DEPENDENCE OF ACETYLCHOLINE-RECEPTOR CHANNEL KINETICS ON AGONIST CONCENTRATION IN CULTURED MOUSE MUSCLE-FIBERS
DEPENDENCE OF ACETYLCHOLINE-RECEPTOR CHANNEL KINETICS ON AGONIST CONCENTRATION IN CULTURED MOUSE MUSCLE-FIBERS
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DOI：
10.1113/jphysiol.1988.sp017019
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发表时间：
1988-03-01
影响因子：
5.5
通讯作者：
JACKSON, MB
中科院分区：
文献类型：
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作者：
JACKSON, MB
1.应用膜片钳技术研究乙酰胆碱受体通道门控动力学。激动剂卡巴胆碱以0.10 - 20 μ M的浓度使用。2.借助将多通道系统的随机行为与单个通道的动力学参数联系起来的数学表达式，对许多通道经常同时打开的数据进行了分析。这些方法提供了一致的参数估计值。这种一致性表明，通道关闭的动力学和膜补丁中通道的密度之间没有相关性。3.关闭时间与两个指数之和很好地拟合。添加第三个指数分量从未显着提高拟合的质量。4.两个指数的和通常提供了最适合的开放时间。缓慢关闭和快速关闭通道的分数的比率与激动剂浓度呈线性增加，以与单配体和双配体受体通道的开放一致的方式。5.封闭形成后不同时间的封闭时间密度分析用于跟踪脱敏的时间过程。在脱敏过程中没有检测到通道关闭动力学的变化。6.在0.10 μ M-卡巴胆碱下，观察到开口的频率仅略高于自发开口的背景频率。在20 μ M-卡巴胆碱下，在密封形成之后和脱敏开始之前，打开的频率高约300倍。7.持续时间的开口的出现频率随卡巴胆碱浓度线性增加，并在约5 μ M时达到饱和。长时间开口的出现频率随着激动剂浓度的平方而增加，只有轻微的饱和迹象。8.这里提出的结果进行了讨论的框架内的两个结合位点模型的乙酰胆碱受体的变构激活。所有的平衡常数和许多相关的反应方案的速率常数的估计。这两个配体结合位点被发现是非常不同的，在它们的解离常数和它们对通道门控转换的影响。这些结果对受体活化的能量学和受体对结合能的利用具有影响。
1. The patch-clamp technique was used to study channel gating kinetics of the acetylcholine receptor. The agonist carbachol was used at concentrations varying from 0.10 to 20 .mu.M. 2. Data in which many channels were often open at the same time were analysed with the aid of mathematical expressions that relate the stochastic behaviour of a many-channel system to the kinetic parameters of a single channel. These methods provide consistent estimates of parameters. This consistency suggests that there is no correlation between the kinetics of channel closure and the density of channels in a patch of membrane. 3. Closed times were well fitted by a sum of two exponentials. Addition of a third exponential component never significantly improved the quality of the fit. 4. A sum of two exponentials usually provided the best fit to open times. The ratio of the fractions of slowly closing and rapidly closing channels increased linearly with agonist concentration, in a manner consistent with the opening of singly and doubly liganded receptor channels. 5. Analysis of closed-time densities at various times after seal formation was used to follow the time course of desensitization. No changes in the kinetics of channel closure were detected during desensitization. 6. At 0.10 .mu.M-carbachol the frequency at which openings were observed was only slightly more than the background frequency of spontaneous opening. At 20 .mu.M-carbachol, immediately after seal formation and before the onset of desensitization, the frequency of opening was approximately 300 times higher. 7. The frequency of appearance of brief-duration openings increased linearly with carbachol concentration and saturated at approximately 5 .mu.M. The frequency of appearance of long-duration openings increased as the square of the agonist concentration, with only a slight hint of saturation. 8. The results presented here are discussed within the framework of a two-binding-site model for the allosteric activation of the acetylcholine receptor. Estimates are made of all of the equilibrium constants and many of the rate constants of the relevant reaction scheme. The two ligand binding sites are found to be very different in terms of their dissociation constants and their influence on the channel gating transitions. These results have implications for the energetics of receptor activation and for the utilization of binding energy by the receptor.