A unique amino acid of the Drosophila GABA receptor with influence on drug sensitivity by two mechanisms.
A unique amino acid of the Drosophila GABA receptor with influence on drug sensitivity by two mechanisms.
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DOI：
10.1113/jphysiol.1994.sp020278
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发表时间：
1994-08
期刊：
影响因子：
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通讯作者：
H. G. Zhang;R. ffrench-Constant;M. Jackson
中科院分区：
文献类型：
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作者：
H. G. Zhang;R. ffrench-Constant;M. Jackson
1.果蝇基因Rdl（狄氏剂抗性）编码GABA受体。在该基因中，在残基302处的丙氨酸至丝氨酸突变赋予对环二烯杀虫剂和印防己毒素的抗性。采用膜片钳技术对野生型和突变型果蝇神经元GABA受体进行了分析，以探讨抗性的生物物理基础。2.在野生型和突变株的培养神经元中，GABA激活了一个在对称氯化物中接近0 mV的通道。野生型和突变型受体的GABA剂量反应特征非常相似。3.来自突变株的神经元中的GABA反应显示出对GABA拮抗剂印防己毒素、林替卡松和叔丁基双环硫代磷酸酯的敏感性降低。三种阻滞剂的耐药率分别为116、970和9。抑制增加阻滞剂浓度的方式与饱和的一个单一的结合位点。4.该突变使内向电流的单通道电导降低了5%，外向电流降低了17%。在野生型和突变体中，外向电流的单通道电流比内向电流低约60%。5.开放和关闭时间都很好地拟合了两个指数的总和。阻力与较长的开放时间和较短的关闭时间相关，反映了通道开放状态的净稳定性约为5倍。6.该突变与GABA诱导的脱敏率显著降低相关，脱敏构象的净不稳定性为29倍。7. Rdl突变通过两种不同的机制表现出抗性。(a)该突变通过药物结合位点的结构变化减弱药物与拮抗剂偏好（脱敏）构象的结合。(b)该突变在变构意义上使拮抗剂有利的构象不稳定。单一氨基酸置换与环二烯抗性的全局关联表明抗性表型取决于这两种性质的变化，并且杀虫剂选择Rdl的残基302进行置换，因为其具有影响这两种功能的独特能力。8.丙氨酸302在Rdl基因产物的序列中的位置支持了一种作用机制，其中惊厥剂如印防己毒素结合在通道腔内，在那里它们诱导快速构象变化至脱敏状态。
1. The Drosophila gene Rdl (resistance to dieldrin) encodes a GABA receptor. An alanine‐to‐serine mutation in this gene at residue 302 confers resistance to cyclodiene insecticides and picrotoxin. Patch clamp analysis of GABA receptors in cultured neurons from wild type and mutant Drosophila was undertaken to investigate the biophysical basis of resistance. 2. In cultured neurons from both wild type and mutant strains, GABA activated a channel that reversed near 0 mV in symmetrical chloride. GABA dose‐response characteristics of wild type and mutant receptors were very similar. 3. GABA responses in neurons from the mutant strains showed reduced sensitivity to the GABA antagonists picrotoxin, lindane and t‐butyl‐bicyclophosphorothionate. Resistance ratios were 116, 970 and 9 for the three blockers, respectively. Inhibition increased with blocker concentration in a manner consistent with saturation of a single binding site. 4. The mutation reduced the single channel conductance by 5% for inward current and 17% for outward current. The single channel current was approximately 60% lower for outward current than for inward current in both wild type and mutant. 5. Open and closed times were both well fitted by the sum of two exponentials. Resistance was associated with longer open times and shorter closed times, reflecting a net stabilization of the channel open state by a factor of approximately five. 6. The mutation was associated with a marked reduction in the rate of GABA‐induced desensitization, and a net destabilization of the desensitized conformation by a factor of 29. 7. The Rdl mutation manifests resistance through two different mechanisms. (a) The mutation weakens drug binding to the antagonist‐favoured (desensitized) conformation by a structural change at the drug binding site. (b) The mutation destabilizes the antagonist‐favoured conformation in an allosteric sense. The global association of a single amino acid replacement with cyclodiene resistance suggests that the resistance phenotype depends on changes in both of these properties, and that insecticides have selected residue 302 of Rdl for replacement because of its unique ability to influence both of these functions. 8. The location of alanine 302 in the sequence of the Rdl gene product supports a mechanism of action in which convulsants such as picrotoxin bind within the channel lumen, where they induce a rapid conformational change to the desensitized state.