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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.

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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

Ichida

Abe

Komoike

et al. 2005

Neurochem Res

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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

et al. 2005

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Characteristics for the specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to crude membranes from BHKN101 cells expressing the alpha1B subunits of Cav2.2 channels and from mice brain lacking the alpha1B 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-omega-CTX GVIA to the crude membranes from BHKN101 cells was observed, but not from control BHK6 cells. omega-CTX GVIA, omega-CTX MVIIC and omega-CTX SVIB inhibited the specific binding of 125I-omega-CTX GVIA to crude membranes from BHKN101 cells, and the IC50 values for omega-CTXGVIA, omega-CTX MVIIC and omega-CTX SVIB were 0.07, 8.5 and 1.7 nM, respectively. However, omega-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 microg protein/ml CaM. The amount of 125I-omega-CTX GVIA (30 pM) specifically bound to membranes from brain of knockout mice lacking alpha1B 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-omega-CTX MVIIC (200 pM) was observed on the crude membranes of both BHKN101 and control BHK6 cells. The specific binding of 125I-omega-CTX MVIIC (200 pM) was not inhibited by omega-CTX GVIA and omega-CTX SVIB, and also omega-Aga IVA and calciseptine at concentrations of 10(-9)-10(-7) M, although specific binding was almost completely dose dependently inhibited by non-radiolabeled omega-CTX MVIIC (IC50 value was about 0.1 nM). 20 microg 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 omega-CTX MVIIC, although omega-CTX MVIIC is a blocker for both Cav2.1 (alpha1A; P/Q-type) and Cav2.2 channels.

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Ichida

Abe

Zhang

et al. 2003

Neurochemical Research

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To clear the structural specificity of calmodulin (CaM) on the specific 125I-omega-CTX binding to crude membranes from whole chick brain, the following experiments were investigated in this study: (i) the attenuating effect of semisynthetic tetrahydroisoquinoline derivatives on the inhibitory effect of Ca2+/CaM, (ii) the effects of chimeras of yeast and chicken Ca2+/CaM, and (iii) the effects of Ca2+-binding proteins (such as troponin c, S 100 a and b, and annexin I, III-V). The inhibitory effect of Ca2+/CaM was attenuated by isoquinoline derivatives (PX 28, 34, 216, 224, and CPU57) and a CaM antagonist W-7. PX 34, a typical synthesized isoquinoline derivative, showed the attenuating effect in a dose-dependent manner. The ED50 value for the attenuating effect of PX 34 was about 20 microM, which is similar to that of W-7 reported previously. Some chimeric CaMs such as YC 51-53 (which are close to the properties of vertebrate CaM) showed a significant inhibitory effect on the specific 125I-omega-CTX binding, but YC 129 and 130 (which retain the properties of yeast CaM), troponin c, S100 a, b, and annexin I, III-V had no effect on the specific 125I-omega-CTX binding. These results suggest that the characteristic structure containing the EF-hand structure of CaM itself is needed to cause the inhibitory effect on the specific 125I-omega-CTX binding.

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Cited by 4 publications

References 28 publications

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

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

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

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