Effects of Calmodulin and Ca2+ Channel Blockers on ω-conotoxin GVI A Binding to Crude Membranes from α1B Subunit (Cav2.2) Expressed BHK Cells and Mice Brain Lacking the α1B Subunits

Characteristics for the specific binding of 125I-ω-CTX GVIA and 125I-ω-CTX MVIIC to crude membranes from BHKN101 cells expressing the α1B subunits of Cav2.2 channels and from mice brain lacking the α1B subunits of Cav2.2 channels, particularly, the effects of CaM and various Ca2+ channel blockers on these specific bindings were investigated. Specific binding of 125I-ω-CTX GVIA to the crude membranes from BHKN101 cells was observed, but not from control BHK6 cells. ω-CTX GVIA, ω-CTX MVIIC and ω-CTX SVIB inhibited the specific binding of 125I-ω-CTX GVIA to crude membranes from BHKN101 cells, and the IC50 values for ω-CTXGVIA, ω-CTX MVIIC and ω-CTX SVIB were 0.07, 8.5 and 1.7 nM, respectively. However, ω-agatoxin IVA and calciseptine at concentrations of 10−9–10−6 M did not inhibit specific binding. Specific binding was also about 80% inhibited by 20 μg protein/ml CaM. The amount of 125I-ω-CTX GVIA (30 pM) specifically bound to membranes from brain of knockout mice lacking α1B subunits of Cav2.2 channels was about 30% of that to the crude membranes from brain of wild-type. On the other hand, specific binding of 125I-ω-CTX MVIIC (200 pM) was observed on the crude membranes of both BHKN101 and control BHK6 cells. The specific binding of 125I-ω-CTX MVIIC (200 pM) was not inhibited by ω-CTX GVIA and ω-CTX SVIB, and also ω-Aga IVA and calciseptine at concentrations of 10−9–10−7 M, although specific binding was almost completely dose dependently inhibited by non-radiolabeled ω-CTX MVIIC (IC50 value was about 0.1 nM). 20 μg protein/ml CaM did not inhibit specific binding. Therefore, these results suggest that BHKN101 cells have a typical Cav2.2 channels which are also inhibited by CaM and have not specific binding sites for ω-CTX MVIIC, although ω-CTX MVIIC is a blocker for both Cav2.1 (α1A; P/Q-type) and Cav2.2 channels.