昆虫感觉气味的细胞与分子机制研究进展

昆虫作为地球上最为成功的类群,已经成功地进化了精细的化学感受系统,通过化学感受系统适应各种复杂的环境,保持种群的繁荣。自1991年在动物中发现嗅觉受体基因以来,关于昆虫感受化学信息的周缘神经系统的分子和细胞机制方面的进展十分迅速。文章主要就昆虫周缘神经系统的感受化学信息的分子和细胞机制进行综述。首先对昆虫感觉气味的细胞机制的研究进展进行简要介绍。昆虫嗅觉神经元在感受化学信息过程中起着极为重要的作用,昆虫嗅觉神经元上表达的嗅觉受体不同而执行着各异的功能。各种嗅觉神经元对于化学信息的感受谱有较大的区别;嗅觉神经元对化学信息类型、浓度、流动动态等产生相应的电生理特征反应。研究表明同一种神经原可以感受多种化学信息,而一种化学信息也可以被多种神经原所感受。由神经原对化学信息感受所形成的特征组合就是感受化学信息的编码。其次较为详细地论述与昆虫感受气味分子相关的一些蛋白质的研究进展。气味分子结合蛋白是一类分子量较小、水溶性的蛋白,主要位于化学感受器神经原树突周围的淋巴液中。在结构上的主要特征是具有6个保守的半光氨酸和由6个α螺旋组成的结合腔。自1981年发现以来,已经在40余种昆虫中发现上百种。由于研究手段的不断进步,已经对该类蛋白的表达特征、结合特性以及三维结构和结合位点进行了大量的研究,提出了多个可能的功能假说,在诸多的假说中,较为广泛接受的是气味分子结合蛋白在昆虫感觉气味的过程中,是与疏水性的气味分子相结合,并将气味分子运输到嗅觉神经原树突膜上的嗅觉受体上。这些处于树突膜上的嗅觉受体则是昆虫感觉气味过程中的另一个十分重要的蛋白质。目前,已经在果蝇、按蚊、蜜蜂和家蚕等10余个昆虫种类中发现上百个嗅觉受体蛋白基因。这类蛋白是跨膜蛋白,一般具有7个跨膜区,整个蛋白的氨基酸残基在400～600个。昆虫的嗅觉受体蛋白的N-端在胞内,而C-端在胞外,这与G耦联蛋白不同。而且,昆虫的一个嗅觉神经元可以表达1～3个嗅觉受体蛋白,也与哺乳动物的一个神经元只表达一种受体蛋白有所不同。每种嗅觉受体可以感受多种气味分子,而一种气味分子可以被多个嗅觉受体所感知,这样组成了感受化学信息的编码谱。最近采用基因敲除技术和膜片钳技术研究发现,昆虫的嗅觉受体蛋白在信号传导中也有特殊性,即嗅觉受体可以直接作为离子通道,而引起动作电位。还有近来的研究表明,神经膜蛋白对于果蝇的性信息素感受神经元感受性信息素cVA是必要的。实际上,昆虫对于化学信息的感受和信号的转导,并不是上述蛋白单独起作用完成的,而是多种蛋白相互作用的结果。论文最后对该领域研究内容进行了展望。     

A look inside odorant-binding proteins in insect chemoreception

Detection of chemical signals from the environment through olfaction is an indispensable mechanism for maintaining an insect’s life, evoking critical behavioral responses. Among several proteins involved in the olfactory perception process, the odorant binding protein (OBP) has been shown to be essential for a normally functioning olfactory system. This paper discusses the role of OBPs in insect chemoreception. Here, structural aspects, mechanisms of action and binding affinity of such proteins are reviewed, as well as their promising application as molecular targets for the development of new strategies for insect population management and other technological purposes.     

昆虫嗅觉相关蛋白的结构和功能

昆虫在长期进化的过程中形成了复杂的嗅觉系统,气味剂结合蛋白(odorant binding proteins,OBPs)、嗅觉受体(olfactory receptors,ORs)是其最主要的组分.其主要作用是结合外围挥发性的气味分子并将信号传递给细胞内的第二信使.OBPs和ORs的结构、功能、表达、进化是昆虫行为与进化关系的重要研究领域和研究热点.本文主要总结了近年来昆虫OBPs和ORs的结构特点、生理功能、表达特点、遗传进化等方面研究的最新进展,对OBPs和ORs的研究趋势进行了展望,为昆虫嗅觉系统进化研究及寻找害虫防治新途径提供参考信息.     

