Hughes David T, Pelletier Julien, Rahman Suhaila, Chen Sisi, Leal Walter S, Luetje Charles W
Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, R-189, PO Box 016189, Miami, FL 33101, USA and.
Present address: Department of Internal Medicine, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA.
Chem Senses. 2017 May 1;42(4):333-341. doi: 10.1093/chemse/bjx011.
In Culex quinquefasciatus, CquiOR91 is the ortholog of 2 larvae-specific odorant receptors (ORs) from Anopheles gambiae (Agam\Or40, previously shown to respond to several odorant ligands including the broad-spectrum repellent N,N-diethyl-3-methylbenzamide, DEET) and Aedes aegypti (Aaeg\Or40). When we cloned full-length CquiOR91 from a Culex quinquefasciatus larval head RNA sample, we found 2 alleles of this OR, differing at 9 residues. Functional analysis using the Xenopus oocyte expression system and 2-electrode voltage clamp electrophysiology revealed one allele (CquiOR91.1) to be nonfunctional, whereas the other allele (CquiOR91.2) was functional. Receptors formed by CquiOR91.2 and Cqui\Orco responded to (-)-fenchone, (+)-fenchone, and DEET, similar to what has been reported for Agam\Or40. We also identified 5 novel odorant ligands for the CquiOR91.2 + Cqui\Orco receptor: 2-isobutylthiazole, veratrole, eucalyptol, d-camphor, and safranal, with safranal being the most potent. To explore possible reasons for the lack of function for CquiOR91.1, we generated a series of mutant CquiOR91.2 subunits, in which the residue at each of the 9 polymorphic residue positions was changed from what occurs in CquiOR91.2 to what occurs in CquiOR91.1. Eight of the 9 mutant versions of CquiOR91.2 formed functional receptors, responding to (-)-fenchone. Only the CquiOR91.2 Y183C mutant was nonfunctional. The reverse mutation (C183Y) conferred function on CquiOR91.1 , which became responsive to (-)-fenchone and safranal. These results indicate that the "defect" in CquiOR91.1 that prevents function is the cysteine at position 183.
在致倦库蚊中，CquiOR91是冈比亚按蚊（Agam\Or40，先前已证明其对包括广谱驱避剂N，N - 二乙基 - 3 - 甲基苯甲酰胺，即避蚊胺在内的多种气味配体有反应）和埃及伊蚊（Aaeg\Or40）的2种幼虫特异性气味受体（ORs）的直系同源物。当我们从致倦库蚊幼虫头部RNA样本中克隆出全长CquiOR91时，我们发现该OR有2个等位基因，在9个残基处存在差异。使用非洲爪蟾卵母细胞表达系统和双电极电压钳电生理学进行的功能分析表明，其中一个等位基因（CquiOR91.1）无功能，而另一个等位基因（CquiOR91.2）有功能。由CquiOR91.2和Cqui\Orco形成的受体对（-）-葑酮、（+）-葑酮和避蚊胺有反应，这与针对Agam\Or40的报道相似。我们还为CquiOR91.2 + Cqui\Orco受体鉴定了5种新的气味配体：2 - 异丁基噻唑、藜芦醚、桉叶油素、d - 樟脑和藏红花醛，其中藏红花醛的活性最强。为了探究CquiOR91.1缺乏功能的可能原因，我们构建了一系列突变的CquiOR91.2亚基，其中9个多态性残基位置上的每个残基都从CquiOR91.2中的状态改变为CquiOR91.1中的状态。CquiOR91.2的9个突变版本中有8个形成了功能性受体，对（-）-葑酮有反应。只有CquiOR91.2 Y183C突变体无功能。反向突变（C183Y）赋予了CquiOR91.1功能，使其对（-）-葑酮和藏红花醛有反应。这些结果表明，CquiOR91.1中阻止其功能的“缺陷”是183位的半胱氨酸。
