Mechanism of muscarinic receptor-induced K+ channel activation as revealed by hydrolysis-resistant GTP analogues.
Mechanism of muscarinic receptor-induced K+ channel activation as revealed by hydrolysis-resistant GTP analogues.
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DOI：
10.1085/jgp.91.4.469
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发表时间：
1988-04
期刊：
影响因子：
--
通讯作者：
Szabo G
中科院分区：
文献类型：
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作者：
Breitwieser GE;Szabo G
用抗水解GTP类似物在心房肌细胞中研究了鸟嘌呤核苷酸结合蛋白（Gk）在毒蕈碱受体激活和内向整流K+通道[IK（M）]开放之间的偶联中的作用。在没有毒蕈碱激动剂的情况下，GTP类似物产生IK（M）特征性的膜电流。为了探索IK（M）激活的动力学特性，测量了各种类似物的这种受体非依赖性IK（M）的初始出现率。我们发现，IK（M）的激活是由细胞内类似物/GTP的比例，而不是由核苷酸的绝对浓度控制。类似物与GTP竞争结合Gk，具有以下相对亲和力：GTP γ S大于GTP大于GppNHp大于GppCH 2 p。然而，在足够高的细胞内浓度下，所有GTP类似物产生相同速率的IK（M）激活。这种与类似物无关的限制速率可能对应于GDP从非活性的GDP结合的Gk释放的速率。在对照条件下不引起IK（M）的纳摩尔浓度乙酰胆碱（ACh）的毒蕈碱受体刺激，在GTP类似物存在下催化IK（M）活化。乙酰胆碱（kACh）激活Gk的速率可以用简单的关系式kACh = 8.4 × 10（8）min- 1 M-1来描述。[ACh]± 0.44 min-1，其第一项大概反映激动剂催化的GDP从Gk.GDP复合物释放的速率，而第二项对应于受体非依赖性GDP释放的基础速率。结合IK（M）- [ACh]剂量效应关系的估计K0.5（160 nM），该结果还使我们能够估计Gk.GTP水解速率kcat接近135 min-1。这些结果提供了，第一次，在体内的G蛋白功能的显着特点的定量描述。
The role of a guanine nucleotide-binding protein (Gk) in the coupling between muscarinic receptor activation and opening of an inwardly rectifying K+ channel [IK(M)] was examined in cardiac atrial myocytes, using hydrolysis-resistant GTP analogues. In the absence of muscarinic agonist, GTP analogues produced a membrane current characteristic of IK(M). The initial rate of appearance of this receptor-independent IK(M) was measured for the various analogues in order to explore the kinetic properties of IK(M) activation. We found that IK(M) activation is controlled solely by the intracellular analogue/GTP ratio and not by the absolute concentrations of the nucleotides. Analogues competed with GTP for binding to Gk with the following relative affinities: GTP gamma S greater than GTP greater than GppNHp greater than GppCH2p. At sufficiently high intracellular concentrations, however, all GTP analogues produced the same rate of IK(M) activation. This analogue- independent limiting rate is likely to correspond to the rate of GDP release from inactive, GDP-bound Gk. Muscarinic receptor stimulation by nanomolar concentrations of acetylcholine (ACh), which do not elicit IK(M) under control conditions, catalyzed IK(M) activation in the presence of GTP analogues. The rate of Gk activation by ACh (kACh) was found to be described by the simple relationship kACh = 8.4 X 10(8) min- 1 M-1.[ACh] + 0.44 min-1, the first term of which presumably reflects the agonist-catalyzed rate of GDP release from the Gk.GDP complex, while the second term corresponds to the basal rate of receptor- independent GDP release. Combined with the estimated K0.5 of the IK(M)- [ACh] dose-effect relationship, 160 nM, this result also allowed us to estimate the rate of Gk.GTP hydrolysis, kcat, to be near 135 min-1. These results provide, for the first time, a quantitative description of the salient features of G-protein function in vivo.