ALCOHOL MODULATORY SITES IN THE 5HT3A RECEPTOR

乙醇和高级n-链醇刺激同质5-羟色胺3A受体(5-HT3A)的功能。对该受体的研究以及其他实验室对相关配体门控离子通道的研究表明，这些受体的第二跨膜结构域(TM2)在酒精作用中发挥着关键作用。然而，目前尚不清楚TMZ中已识别的残基是否是醇的关键结合域的一部分，或者它们是否在n-链醇作用的转导中发挥关键作用。第三种可能性是，TM2的突变可能会通过动力学机制掩盖酒精的影响。这项建议的主要目的是描述这三种机制中的哪一种负责改变TM2区域突变的5-HT3A受体的酒精敏感性。野生型和突变型5-HT3A受体的酒精敏感性将用双电极电压钳技术在卵母细胞中和在HEK293细胞中用膜片钳技术和标准灌流和超快速灌流药物应用方法进行评估。通道孔内的残基将通过丙氨酸扫描诱变和半胱氨酸替代方法进行鉴定并从研究中消除。二级结构的变化将通过色氨酸扫描突变和离散傅立叶变换分析进行评估。面对其他跨膜结构域的残基将被突变为不同物理化学性质的氨基酸，并将检查酒精作用的差异。假阳性受体的正常门控特性被突变改变，如果它们改变了对作用于受体内不同位点的5-HT3A受体药物的反应性，则将被识别和消除。通过酒精敏感性的变化与取代氨基酸的物理化学性质的变化、通道动力学的变化以及酒精切断的改变，可以初步将候选的酒精结合区域从酒精转导位点分离出来。野生型/突变滴定实验将被用来确定每个受体获得突变酒精表型所需的最小突变亚基数量；显性突变表型表明已经确定了酒精转导位点。

Ethanol and higher n-chain alcohols stimulate homomeric 5- Hyrdroxytryptamine3A receptor (5-HT3A) function. Work on this receptor, as well as studies in other laboratories on related ligand-gated ion channels, suggests that the second transmembrane domains (TM2) of these receptors play a critical role in alcohol action. However, it remains unknown whether identified residues in TMZ are part of a critical binding domain for alcohols or whether they play a key role in the transduction of n-chain alcohol action. A third possibility is that a mutation in TM2 could obscure the effects of alcohols through a kinetic mechanism. The major goal of this proposal is to delineate which of these three mechanisms is responsible for altering the alcohol sensitivity of 5-HT3A receptors mutated in the TM2 domain. The alcohol sensitivity of wild-type and mutant 5-HT3A receptors will be assessed in oocytes with the two-electrode voltage clamp technique and in HEK293 cells with patch-clamp technology and standard perfusion and ultra-fast superfusion drug application methods. Residues lining the channel pore will be identified and eliminated from study through alanine scanning mutagenesis and cysteine substitution methods. Changes in secondary structure will be assessed with tryptophan scanning mutagenesis and discrete Fourier transformation analysis. Residues facing other transmembrane domains will be mutated to amino acids differing in physicochemical properties and will be examined for differences in alcohol action. False positive receptors, whose normal gating properties are changed by mutagenesis, will be identified and eliminated if they have altered responsiveness to battery of 5-HT3A receptor drugs acting at different loci within the receptor. Candidate alcohol binding domains can be preliminarily separated from alcohol transduction sites through correlation of changes in alcohol sensitivity with physicochemical properties of substituted amino acids, changes in channel kinetics, and alteration in alcohol cut-off. Wild-type/mutant titration experiments will be used to determine the minimum number of mutant subunits required per receptor to achieve the mutant alcohol phenotype; a dominant mutant phenotype would suggest that an alcohol transduction site has been identified.