Isolation and reconstitution of <Ca^(2+)> -dependent <K^+> channel

<Ca^(2)> 依赖的 <K^> 通道的分离和重建

• 批准号: 60570055
• 负责人: ABE Teruo
• 金额: $ 1.02万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1985
• 资助国家: 日本
• 起止时间: 1985 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-60570055/
• 关键词: Apamin; <Ca^(2+)> -dependent <K^+> channel; 後過分極

Apamin is a neurotoxic peptide present in the venom of honey bee. When applied to the sympathetic ganglion cells of bullfrog, apamin did not affect either the <Ca^(2+)> channel or the <Ca^(2+)> -dependent <K^+> channel involved in the falling phase of the action potential. Apamin specifically blocked the <Ca^(2+)> -dependent <K^+> channel that genarates the afterhyperpolarization. Voltage clamp studies showed that the <Ca^(2+)> -dependent <K^+> current consists of at least 2 components responsible for the afterhyperpolarization; fast decaying component and slowly decaying component. The time constant of the two components were about 50 and 250 msec. Apamin decreased the time constant of the former without changing its amplitude and decreased the amplitude of the latter without changing its time constant. These results indicate that apamin is a specific probe for certain types of <Ca^(2+)> -dependent <K^+> channel.Synaptic plasma membrane (SPM) prepared from rat brain was incubated with <^(125)I> -apamin and then bifunctional cross-linker was added to cross-linke the apamin-receptor complex. Autoradiographs of two-dimensional gels gave a single acidic protein of <M_r> about 30,000. The density of the apamin receptor was of the order of 10 fmol/mg protein, too low to be purified by ordinary biochemical means. We extracted the radiolabeled spot from the gel and concentrated it to immunize a BALB/c mouse to obtain polyclonal and monoclonal antibodies to the receptor. However, it has not been successful so far.

蜂毒肽是一种存在于蜜蜂毒液中的神经毒性肽。当应用于牛蛙交感神经节细胞时，蜂毒肽既不影响参与动作电位下降相的<Ca^（2+）>通道，也不影响依赖于<Ca^（2+）>的<K^+>通道。Apamin特异性阻断产生后超极化的依赖于<Ca^（2+）>的<K^+>通道。电压钳研究表明，依赖于<Ca^（2+）>的<K^+>电流至少由两个负责后超极化的成分组成：快速衰减成分和缓慢衰减成分。两个分量的时间常数约为50和250毫秒。Apamin降低前者的时间常数而不改变其振幅，降低后者的振幅而不改变其时间常数。用~（125）I-apamin孵育大鼠脑突触质膜（SPM），然后加入双功能交联剂，使apamin-受体复合物交联。二维凝胶的放射自显影给出了约30，000的单一酸性蛋白质<M_r>。蜂毒肽受体的密度约为10 fmol/mg蛋白质，太低而不能用普通的生物化学方法纯化。我们从凝胶中提取放射性标记的斑点并将其浓缩以免疫BALB/c小鼠，以获得针对该受体的多克隆和单克隆抗体。然而，迄今为止并没有成功。

项目成果

Konomi Koyano and Teruo Abe: "Pharmacological evidence for two types of <Ca^(2+)> -dependent <K^+> -conductance in bullfrog sympathetic ganglion cells" Neuroscience Research. 3. 162-166 (1985)

Konomi Koyano 和 Teruo Abe：“牛蛙交感神经节细胞中两种 <Ca^(2)> 依赖性 <K^> 电导的药理学证据”神经科学研究。

Kohichi Tanaka, Shoichi Minota, Kenji Kuba, Konomi Koyano and Teruo Abe: "Differential effects of apamin on <Ca^(2+)> -dependent <K^+> currents in bullfrog sympathetic ganglion cells" Neuroscience Letters. 69. 233-238 (1986)

Kohichi Tanaka、Shoichi Minota、Kenji Kuba、Konomi Koyano 和 Teruo Abe：“apamin 对牛蛙交感神经节细胞中 <Ca^(2)> 依赖性 <K^> 电流的不同影响”《神经科学快报》。

Konomi Koyano;Teruo Abe: Neuroscience Research. 3. 162-166 (1985)

Koyano Konomi；Abe Teruo：神经科学研究。

Kohichi Tanaka;Shoichi Minota;Kenji Kuba;Koni Konomi Koyano;Teruo Abe: Neuroscience Letters. 69. 233-238 (1986)

田中浩一；美田翔一；久场健二；小矢野小乃；阿部辉夫：神经科学快报。