Interaction of hydrolysis-resistant analogs of cyclic GMP with the phosphodiesterase and light-sensitive channel of retinal rod outer segments.
Interaction of hydrolysis-resistant analogs of cyclic GMP with the phosphodiesterase and light-sensitive channel of retinal rod outer segments.
复制标题
环 GMP 的抗水解类似物与磷酸二酯酶和视网膜杆外节的光敏通道的相互作用。
DOI：
10.1073/pnas.82.24.8813
复制
发表时间：
1985
影响因子：
11.1
通讯作者：
Stryer,L
中科院分区：
文献类型：
--
作者：
Zimmerman,AL;Yamanaka,G;Eckstein,F;Baylor,DA;Stryer,L
当应用于视网膜杆外段（ROS）切除膜片的细胞质侧时，cGMP打开阳离子选择通道。如果完整棒中的光敏通道仅由cGMP控制，则应该有可能找到cGMP的抗水解类似物，通过保持通道开放而独立于光照程度来阻止对光的正常反应。我们研究了8-溴-cGMP （8-Br-cGMP）和cGMP的SP和RP磷酸化衍生物[(SP)-cGMP[S]和(RP)-cGMP[S]]与ROS的cGMP磷酸二酯酶（PDEase）、切除ROS斑块的cGMP敏感通道和完整杆的光敏通道的相互作用。这三种类似物被PDEase水解的速度比cGMP慢得多。8-Br-cGMP、(SP)-cGMP[S]和(RP)-cGMP[S]的最大水解速率分别为7.3、3.7和小于0.2 S -1，而cGMP的最大水解速率为4000 S -1。这些类似物是PDEase的有效竞争性抑制剂，Ki值分别为48、25和90微米。在浓度分别为1.6、210和1200微米时，与浓度为17微米的cGMP相比，切除斑块的核苷酸激活电导是最大的一半。因此，8-Br-cGMP是一种高效的通道激动剂。这些类似物对完整杆细胞的暗电流和光响应的影响也被测量。吸入电极监测ROS上的膜电流，而密封在内段的贴片电极用于引入cGMP模拟物并控制膜电位。这三种类似物都增加了暗电流，并显著减缓了对闪光的反应。8-Br-cGMP可使外段暗电流增加48倍。当这种类似物的浓度上升到一定程度后，光就会切断很少的电流，这是对质膜的光敏通道的直接影响。这些结果与以下观点一致：(i)棒体的光敏通道完全由cGMP的瞬时浓度控制，（ii）切除斑块的cGMP敏感通道与完整棒体的光敏通道相同。
cGMP opens cation-selective channels when applied to the cytoplasmic side of excised patches of membrane from retinal rod outer segments (ROS). If the light-sensitive channel in intact rods is gated only by cGMP, it should be possible to find a hydrolysis-resistant analog of cGMP that blocks the normal response to light by holding the channel open independent of the degree of illumination. We have studied the interaction of 8-bromo-cGMP (8-Br-cGMP) and the SP and RP phosphorothioate derivatives of cGMP [(Sp)-cGMP[S] and (RP)-cGMP[S]) with the cGMP phosphodiesterase (PDEase) of ROS, the cGMP-sensitive channel of excised ROS patches, and the light-sensitive channel of intact rods. All three analogs were hydrolyzed by PDEase much more slowly than was cGMP. The maximal rates of hydrolysis of 8-Br-cGMP, (SP)-cGMP[S], and (RP)-cGMP[S] were 7.3, 3.7, and less than 0.2 s-1, respectively, compared with 4000 s-1 for cGMP. These analogs are effective competitive inhibitors of the PDEase, with Ki values of 48, 25, and 90 microM, respectively. The nucleotide-activated conductances of excised patches were half-maximal at concentrations of 1.6, 210, and 1200 microM, respectively, compared with 17 microM for cGMP. Thus, 8-Br-cGMP is a highly potent channel agonist. The effects of these analogs on the dark current and photoresponses of intact rod cells were also measured. A suction electrode monitored membrane current across the ROS, while a patch electrode sealed on the inner segment was used to introduce a cGMP analog and to control membrane potential. All three analogs increased the dark current and markedly slowed the response to light flashes. 8-Br-cGMP increased the dark current of the outer segment as much as 48-fold. After the concentration of this analog had risen sufficiently, little of the current could be shut off by light, as expected of a direct effect on the light-sensitive channel of the plasma membrane. These results are consistent with the notions that (i) the light-sensitive channel of rods is controlled solely by the instantaneous concentration of cGMP and (ii) the cGMP-sensitive channel of excised patches is identical to the light-sensitive channel of intact rods.