Ichida Seiji, Abe Junichi, Komoike Kuniyo, Imanishi Takashi, Wada Tetsuyuki, Masuko Takashi, Minami Takeshi
Department of Biological Chemistry, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, 577-8502, Higashio-saka, Japan.
Neurochem Res. 2005 Apr;30(4):457-66. doi: 10.1007/s11064-005-2681-5.
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.
采用一种新的结合方法（NBM）来研究125I-ω-芋螺毒素（ω-CTX）GVIA和125I-ω-CTX MVIIC与通过抗B1Nt（Cav2.1和Cav2.2通道中的一个肽序列）抗体从鸡脑膜捕获的Cav2.1和Cav2.2通道的特异性结合特性。NBM的结果如下：（1）125I-ω-CTX GVIA和125I-ω-CTX MVIIC与Cav2.1和Cav2.2通道特异性结合的半数有效剂量（ED50）值分别约为68和60 pM，与它们与鸡脑粗膜结合的ED50值（分别为87和35 pM）非常相似。（2）ω-CTX GVIA（0.5 nM）、强啡肽A（Dyn）、庆大霉素（Gen）、新霉素（Neo）和妥布霉素（Tob）（各100 μM）可抑制125I-ω-CTX GVIA（100 pM）的特异性结合，但ω-阿加芋螺毒素（Aga）IVA、钙调蛋白（calciseptine）、ω-CTX SVIB、ω-CTX MVIIC（各0.5 nM）、PN200-110（PN）、地尔硫䓬（Dil）或维拉帕米（Ver）（各100 μM）则不能。钙调蛋白（CaM）以剂量依赖性方式抑制特异性结合（半数抑制浓度（IC50）值约为100 μg蛋白/ml）。（3）ω-CTX MVIIC、ω-CTX GVIA、ω-CTX SVIB（各0.5 nM）、Dyn、Neo和Tob（各100 μM）可抑制125I-ω-CTX MVIIC（60 pM）的特异性结合，但ω-Aga IVA、钙调蛋白（各为0.5 nM）、PN、Dil、Ver（各100 μM）或100 μg蛋白/ml CaM则不能。这些结果表明，在NBM中，125I-ω-CTX GVIA和125I-ω-CTX MVIIC与Cav2.1和Cav2.2通道的特异性结合特性与它们与鸡脑粗膜的结合特性非常相似，尽管CaM和CaM游离Ca2+的IC50值分别比125I-ω-CTX GVIA和125I-ω-CTX MVIIC与粗膜特异性结合的IC50值高约33倍和5000倍。
