ACTIVATION OF ION CHANNELS IN THE FROG ENDPLATE BY HIGH-CONCENTRATIONS OF ACETYLCHOLINE
ACTIVATION OF ION CHANNELS IN THE FROG ENDPLATE BY HIGH-CONCENTRATIONS OF ACETYLCHOLINE
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DOI：
10.1113/jphysiol.1988.sp016912
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发表时间：
1988-01-01
影响因子：
5.5
通讯作者：
OGDEN, DC
中科院分区：
文献类型：
--
作者：
COLQUHOUN, D;OGDEN, DC
1.本文用单离子通道记录法研究了成年蛙骨骼肌神经肌肉接头处乙酰胆碱（ACh）浓度与反应（通道开放分数）之间的平衡关系，并对脱敏效应进行了校正。在高ACh浓度下，通道开口发生在由长脱敏间隔分开的定义明确的簇中。响应po被估计为在群集期间单个通道打开的时间的比例。2.在负膜电位（-120 mV）下，po在100 μ M-ACh时达到最大值0.9，在15 μ M时为半最大值，此时希尔斜率为1.6。在高于200 μ M-ACh的浓度下，由于游离ACh本身的开放通道阻断，po下降。3.在正膜电位（+100 mV）下，几乎没有通道被ACh阻断; po在500 μ M-ACh下达到0.41的最大值，在50 μ M下具有半最大活化，此时Hill斜率为1.2。4.通道激活乙酰胆碱的具体机制，拟合数据的最小二乘法。拟合完全确定，只有当两个结合位点的乙酰胆碱被假定为是等效的乙酰胆碱结合反应中没有协同性。在负电位下，结合的微观平衡常数为K1 = K2 = 77 μ M，通道开放的平衡常数（开放/关闭速率，β/.（alpha.）为32.在正电位下，亲和力稍高，K = 32 μ M，这证实了ACh的结合位点在膜电场之外的观点。通道打开的平衡常数降低到0.7，这主要是由于打开寿命短得多（增加的关闭速率α）。正电位。5.该数据也通过非常高的β/值很好地拟合。α的以及ACh结合亲和力的高度负协同性或不等价性（K2. mchgt. K1）。一个很好的拟合也可以得到适度的正协同结合结合位点的非等效性。6.一个机制，假定受体与两个独立的门控亚基提供了一个不良的拟合数据在负电位。7.通道开放和ACh解离的速率常数估计通过约束的拟合参数，使通道开放的突发长度等于其在低浓度的ACh的观察值。结果与Colquhoun和Sakmann（1985）的结果一致，支持他们对低ACh浓度下爆发精细结构的解释。估算的速率常数给出了ACh结合和解离速率的高值（8 × 104）。107 M-1 s-1和6000 s-1）和通道开放率（20 000 s-1）。这些值与Colquhoun和Sakmann在低浓度下获得的值相似。
1. The equilibrium relationship between acetylcholine (ACh) concentration and response (fraction of channels open), corrected for the effects of desensitization, has been estimated by single-ion-channel recording at the adult frog skeletal neuromuscular junction. At high ACh concentration channel openings occur in well-defined clusters separated by long desensitized intervals. The response, po, was estimated as the proportion of time for which a single channel was open during a cluster. 2. At negative membrane potential (-120 mV) po reached a maximum value of 0.9 at 100 .mu.M-ACh and was half-maximum at 15 .mu.M with a Hill slope of 1.6 at this point. At concentrations higher than 200 .mu.M-ACh, po declined as a result of open-channel block by free ACh itself. 3. At positive membrane potentials (+100 mV) there was little channel block by ACh; po reached a maximum value of 0.41 at 500 .mu.M-ACh, with half-maximum activation at 50 .mu.M and Hill slope of 1.2 at this point. 4. Particular mechanisms for channel activation by ACh were fitted to the data by the method of least squares. Fits were fully determinate only if the two binding sites for ACh were assumed to be equivalent with no co-operativity in the ACh binding reactions. At negative potential the microscopic equilibrium constant for binding was K1 = K2 = 77 .mu.M and the equilibrium constant for channel opening (opening/closing rates, .beta./.alpha.) was 32. At positive potential the affinity was slightly higher, K = 32 .mu.M, which confirms the view that the binding sites for ACh are outside the membrane electric field. The equilibrium constant for channel opening was reduced to 0.7 mainly as a result of the much shorter open lifetime (increased closing rate .alpha.) at positive potentials. 5. The data were also fitted well by very high values of .beta./.alpha. together with a high degree of negative co-operativity or non-equivalence in ACh binding affinity (K2 .mchgt. K1). A good fit could also be obtained with moderate positive co-operativity combined with non-equivalence of the binding sites. 6. A mechanism that postulates a receptors with two independent gating subunits provided a poor fit to the data at negative potential. 7. The rate constants for channel opening and ACh dissociation were estimated by constraining the fitted parameters so that the burst length for channel opening was equal to its observed value at low concentrations of ACh. The results were consistent with those of Colquhoun and Sakmann (1985) and support their interpretation of the fine structure of bursts at low ACh concentration. The rate constants estimated give high values for the ACh association and dissociation rates (8 .times. 107 M-1 s-1 and 6000 s-1) and for the channel opening rate (20 000 s-1). These are similar to the values obtained by Colquhoun and Sakmann at low concentration.