Biochemical properties and subcellular distribution of the class A calcium channel α1 subunits (α(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 α(1A). In immunoblotting experiments, anti-CNA3 specifically identifies multiple α(1A) polypeptides with apparent molecular masses of 210, 190, and 160 kD, and anti-NBI-1 and anti-NBI-2 specifically recognize 190-kD α(1A) polypeptides in rat brain membrane. In rabbit brain, anti-NBI-1 or anti-NBI-2 specifically detect α(1A) polypeptides with apparent molecular masses of 220, 200, and 190 kD, while anti-CNA3 specifically recognizes 190-kD α(1A) polypeptides. These polypeptides evidently represent multiple isoforms of α(1A) present in both rat and rabbit brain. Anti-CNA3 specifically immunoprecipitates high affinity receptor sites for ω-conotoxin MVIIC (K(d) ~ 100 pM), whereas anti-NBI-2 immunoprecipitates two distinct affinity receptor sites for ω- conotoxin MVIIC (K(d) ~ 100 pM and ~1 μM). Coimmunoprecipitation experiments indicate that α(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 α(1A) are expressed in rat and rabbit brain and form calcium channels having α(1A) subunits with distinct molecular mass, pharmacology, and subcellular localization.
ASJC Scopus subject areas
- Cell Biology