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

蜂胺是存在于蜜蜂毒液中的一种神经毒性肽。当应用于牛蛙交感神经节细胞时，apamin既不影响<Ca^(2+)>通道，也不影响<Ca^(2+)>依赖的<K^+>通道参与动作电位下降期。Apamin特异性阻断了产生后超极化的<Ca^(2+)> -依赖的<K^+>通道。电压箝位研究表明，<Ca^(2+)>依赖性<K^+>电流由至少2个负责后超极化的分量组成；快衰成分和慢衰成分。两组分的时间常数分别为50和250 msec。Apamin降低了前者的时间常数，但不改变其振幅；降低了后者的振幅，但不改变其时间常数。这些结果表明，维生素a是特定类型的<Ca^(2+)> -依赖性<K^+>通道的特异性探针。用<^(125)I> -apamin孵育大鼠脑突触质膜（SPM），加入双功能交联剂交联apamine -receptor复合物。二维凝胶的射线自照相显示单个酸性蛋白<M_r>约为30,000。维生素a受体的密度约为10 fmol/mg蛋白质，太低，无法用普通生化方法纯化。我们从凝胶中提取放射性标记点，浓缩后免疫BALB/c小鼠，获得针对受体的多克隆和单克隆抗体。然而，到目前为止，它还没有成功。

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.