Single channel properties of P2X2 purinoceptors
Single channel properties of P2X2 purinoceptors
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DOI：
10.1085/jgp.113.5.695
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发表时间：
1999-05-01
影响因子：
3.8
通讯作者：
Sachs, F
中科院分区：
文献类型：
--
作者：
Ding, SH;Sachs, F
在由外向外的贴片中研究了克隆的P2 X（2）、在人胚肾（HEK;）293细胞和爪蟾卵母细胞中表达的嘌呤受体的单通道特性。平均单通道电流-电压关系在对称溶液中表现出内向整流，在145 mM NaCl中-100 mV时弦电导类似于30 pS。通道打开状态表现出快速闪烁，功率超过10 kHz。构象变化，而不是离子阻断，似乎负责闪烁。Na+结合在孔TMS中的平衡常数类似于150 mM I（0 mV）和电压依赖性。结合位点似乎与细胞外表面的电距离的0.2相似。平均通道电流和过量噪声具有选择性：K+ > Rb+ > Cs+ > Na+ > Li+。ATP增加了开放的概率（P-O），最大值为0.6，EC 50为11.2 μ M，希尔系数为2.3。Lowe环细胞外pH增强了通道对ATP的表观亲和力，pK（a）接近7.9，但不会导致开放通道的质子阻断。高pH值使艾德的上升时间减慢到ATP的台阶，而不影响秋霜。平均单通道振幅是独立的pH值，但过量的噪音增加，pH值降低。动力学分析表明，ATP缩短平均关闭时间，但不影响平均开放时间。最大似然动力学拟合的理想化的单通道电流在不同的ATP浓度产生了一个模型，有四个连续的封闭状态：（三个结合步骤）分支到两个开放状态，收敛于最终的封闭状态。ATP结合率随ATP的顺序结合而增加，表明结合位点不是独立的，而是正协同的。部分配体化的通道似乎没有打开。预测的P-o-ATP浓度与单通道电流剂量-反应曲线非常吻合。
The single channel properties of cloned P2X(2), purinoceptors expressed in human embryonic kidney (HEK;) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current-voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of similar to 30 pS at -100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore TMS similar to 150 mM I at 0 mV and voltage dependent. The binding site appeared to be similar to 0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity : K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (P-o) to a maximum of 0.6 with an EC50 of 11.2 mu M and a Hill coefficient of 2.3. Lowe ring extracellular pH enhanced the apparent affinity of the channel for ATP with a pK(a) of similar to 7.9 but did not cause a proton block; of the open channel. High pH slow ed the rise time to steps of ATP without affecting the fall rime. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states: (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates in creased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted P-o vs. ATP concentration closely matches th single channel curl-ent dose-response curve.