 are associated with syntaxin in a stable, SDS-resistant complex and with synaptotagmin. Immunofluorescence studies reveal that calcium channels recognized by anti-NBI-2 are localized predominantly in dendrites and nerve terminals forming synapses on them, while calcium channels recognized by anti-CNA3 are localized more prominently in cell bodies and in nerve terminals. The mossy fiber terminals in hippocampus and the terminals of climbing and parallel fibers in cerebellum are differentially stained by these isoform-specific antibodies. These results indicate that both rbA and BI isoforms of alpha 1A are expressed in rat and rabbit brain and form calcium channels having alpha 1A subunits with distinct molecular mass, pharmacology, and subcellular localization.