Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of vertebrate pentameric ligand-gated ion channels (pLGICs) and has proven to be a valuable model for understanding the structure and function of this important protein family. There is nevertheless still a question about whether molecular details can be accurately extrapolated from this protein to those found in eukaryotes. Here we explore the role of proline residues (Pros) in ELIC by creating mutant receptors, expressing them in Xenopus laevis oocytes, and using whole-cell voltage-clamp electrophysiology to monitor channel activity. In contrast to eukaryotic pLGICs, proline-to-alanine (Pro-to-Ala) substitution in ELIC mostly resulted in gain of function, and even altering highly conserved Pro residues in M1 and the M2–M3 loop did not ablate function. These substitutions also mostly resulted in ablation of the modulation by Ca²⁺ observed in wild-type receptors. Substitution of the Pro in the “Cys loop”, however, did result in nonfunctional receptors. Probing this residue with noncanonical amino acids revealed a requirement for a substituted amine at this position, as well as a general preference for Pro analogues with greater intrinsic cis biases. We propose there is likely a cis bond at the apex of the Cys loop in this protein, which is consistent with some, but not all, findings from other pLGICs. Overall, the data show that the roles of proline residues are less critical in ELIC than in other pLGICs, supporting other studies that suggest caution must be applied in using data from this prokaryotic receptor to understand molecular details of eukaryotic pLGIC receptor function.

中文翻译：

---

探测原核配体门控离子通道中的脯氨酸残基，ELIC

欧文氏菌配体门控离子通道 (ELIC) 是脊椎动物五聚体配体门控离子通道 (pLGIC) 的细菌同源物，已被证明是了解这一重要蛋白质家族结构和功能的有价值模型。然而，关于分子细节是否可以从这种蛋白质准确地推断到真核生物中发现的那些，仍然存在一个问题。在这里，我们通过创建突变受体来探索脯氨酸残基 (Pros) 在 ELIC 中的作用，在非洲爪蟾中表达它们卵母细胞，并使用全细胞电压钳电生理学监测通道活动。与真核 pLGIC 相比，ELIC 中的脯氨酸到丙氨酸（Pro-to-Ala）取代主要导致功能获得，甚至改变 M1 和 M2-M3 环中高度保守的 Pro 残基也不会消除功能。这些取代还主要导致在野生型受体中观察到的Ca ^{2+调节的消融。}然而，在“Cys 环”中取代 Pro 确实导致受体无功能。用非规范氨基酸探测这个残基揭示了在这个位置需要一个取代的胺，以及对具有更大内在顺式偏差的 Pro 类似物的普遍偏好。我们建议可能有一个顺式在该蛋白质中 Cys 环的顶端结合，这与其他 pLGIC 的一些但不是全部发现一致。总体而言，数据表明，脯氨酸残基在 ELIC 中的作用不如在其他 pLGIC 中重要，这支持其他研究表明，在使用来自该原核受体的数据来了解真核 pLGIC 受体功能的分子细节时必须谨慎行事。