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 ¹²⁵I-omega-conotoxin (ω-CTX) GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels captured from chick brain membranes by antibodies against B1Nt (a peptide sequence in Ca_v2.1 and Ca_v2.2 channels). The results for the NBM were as follows. (1) The ED₅₀ values for specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 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-ω-CTX GVIA (100 pM) binding was inhibited by ω-CTX GVIA (0.5 nM), dynorphine A (Dyn), gentamicin (Gen), neomycin (Neo) and tobramicin (Tob) (100 μM each), but not by ω-agaconotoxin (Aga) IVA, calciseptine, ω-CTX SVIB, ω-CTX MVIIC (0.5 nM each), PN200-110 (PN), diltiazem (Dil) or verapamil (Ver) (100 μM each). Calmodulin (CaM) inhibited the specific binding in a dose-dependent manner (IC₅₀ value of about 100 μg protein/ml). (3) The specific ¹²⁵I-ω-CTX MVIIC (60 pM) binding was inhibited by ω-CTX MVIIC, ω-CTX GVIA, ω-CTX SVIB (0.5 nM each), Dyn, Neo and Tob (100 μM, each), but not by ω-Aga IVA, calciseptine (0.5 nM each), PN, Dil, Ver (100 μM each) or 100 μg protein/ml CaM. These results suggested that the characteristics of the specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 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-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to crude membranes.