Gating of maxi K+ channels studied by Ca2+ concentration jumps in excised inside-out multi-channel patches (myocytes from guinea pig urinary bladder).
Gating of maxi K+ channels studied by Ca2+ concentration jumps in excised inside-out multi-channel patches (myocytes from guinea pig urinary bladder).
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DOI：
10.1085/jgp.99.6.841
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发表时间：
1992-06
期刊：
影响因子：
--
通讯作者：
Isenberg G
中科院分区：
文献类型：
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作者：
Markwardt F;Isenberg G
在由内向外的宏片上记录通过最大钾通道的电流。使用液体灯丝开关(Franke，C.，H.Hatt和J.Dudel)。1987年。神经科，莱特。77：199-204)膜电极尖端的钙离子浓度([Ca~(2+)]_i)在不到1ms的时间内发生变化。当[Ca~(2+)]i从不到10 nM升高到3、6、20、50、320或1,000微米时，激活了斑块中的几个最大K+通道，而回到低于10 nM则使它们失活。以10-50次扫描的平均值评价钙依赖激活和失活的时程。平均电流开始了大约10毫秒的延迟，这归因于钙离子从尖端向K+通道蛋白的扩散。激活和去激活的时间过程用指数项的三次幂来拟合。[Ca~(2+)]i越高、正性电位越高，激动率越高。失活速率不依赖于之前的[Ca~(2+)]_i，且在较高的正电位下降低。在16-37℃的五个温度下测量了失活速率；用Arrhenius方程对结果进行了拟合，得到了0 mV下钙离子解离的势垒为16kcal/mol。200ms后，依赖时间的过程处于稳定状态，即没有失活迹象。在稳态(200ms)，通道开放Np(O)与[Ca~(2+)]_i的关系为Hill系数约为3，表观解离常数Kd由-50 mV时的13微米降至+70 mV时的0.5微米。Np(O)与电压的关系符合Boltzmann分布，最大P(O)为0.8，斜率因子约为39 mV。结果由一个模型总结，该模型描述了钙离子和电压相关的激活和失活，以及在与细胞质侧电距离为0.31的情况下，钙离子与细胞膜电场中三个相等而独立的位置结合的稳态开放概率。
Currents through maxi K+ channels were recorded in inside-out macro- patches. Using a liquid filament switch (Franke, C., H. Hatt, and J. Dudel. 1987. Neurosci, Lett. 77:199-204) the Ca2+ concentration at the tip of the patch electrode ([Ca2+]i) was changed in less than 1 ms. Elevation of [Ca2+]i from less than 10 nM to 3, 6, 20, 50, 320, or 1,000 microM activated several maxi K+ channels in the patch, whereas return to less than 10 nM deactivated them. The time course of Ca(2+)- dependent activation and deactivation was evaluated from the mean of 10- 50 sweeps. The mean currents started a approximately 10-ms delay that was attributed to diffusion of Ca2+ from the tip to the K+ channel protein. The activation and deactivation time courses were fitted with the third power of exponential terms. The rate of activation increased with higher [Ca2+]i and with more positive potentials. The rate of deactivation was independent of preceding [Ca2+]i and was reduced at more positive potentials. The rate of deactivation was measured at five temperatures between 16 and 37 degrees C; fitting the results with the Arrhenius equation yielded an energy barrier of 16 kcal/mol for the Ca2+ dissociation at 0 mV. After 200 ms, the time-dependent processes were in a steady state, i.e., there was no sign of inactivation. In the steady state (200 ms), the dependence of channel openness, N.P(o), on [Ca2+]i yielded a Hill coefficient of approximately 3. The apparent dissociation constant, KD, decreased from 13 microM at -50 mV to 0.5 microM at +70 mV. The dependence of N.P(o) on voltage followed a Boltzmann distribution with a maximal P(o) of 0.8 and a slope factor of approximately 39 mV. The results were summarized by a model describing Ca2+- and voltage-dependent activation and deactivation, as well as steady-state open probability by the binding of Ca2+ to three equal and independent sites within the electrical field of the membrane at an electrical distance of 0.31 from the cytoplasmic side.