BIOCHEMICAL-PROPERTIES AND SUBCELLULAR-DISTRIBUTION OF THE BI AND RBAISOFORMS OF ALPHA(1A) SUBUNITS OF BRAIN CALCIUM CHANNELS

Biochemical properties and subcellular distribution of the class A cal cium channel alpha 1 subunits (alpha(1A)) from rat and rabbit brain we re examined using site-directed anti-peptide antibodies specific for r at rbA (anti-CNA3) and for rabbit BI (anti-NBI-1 and anti-NBI-2) isofo rms of alpha(1A). In immunoblotting experiments, anti-CNA3 specificall y identifies multiple alpha(1A) polypeptides with apparent molecular m asses of 210, 190, and 160 kD, and anti-NBI-1 and anti-NBI-2 spe cific ally recognize 190-kD alpha(1A) polypeptides in rat brain membrane. In rabbit brain, anti-NBI-1 or anti-NBI-2 specifically detect al, polype ptides with apparent molecular masses of 220, 200, and 190 kD, while a nti-CNA3 specifically recognizes 190-kD oil, polypeptides. These polyp eptides evidently represent multiple isoforms of oil, present in both rat and rabbit brain. Anti-CNA3 specifically immunoprecipitates high a ffinity receptor sites for omega-conotoxin MVIIC (K-d similar to 100 p M), whereas anti-NBI-2 immunoprecipitates two distinct affinity recept or sites for omega-conotoxin MVIIC (K-d similar to 100 pM and similar to 1 mu M) Coimmunoprecipitation experiments indicate that alpha(1A) s ubunits recognized by anti-CNA3 and anti-NBI-2 are associated with syn taxin in a stable, SDS-resistant complex and with synaptotagmin. Immun ofluorescence studies reveal that calcium channels recognized by anti- NBI-2 are localized predominantly in dendrites and nerve terminals for ming synapses on them, while calcium channels recognized by anti-CNA3 are localized more prominently in cell bodies and in nerve terminals. The messy 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 a,, are expressed in rat and rabbit brain and form cal cium channels having alpha(1A) subunits with distinct molecular mass, pharmacology, and subcellular localization.