Study of electrophysiological properties of cloned N-type Ca^<2+> channel

为了确定n型Ca^<2+>通道的离子传导特性，我们使用传统的全细胞膜片钳技术检测了在幼鼠肾细胞中表达的重组n型Ca^<2+>通道。重组的n型Ca^<2+>通道由α _<1B>、α _<1*>和β _<1*>亚基组成，显示出高压激活的Ba^<2+>电流，并被n型通道阻滞剂ω -conotoxin- gvia强烈阻断。在110mM Ba^<2+>存在下，单一电流的斜率电导为18.2pS，具有n型通道的特征。Ca^<2+>和Sr^<2+>产生的离子通量小于Ba^<2+>, Ba^<2+>、Ca^<2+>和Sr^<2+>电流的最大电导比分别为1.0:0.72:0.75。在Ba^<2+>和Ca^<2+>的混合物中，Ca^<2+>浓度代替Ba^<2+>稳步增加，Ba^<2+>和Ca^<2+>的总浓度保持在3mM不变，电流振幅为…，当20%的外部Ba^<2+>被Ca^<2+>取代时，电流振幅达到明显的最小值。这种反常的摩尔分数效应表明存在一个离子结合位点，其中两个或多个渗透离子可以同时存在。在不含多价阳离子的110mM Na^+外溶液中，测试电位大于-5OmV可诱发向内Na^+电流。这些电流以类似于Ba^<2+>电流的动力学方式激活和灭活。无机Ca^<2+>拮抗剂的应用以浓度依赖的方式阻断了通过n型通道的Ba^<2+>电流。抑制等级为La^<3+> <大于等于> Cd^<2+> * Zn^<2+> * Ni^<2+> <大于等于> Co^<2+>。在中等去极化电位的测试脉冲前施加短时间强去极化，可以观察到La^<3+>和Cd^<2+>对通道阻塞的缓解，随后通道重新阻塞。测量到的再阻塞速率（2 × 10^8M^<-1> ^<-1>）接近扩散控制极限。这些结果表明，n型Ca^<2+>通道与l型Ca^<2+>通道具有高亲和力离子结合位点的一般特征，并且该位点易于从通道孔外接近。通道钙膜片钳- omega-conotoxin-GVIA Less

To establish ion conducting properties of the N-type Ca^<2+> channel, we examined a recombinant N-type Ca^<2+> channels expressed in baby hamster kidney cells, using a conventional whole-cell patch-clamp technique.The recombinant N-type Ca^<2+> channel, composed of the alpha_<1B>, alpha_<1*> and beta_<1*> subunits, displayed high-voltage-activated Ba^<2+> currents, and were strongly blocked by the N-type channel blocker omega-conotoxin-GVIA.In the presence of 110mM Ba^<2+>, the unitary current showed a slope conductance of 18.2pS, characteristic of N-type channels. Ca^<2+> and Sr^<2+> resulted in smaller ion fluxes than Ba^<2+>, with the ratio 1.0 : 0.72 : 0.75 of maximum conductance in current-voltage relationships of Ba^<2+>, Ca^<2+> and Sr^<2+> currents, respectively. In mixtures of Ba^<2+> and Ca^<2+>, where the Ca^<2+> concentration was steadily increased in place of Ba^<2+>, with the total concentration of Ba^<2+> and Ca^<2+> held constant at 3mM, the current amplitude went throu … More gh a clear minimum when 20% of the external Ba^<2+> was replaced by Ca^<2+>.This anomalous mole fraction effect suggests an ion-binding site where two or more permeant ions can sit simultaneously.Using an external solution containing 110mM Na^+ without polyvalent cations, inward Na^+ currents were evoked by test potentials more positive than -5OmV.These currents were activated and inactivated in a kinetic manner similar to that of Ba^<2+> currents.Application of inorganic Ca^<2+> antagonists blocked Ba^<2+> currents through N-type channels in a concentration-dependent manner.The rank order of inhibition was La^<3+> <greater than or equal> Cd^<2+> * Zn^<2+> * Ni^<2+> <greater than or equal> Co^<2+>.When a short strong depolarization was applied before test pulses of moderate depolarizing potentials, relief from channel blockade by La^<3+> and Cd^<2+>, and subsequent channel reblocking was observed.The measured rate (2x10^8M^<-1>s^<-1>) of reblocking approached the diffusion-controlled limit.These results suggest that N-type Ca^<2+> channels share general features of a high affinity ion-binding site with the L-type Ca^<2+> channel, and that this site is easily accessible from the outside of the channel pore.channel calcium patch-clamp omega-conotoxin-GVIA Less