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

为了确定N型钙离子通道的离子传导特性，我们用传统的全细胞膜片钳技术检测了在幼年仓鼠肾脏细胞中表达的重组N型钙通道。重组N型钙通道由α_1B；lt；1*和β_lt；1*&gt；亚基组成，呈现高压激活的Ba^&lt；在110 mM的Ba2+存在下，单位电流的斜率电导为18.2pS，为N型通道的特征。在Ba^&lt；2+&gt；、Ca^&lt；2+&gt；和Sr^&lt；2+&gt；电流的电流-电压关系中，Ca^&lt；2+&gt；和Sr^&lt；2+&gt；电流的最大电导比分别为1.0：0.72：0.75。在Ba^&lt；2+和Ca^&lt；2+&gt；的混合物中，Ca^&lt；2+&gt；浓度代替Ba^&lt；2+&gt；的浓度稳步增加，而Ba^&lt；2+&gt；和Ca^&lt；2+&gt；的总浓度在3 mM时保持不变，电流幅度在…这种异常的摩尔分数效应表明有一个离子结合部位，两个或两个以上的离子可以同时存在。使用不含多价阳离子的含有110 mM Na+的外部溶液，内向Na+电流被比-5 OMV更正的测试电位诱发。这些电流被激活和失活的动力学方式类似于Ba^lt；2+和gt；电流的动力学方式。无机Ca^&lt；2+和gt；拮抗剂的应用阻断了Ba^电流以浓度依赖的方式通过N型通道。抑制的等级顺序是La^；lt；3+>lt；大于或等于；*Zn^；；lt；2+&gt；*Ni^；lt；2+&gt；；大于或等于。当在中等去极化电位的测试脉冲之前施加短而强烈的去极化时，解除了La^；结果表明，N型钙通道与L型钙通道具有高亲和力离子结合部位的一般特征，且该部位很容易从通道孔外进入。

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