Ethanol Stabilizes the Open State of Single 5-Hydroxytryptamine3A(QDA) Receptors
Ethanol Stabilizes the Open State of Single 5-Hydroxytryptamine3A(QDA) Receptors
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DOI：
10.1124/jpet.109.164863
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发表时间：
2010-06-01
影响因子：
3.5
通讯作者：
Davies, Paul A.
中科院分区：
文献类型：
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作者：
Feinberg-Zadek, Paula L.;Davies, Paul A.
乙醇增强 5-羟色胺 (5-HT)(3A) 受体介导的反应可能对乙醇的中毒和成瘾特性产生重要影响。尽管确切的机制尚不清楚，但乙醇介导的 5-HT3 受体电流增强已被认为是由于开放通道状态的稳定而发生的。由于5-HT3A通道的单通道电导极低，无法直接测量通道的开放状态。最近，5-HT3A 亚基的大细胞内环内的三个精氨酸残基被 5-HT3B 亚基的等效残基（谷氨酰胺、天冬氨酸和丙氨酸）取代，产生 5-HT3A(QDA) 亚基，该亚基形成具有可测量的单通道电导的功能同聚通道。使用全细胞快速激动剂应用技术和细胞附着单通道记录配置，我们检查了人胚胎肾 293 细胞中表达的人 5-HT3A（QDA）受体。突变型和野生型通道的激动剂敏感性、宏观动力学和乙醇调节相似，表明取代没有改变这些通道的结构功能特性。在存在最大 5-HT 的情况下，由 5-HT3A(QDA) 受体介导的单通道事件的开放时间直方图最适合三个指数，但在存在乙醇的情况下，第四个开放状态是明显的。总之，QDA 取代极大地增强了单通道电导，对 5-HT3A 通道的动力学特性几乎没有影响，并且乙醇通过诱导新的、长寿命的开放通道状态来增强对 5-HT3A 受体的激动作用。此外，5-HT3A(QDA)受体似乎适合在单通道水平上进行5-HT3A受体调节的药理学研究。
Ethanol enhancement of 5-hydroxytryptamine (5-HT)(3A) receptor-mediated responses may have important consequences in the intoxicating and addictive properties of ethanol. Although the exact mechanism is unknown, ethanol-mediated enhancement of 5-HT3 receptor current has been proposed to occur due to stabilization of the open-channel state. It has not been possible to directly measure the open state of the channel due to the extremely low single-channel conductance of 5-HT3A channels. Recently, three arginine residues within the large intracellular loop of the 5-HT3A subunit were substituted by their equivalent residues (glutamine, aspartate, and alanine) of the 5-HT3B subunit to produce a 5-HT3A(QDA) subunit that forms functional homomeric channels exhibiting a measurable single-channel conductance. Using whole-cell rapid-agonist application techniques and the cell-attached single-channel recording configuration, we examined human 5-HT3A(QDA) receptors expressed in human embryonic kidney 293 cells. The agonist sensitivity, macroscopic kinetics, and modulation by ethanol were similar between mutant and wild-type channels, suggesting the substitutions had not altered these channel structure-function properties. The open time histogram for single-channel events mediated by 5-HT3A(QDA) receptors in the presence of maximal 5-HT was best fit by three exponentials, but in the presence of ethanol a fourth open state was evident. In summary, the QDA substitution greatly enhanced single-channel conductance with little effect on 5-HT3A channel's kinetic properties and ethanol enhances agonist action on 5-HT3A receptors by inducing a new, long-lived open-channel state. Furthermore, the 5-HT3A(QDA) receptor appears to be suitable for pharmacological studies of 5-HT3A receptor modulation at a single-channel level.