Characteristics of omega-conotoxin GVI A and MVIIC binding to Cav 2.1 and Cav 2.2 channels captured by anti-Ca2+ channel peptide antibodies

A New Binding Method (NBM) was used to investigate the characteristics of the specific binding of I-125-omega-conotoxin (omega-CTX) GVIA and I-125-omega-CTX MVIIC to Ca(v)2.1 and Ca(v)2.2 channels captured from chick brain membranes by antibodies against B1Nt (a peptide sequence in Ca(v)2.1 and Ca(v)2.2 channels). The results for the NBM were as follows. (1) The ED50 values for specific binding of I-125-omega-CTX GVIA and I-125-omega-CTX MVIIC to Ca(v)2.1 and Ca(v)2.2 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 I-125-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 mu M 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 mu M each). Calmodulin (CaM) inhibited the specific binding in a dose-dependent manner (IC50 value of about 100 mu g protein/ml). (3) The specific I-125-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 mu M, each), but not by omega-Aga IVA, calciseptine (0.5 nM each), PN, Dil, Ver (100 mu M each) or 100 mu g protein/ml CaM. These results suggested that the characteristics of the specific binding of I-125-omega-CTX GVIA and I-125-omega-CTX MVIIC to Ca(v)2.1 and Ca(v)2.2 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 I-125-omega-CTX GVIA and I-125-omega-CTX MVIIC to crude membranes.