Tae Hee Han, Rosario Vicidomini, Cathy Isaura Ramos, Mark L. Mayer, Mihaela Serpe
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Here, using outside-out, patch clamp recordings and fast ligand application, we examine for the first time the biophysical properties of native type-A and type-B NMJ receptors in complexes with either Neto-α or Neto-β and compare them with recombinant receptors expressed in HEK293T cells. We found that type-A and type-B receptors have strikingly different gating properties that are further modulated by Neto-α and Neto-β. We captured single-channel events and revealed major differences between type-A and type-B receptors and also between Neto splice variants. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of ionotropic glutamate receptor (iGluR) desensitization. The functional analyses of recombinant iGluRs that we report here should greatly facilitate the interpretation of compound <i>in vivo</i> phenotypes of mutant animals.\n\n <figure>\n <div><picture>\n <source></source></picture><p></p>\n </div>\n </figure>\n </div>\n </section>\n \n <section>\n \n <h3> Key points</h3>\n \n <div>\n <ul>\n \n <li>We report the reconstitution of <i>Drosophila</i> neuromuscular junction ionotropic glutamate receptors (iGluRs) with Neto splice forms.</li>\n \n <li>Using outside-out patches and fast ligand application, we examine the deactivation and desensitization of the four iGluR/Neto complexes found <i>in vivo</i>.</li>\n \n <li>Expression of functional channels is absolutely dependent on Neto.</li>\n \n <li>Single-channel recordings revealed different lifetimes for different receptor complexes.</li>\n \n <li>The decay of synaptic currents is controlled by desensitization.</li>\n </ul>\n </div>\n </section>\n </div>","PeriodicalId":50088,"journal":{"name":"Journal of Physiology-London","volume":"602 24","pages":"7043-7064"},"PeriodicalIF":4.7000,"publicationDate":"2024-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1113/JP287331","citationCount":"0","resultStr":"{\"title\":\"The gating properties of Drosophila NMJ glutamate receptors and their dependence on Neto\",\"authors\":\"Tae Hee Han, Rosario Vicidomini, Cathy Isaura Ramos, Mark L. 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引用次数: 0
摘要
果蝇神经肌肉接头（NMJ）是一个强大的遗传系统，它揭示了突触发育和平衡的许多保守机制。果蝇的 NMJ 使用谷氨酸作为兴奋性神经递质，并依赖于 kainate 型谷氨酸受体及其辅助蛋白 Neto 来实现突触的组装和功能。然而，尽管经过数十年的研究，利用异源系统重组 NMJ 谷氨酸受体的工作直到最近才得以实现，而且目前还没有关于重组受体门控特性的报道。在这里，我们利用外向型膜片钳记录和快速配体应用，首次研究了原生 A 型和 B 型 NMJ 受体与 Neto-α 或 Neto-β 复合物的生物物理特性，并将它们与 HEK293T 细胞中表达的重组受体进行了比较。我们发现，A 型和 B 型受体具有显著不同的门控特性，这些特性受到 Neto-α 和 Neto-β 的进一步调节。我们捕获了单通道事件，发现了 A 型和 B 型受体之间以及 Neto 拼接变体之间的重大差异。令人惊讶的是，我们发现失活速度极快，突触电流的衰减与离子型谷氨酸受体（iGluR）脱敏的速度相似。我们在此报告的重组 iGluR 的功能分析将大大有助于解释突变动物的体内复合表型。要点：我们报告了果蝇神经肌肉接头处离子型谷氨酸受体（iGluRs）与 Neto 拼接形式的重组。通过使用外向型贴片和快速配体应用，我们研究了在体内发现的四种 iGluR/Neto 复合物的失活和脱敏。功能通道的表达完全依赖于 Neto。单通道记录显示，不同的受体复合物有不同的寿命。突触电流的衰减受脱敏控制。
The gating properties of Drosophila NMJ glutamate receptors and their dependence on Neto
The Drosophila neuromuscular junction (NMJ) is a powerful genetic system that has revealed numerous conserved mechanisms for synapse development and homeostasis. The fly NMJ uses glutamate as the excitatory neurotransmitter and relies on kainate-type glutamate receptors and their auxiliary protein Neto for synapse assembly and function. However, despite decades of study, the reconstitution of NMJ glutamate receptors using heterologous systems has been achieved only recently, and there are no reports on the gating properties for the recombinant receptors. Here, using outside-out, patch clamp recordings and fast ligand application, we examine for the first time the biophysical properties of native type-A and type-B NMJ receptors in complexes with either Neto-α or Neto-β and compare them with recombinant receptors expressed in HEK293T cells. We found that type-A and type-B receptors have strikingly different gating properties that are further modulated by Neto-α and Neto-β. We captured single-channel events and revealed major differences between type-A and type-B receptors and also between Neto splice variants. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of ionotropic glutamate receptor (iGluR) desensitization. The functional analyses of recombinant iGluRs that we report here should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals.
Key points
We report the reconstitution of Drosophila neuromuscular junction ionotropic glutamate receptors (iGluRs) with Neto splice forms.
Using outside-out patches and fast ligand application, we examine the deactivation and desensitization of the four iGluR/Neto complexes found in vivo.
Expression of functional channels is absolutely dependent on Neto.
Single-channel recordings revealed different lifetimes for different receptor complexes.
The decay of synaptic currents is controlled by desensitization.
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