ABSTRACT

Nicotinic acetylcholine receptors (nAChRs) are members of the “cys-loop” ligand-gated ion channel superfamily that play important roles in both the peripheral and central system. At the neuromuscular junction, the endplate current is induced by ACh binding and nAChR activation, and then, the current declines to a small steady state, even though ACh is still bound to the receptors. The kinetics of nAChRs with high affinity for ACh but no measurable ion conductance is called desensitization. This adopted desensitization of nAChR channel currents might be an important mechanism for protecting cells against uncontrolled excitation. This study aimed to show that Grammostola spatulata toxin (GsMTx4), which was first purified and characterized from the venom of the tarantula Grammostola spatulata (now genus Phixotricus), can facilitate the desensitization of nAChRs in murine C2C12 myotubes. To examine the details, muscle-type nAChRs, which are expressed heterologously in HEK293T cells, were studied. A single channel current was recorded under the cell-attached configuration, and the channel activity (NP_o) decayed much faster after the addition of GsMTx-4 to the pipette solution. The channel kinetics were further analyzed, and GsMTx-4 affected the channel activity of nAChRs by prolonging the closing time without affecting channel conductance or opening activity. The interaction between nAChRs embedded in the lipid membrane and toxin inserted into the membrane may contribute to the conformational change in the receptor and thus change the channel activity. This new property of GsMTx-4 may lead to a better understanding of the desensitization of ligand-gated channels and disease therapy.

摘要

烟碱型乙酰胆碱受体 (nAChRs) 是“cys-loop”配体门控离子通道超家族的成员，在外周系统和中枢系统中都发挥着重要作用。在神经肌肉接头处，终板电流由 ACh 结合和 nAChR 激活诱导，然后，电流下降到一个小的稳态，即使 ACh 仍然与受体结合。对乙酰胆碱具有高亲和力但没有可测量的离子电导的 nAChR 的动力学称为脱敏。这种采用的 nAChR 通道电流脱敏可能是保护细胞免受不受控制的激发的重要机制。本研究旨在表明，首先从狼蛛的毒液中纯化并表征了匙状革兰氏菌毒素 ( GsMTx4 )（现为 Phixotricus 属）可促进小鼠 C2C12 肌管中 nAChR 的脱敏。为了检查细节，研究了在 HEK293T 细胞中异源表达的肌肉型 nAChR。在细胞附着配置下记录单通道电流，通道活动（NP _o) 在移液管溶液中加入 GsMTx-4 后衰减得更快。进一步分析了通道动力学，GsMTx-4通过延长关闭时间而不影响通道电导或打开活性来影响nAChRs的通道活性。嵌入脂质膜中的 nAChR 与插入膜中的毒素之间的相互作用可能有助于受体的构象变化，从而改变通道活性。GsMTx-4 的这一新特性可能有助于更好地理解配体门控通道的脱敏和疾病治疗。