ALCOHOL MODULATORY SITES IN THE 5HT3A RECEPTOR

乙醇和高n链醇刺激同质5-羟色胺3a受体（5- ht3a）的功能。对这种受体的研究，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）在酒精作用中起着关键作用。然而，尚不清楚TMZ中鉴定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链醇的转导中起关键作用。第三种可能性是，TM2的突变可能通过一种动力学机制掩盖了酒精的作用。本研究的主要目标是描述这三种机制中的哪一种是改变TM2结构域突变的5-HT3A受体的酒精敏感性的原因。采用双电极电压钳技术评估野生型和突变型5-HT3A受体在卵母细胞中的酒精敏感性，采用膜片钳技术以及标准灌注和超快速灌注给药方法评估HEK293细胞中的酒精敏感性。通过丙氨酸扫描诱变和半胱氨酸替代等方法对通道孔内的残基进行识别和清除。二级结构的变化将通过色氨酸扫描诱变和离散傅立叶变换分析来评估。面对其他跨膜结构域的残基将突变为物理化学性质不同的氨基酸，并将检查醇作用的差异。假阳性受体，其正常的门控特性被诱变改变，如果它们改变了对受体内不同位点的5-HT3A受体药物的反应性，将被识别和消除。候选醇结合域可以通过醇敏感性的变化与取代氨基酸的理化性质、通道动力学的变化和醇切断的改变的相关性，从醇转导位点上初步分离出来。野生型/突变体滴定实验将用于确定每个受体实现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.