Mechanism of tetracaine block of cyclic nucleotide-gated channels.
Mechanism of tetracaine block of cyclic nucleotide-gated channels.
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DOI：
10.1085/jgp.109.1.3
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发表时间：
1997-01
期刊：
影响因子：
--
通讯作者：
Zagotta WN
中科院分区：
文献类型：
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作者：
Fodor AA;Gordon SE;Zagotta WN
局部麻醉剂是一组不同的离子通道阻断剂，可用于探测孔中的构象变化。我们研究了局部麻醉剂丁卡因对非洲爪蟾卵母细胞1亚基表达的视杆细胞和嗅环核苷酸门控通道的影响。我们发现，在cGMP饱和浓度下，40 μM丁卡因能有效地阻断牛视杆通道，但不能阻断大鼠嗅通道。通过测试嵌合通道包含区域的序列从杆和嗅觉通道，我们发现，在饱和cGMP丁卡因的表观亲和力的决定因素没有映射到任何一个区域的通道序列。相反，表观亲和力的差异可以通过嵌合体之间的开放变构转变的自由能的差异来解释。如果通道结构（如杆状通道）在cGMP饱和时处于闭合状态的时间可观，则其对丁卡因具有高的表观亲和力。另一方面，如果通道结构（如嗅觉通道）在cGMP饱和时处于关闭状态的时间很少，则它对丁卡因的表观亲和力较低。此外，丁卡因在低浓度的cGMP和饱和浓度的cAMP下变得更有效，这些条件允许通道在闭合构型中花费更多时间。这些结果与丁卡因与封闭通道的结合比与开放通道的结合更紧密的模型很好地拟合。在存在饱和cGMP的情况下丁卡因的剂量-反应曲线与Michaelis-Menten结合图很好地拟合，表明单个丁卡因分子足以产生阻滞。此外，丁卡因阻滞具有电压依赖性，有效zδ为+0.56。这些数据与孔块假说一致。丁卡因是一种状态依赖性的孔道阻断剂，这一发现表明环核苷酸门控通道的孔道内口在通道开放过程中发生了构象变化。
Local anesthetics are a diverse group of ion channel blockers that can be used to probe conformational changes in the pore. We examined the effects of the local anesthetic tetracaine on rod and olfactory cyclic nucleotide-gated channels expressed from subunit 1 in Xenopus oocytes. We found that 40 μM tetracaine effectively blocked the bovine rod channel but not the rat olfactory channel at saturating concentrations of cGMP. By testing chimeric channels containing regions of sequence from both rod and olfactory channels, we found that determinants of apparent affinity for tetracaine at saturating cGMP did not map to any one region of the channel sequence. Rather, the differences in apparent affinity could be explained by differences between the chimeras in the free energy of the opening allosteric transition. If a channel construct (such as the rod channel) spent appreciable time in the closed state at saturating cGMP, then it had a high apparent affinity for tetracaine. If, on the other hand, a channel construct (such as the olfactory channel) spent little time in the closed state at saturating cGMP, then it had a low apparent affinity for tetracaine. Furthermore, tetracaine became more effective at low concentrations of cGMP and at saturating concentrations of cAMP, conditions which permit the channels to spend more time in the closed configuration. These results were well fit by a model in which tetracaine binds more tightly to the closed channel than to the open channel. Dose-response curves for tetracaine in the presence of saturating cGMP are well fit with a Michaelis-Menten binding scheme Indicating that a single tetracaine molecule is sufficient to produce block. In addition, tetracaine block is voltage dependent with an effective zδ of +0.56. These data are consistent with a pore-block hypothesis. The finding that tetracaine is a state-dependent pore blocker suggests that the inner mouth of the pore of cyclic nucleotide-gated channels undergoes a conformational change during channel opening.