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

为了建立N型Ca ^&lt;2 +&gt;通道的离子传导特性，我们用常规的全细胞膜片钳技术检测了在幼仓鼠肾细胞中表达的重组N型Ca ^&lt;2 +&gt;通道，该重组N型Ca ^&lt;2 +&gt;通道由α_，α_&lt;1 *&gt;和β_&lt;1 *&gt;亚基组成，显示高电压激活的Ba ^&lt;2 +&gt;电流，<1B>在110mM Ba ^&lt;2 +&gt;存在下，单位电流的斜率电导为18.2pS，具有N型通道的特征。Ca ^&lt;2 +&gt;和Sr ^&lt;2 +&gt;导致的离子通量小于Ba ^&lt;2 +&gt;，在Ba ^&lt;2 +&gt;、Ca ^&lt;2 +&gt;和Sr ^&lt;2 +&gt;的电流-电压关系中，最大电导之比分别为1.0：0.72：0.75。在Ba ^&lt;2 +&gt;和Ca ^&lt;2 +&gt;的混合物中，当Ca ^&lt;2 +&gt;浓度稳定增加时，Ba ^&lt;2 +&gt;和Ca ^&lt;2 +&gt;的总浓度保持在3mM，电流幅值从0.000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000 关于我们 当20%的外部Ba ^&lt;2 +&gt;被Ca ^&lt;2 +&gt;取代时，出现了一个明显的最小值。这种反常的摩尔分数效应表明存在一个离子结合位点，两个或多个渗透离子可以同时存在。使用含有110 mM Na ^+且不含多价阳离子的外部溶液，当测试电位大于-5OmV时，可诱发内向Na ^+电流，其激活和失活的动力学方式与Ba ^&lt;2 +&gt;类似。应用无机Ca ^&lt;2 +&gt;拮抗剂以浓度依赖性方式阻断N型通道的Ba ^&lt;2 +&gt;电流。抑制的等级顺序为La ^&lt;3 +&gt; Cd ^&lt;2 +&gt;* Zn ^&lt;2 +&gt;* Ni ^&lt;2 +&gt; Co ^&lt;2 +&gt;。当在中等去极化电位的测试脉冲之前施加短的强去极化时，La ^&lt;3 +&gt;和Cd ^&lt;2 +&gt;的通道阻断解除，<greater than or equal>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共同特征，<greater than or equal>而且这个网站可以很容易地从外部通道www.example.com钙膜片钳欧米茄芋螺毒素-GVIA减<-1><-1>

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