A New Binding Method (NBM) was used to investigate the characteristics of the specific binding of 125I-omega-conotoxin (omega-CTX) GVIA and 125I-omega-CTX MVIIC to Cav2.1 and Cav2.2 channels captured from chick brain membranes by antibodies against B1Nt (a peptide sequence in Car2.1 and Cav2.2 channels). The results for the NBM were as follows. (1) The ED50 values for specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to Cav2.1 and Cav2.2 channels were about 68 and 60 pM, respectively, and very similar to those (87 and 35 pM, respectively) to crude membranes from chick brain. (2) The specific 125I-omega-CTX GVIA (100 pM) binding was inhibited by omega-CTX GVIA (0.5 nM), dynorphine A (Dyn), gentamicin (Gen), neomycin (Neo) and tobramicin (Tob) (100 microM each), but not by omega-agaconotoxin (Aga) IVA, calciseptine, omega-CTX SVIB, omega-CTX MVIIC (0.5 nM each), PN200-110 (PN), diltiazem (Dil) or verapamil (Ver) (100 microM each). Calmodulin (CaM) inhibited the specific binding in a dose-dependent manner (IC50 value of about 100 microg protein/ml). (3) The specific 125I-omega-CTX MVIIC (60 pM) binding was inhibited by omega-CTX MVIIC, omega-CTX GVIA, omega-CTX SVIB (0.5 nM each), Dyn, Neo and Tob (100 microM, each), but not by omega-Aga IVA, calciseptine (0.5 nM each), PN, Dil, Ver (100 microM each) or 100 microg protein/ml CaM. These results suggested that the characteristics of the specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to Cav2.1 and Cav2.2 channels in the NBM were very similar to those to crude membranes from chick brain, although the IC50 values for CaM and free Ca2+ of CaM were about 33- and 5000-fold higher, respectively, than those for the specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to crude membranes.

The antibodies against omega-conotoxin GVIA (omega-CTX GVIA; N-type voltage-dependent calcium channel [VDCC] blocker) and B1Nt (N-terminal segment [residues 1-13] of BI alpha1 subunits of VDCCs) were prepared, and the selectivity for each antigen omega-CTX GVIA and B1Nt was investigated. For the antigen selectivity of anti-omega-CTX GVIA antibody against omega-CTX GVIA, ELISA, and immunoprecipitation were used. The reactions for ELISA and immunoprecipitation were observed except when antibody IgG purified by Protein A-Sepharose CL-4B from nonimmunized serum (purified NI-Ab) was used. The specific reactions were inhibited by 10 nM omega-CTX GVIA, but not by omega-CTX SVIB (N-type VDCC blocker), omega-CTX MVIIC (N- and P-type VDCC blocker), or omega-Aga IVA (P-type VDCC blocker). For the antigen selectivity of the anti-B1Nt antibody, analyses by ELISA, immunoprecipitation, and Western blotting were conducted. The reactions were observed except when NI-Ab was used. The ELISA and immunoprecipitation reactions were inhibited by the antigen peptide B1Nt, and the IC50 values were about 1.2 x 10(-8) and 1.3 x 10(-8) M, respectively. The bands of 210 and 190 kD by Western blotting of crude membranes from chick brain were also inhibited by 1 microM B1Nt. These results suggest that the antibodies prepared against omega-CTX GVIA and B1Nt in this work have high selectivity for their antigen. Therefore we assume that the antibodies against omega-CTX GVIA and B1Nt are useful tools for the analyses of the function and distribution of N-type VDCCs. The anti omega-CTX GVIA antibody must also be useful for the radioimmunoassay of omega-CTX GVIA.