Antagonistic postsynaptic and presynaptic actions of cyclohexanol on neuromuscular synaptic transmission and function
Antagonistic postsynaptic and presynaptic actions of cyclohexanol on neuromuscular synaptic transmission and function
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DOI：
10.1113/jp281921
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发表时间：
2021-11-26
影响因子：
5.5
通讯作者：
Ribchester，Richard R.
中科院分区：
文献类型：
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作者：
Dissanayake，Kosala N.;Margetiny，Filip;Ribchester，Richard R.
农业有机磷杀虫剂的故意摄入是亚洲农村地区一个重要的公共卫生问题，每年造成数千人死亡。一些幸存者发展为严重的、急性的或迟发性肌无力综合征。在动物模型中，类似的肌无力与一种杀虫剂溶剂代谢物环己醇的血浆浓度增加有关。我们使用小鼠神经肌肉接头（NMJ）和转染的人细胞系的电压和电流记录研究了可能的机制。环己醇（10-25 mM）使终板电位（EPP）振幅降低10-40%，并使重复（2-20 Hz）刺激期间的抑制增强高达60%。EPP衰减延长两倍以上。微型EPP衰减超过50%。环己醇抑制表达人连接后乙酰胆碱受体（hnAChR）的CN 21细胞记录的全细胞电流，IC 50为3.74 mM。环己醇（10-20 mM）还导致转染HEK 293 T细胞中表达的hnAChR的外向斑片记录中的电流降低、多通道爆发的长时间发作，使电荷转移减少50%以上。分子模拟表明，环己醇（−6 kcal/mol）与烟碱AChR α亚基上先前确定的醇结合位点结合。环己醇也使诱发的递质释放的量子含量增加约50%。在神经束膜记录中，环己醇选择性抑制突触前K+电流。建模表明环己醇（−3.8 kcal/mol）在与四乙基铵（TEA）相同的位点与电压敏感性K+通道结合。TEA（10 mM）比环己醇更有效地阻断K+通道，但EPP在20 mM环己醇中更延长。结果解释了摄入含有环己醇前体的有机磷杀虫剂后神经肌肉功能障碍的模式，并表明环己醇可能有助于调查NMJs.Key pointsIntentional摄入农业有机磷杀虫剂是一个重要的公共卫生问题，在亚洲农村，每年造成数千人死亡的机制。幸存者可能发展为严重的肌无力综合征或瘫痪，与血浆中环己醇（一种杀虫剂溶剂代谢物）水平升高有关。用等长张力记录和终板电压和电流的微电极记录分析离体小鼠骨骼肌神经肌肉接头处的突触传递，表明环己醇降低了突触后对乙酰胆碱神经递质的敏感性（减少量子大小），同时增强诱发递质释放来自转染细胞系的斑片记录，连同分子建模，表明环己醇引起选择性，突触后烟碱乙酰胆碱受体的别构拮抗作用和突触前K+的阻滞这些数据提供了对有意摄入以下神经肌肉无力的细胞和分子机制的深入了解：农用有机磷杀虫剂我们的发现也扩展了对酒精对突触传递和稳态突触功能的影响的理解。
AbstractIntentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Some survivors develop a severe, acute or delayed myasthenic syndrome. In animal models, similar myasthenia has been associated with increasing plasma concentration of one insecticide solvent metabolite, cyclohexanol. We investigated possible mechanisms using voltage and current recordings from mouse neuromuscular junctions (NMJs) and transfected human cell lines. Cyclohexanol (10–25 mM) reduced endplate potential (EPP) amplitudes by 10–40% and enhanced depression during repetitive (2–20 Hz) stimulation by up to 60%. EPP decay was prolonged more than twofold. Miniature EPPs were attenuated by more than 50%. Cyclohexanol inhibited whole‐cell currents recorded from CN21 cells expressing human postjunctional acetylcholine receptors (hnAChR) with an IC50of 3.74 mM. Cyclohexanol (10–20 mM) also caused prolonged episodes of reduced‐current, multi‐channel bursting in outside‐out patch recordings from hnAChRs expressed in transfected HEK293T cells, reducing charge transfer by more than 50%. Molecular modelling indicated cyclohexanol binding (−6 kcal/mol) to a previously identified alcohol binding site on nicotinic AChR α‐subunits. Cyclohexanol also increased quantal content of evoked transmitter release by ∼50%. In perineurial recordings, cyclohexanol selectively inhibited presynaptic K+currents. Modelling indicated cyclohexanol binding (−3.8 kcal/mol) to voltage‐sensitive K+channels at the same site as tetraethylammonium (TEA). TEA (10 mM) blocked K+channels more effectively than cyclohexanol but EPPs were more prolonged in 20 mM cyclohexanol. The results explain the pattern of neuromuscular dysfunction following ingestion of organophosphorus insecticides containing cyclohexanol precursors and suggest that cyclohexanol may facilitate investigation of mechanisms regulating synaptic strength at NMJs.Key pointsIntentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Survivors may develop a severe myasthenic syndrome or paralysis, associated with increased plasma levels of cyclohexanol, an insecticide solvent metabolite.Analysis of synaptic transmission at neuromuscular junctions in isolated mouse skeletal muscle, using isometric tension recording and microelectrode recording of endplate voltages and currents, showed that cyclohexanol reduced postsynaptic sensitivity to acetylcholine neurotransmitter (reduced quantal size) while simultaneously enhancing evoked transmitter release (increased quantal content).Patch recording from transfected cell lines, together with molecular modelling, indicated that cyclohexanol causes selective, allosteric antagonism of postsynaptic nicotinic acetylcholine receptors and block of presynaptic K+‐channel function.The data provide insight into the cellular and molecular mechanisms of neuromuscular weakness following intentional ingestion of agricultural organophosphorus insecticides. Our findings also extend understanding of the effects of alcohols on synaptic transmission and homeostatic synaptic function.