昆虫气味结合蛋白的研究进展

摘要: 昆虫主要依赖其复杂且灵敏的化学感受系统来识别并区分外界环境中的各种化学信号。嗅觉是负责嗅觉信号传导的感官方式,能够引起昆虫觅食、产卵、交配和躲避天敌等对生存和繁殖至关重要的行为反应。在嗅觉感知过程中,气味结合蛋白(odorant binding proteins, OBPs)最先与外界脂溶性化学物质相互作用,并将其转运至化学受体神经元上,激活树突膜表面分布的嗅觉受体(olfactory receptors, ORs),是嗅觉系统正常运行的必需蛋白。近年来,随着高通量测序和分子生物学技术的快速发展,越来越多的昆虫OBPs相继得以鉴定并开展功能研究。昆虫OBPs是一类可溶性的小分子蛋白,一般由6个α-螺旋构成一个稳定、紧密的疏水性结合腔,其构象变化因昆虫种类和配体结构不同而有所差异。OBPs的分布不受限于嗅觉器官,还在口器、足、中肠、腺体等非嗅觉组织中表达,具有嗅觉识别、味觉感受、营养物质转运、信息素合成与释放、组织发育与分化等生理功能。OBPs行使以上功能的共同特性为结合和溶解包括信息素组分、普通气味分子和非挥发性物质等的疏水性小分子物质。昆虫OBPs的稳定性和多功能性暗示其可广泛应用于害虫防治、生物传感器、分析化学、生态学等多个领域。本文对过去20多年来昆虫OBPs的相关研究进行综述,为进一步深入开展OBPs的功能研究提供理论参考。     

果蝇嗅觉分子机理研究进展

黑腹果蝇Drosophila melanogaster是生物学研究的重要模式生物,也是探索研究生物体嗅觉奥秘的理想材料。近年来,由于分子生物学技术在神经科学领域的广泛应用,黑腹果蝇嗅觉机理研究取得了许多重大突破, 对气味分子受体及其识别机理、 嗅觉神经电信号的产生和传递、嗅觉信息的加工、编码以及记忆等方面都有了深入的了解。研究表明, 果蝇约1 300个嗅神经元(olfactory receptor neurons, ORNs)共表达62种不同的气味受体蛋白（olfactory receptor proteins, ORs）, 用以检测和识别其所感受的所有化学气味分子。许多OR所识别的气味分子配体已鉴定出来,普通的气味（如水果的气味）由数种不同的OR组合来识别,而信息素（pheromone）分子则由单种特定的OR来检测。气味信息在嗅神经元内转换成神经电信号,嗅觉电信号沿嗅神经元的轴突传递到触角叶, 再经投射神经元（projection neurons, PNs）将信息送至高级中枢如蘑菇体（mushroom body, MB）和侧角（lateral horn, LH）,最终引发行为反应。在黑腹果蝇嗅觉信息传递通路中,某些蛋白如Dock,N-cadherin,Fruitless等起着重要作用,缺失这些蛋白会导致嗅觉异常。本文对这些研究进展作一综述。     

Functional dissection of Odorant binding protein genes in Drosophila melanogaster

Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial.     

How Far Does a Receptor Influence Vibrational Properties of an Odorant?

The biophysical mechanism of the sense of smell, or olfaction, is still highly debated. The mainstream explanation argues for a shape-based recognition of odorant molecules by olfactory receptors, while recent investigations suggest the primary olfactory event to be triggered by a vibrationally-assisted electron transfer reaction. We consider this controversy by studying the influence of a receptor on the vibrational properties of an odorant in atomistic details as the coupling between electronic degrees of freedom of the receptor and the vibrations of the odorant is the key parameter of the vibrationally-assisted electron transfer. Through molecular dynamics simulations we elucidate the binding specificity of a receptor towards acetophenone odorant. The vibrational properties of acetophenone inside the receptor are then studied by the polarizable embedding density functional theory approach, allowing to quantify protein-odorant interactions. Finally, we judge whether the effects of the protein provide any indications towards the existing theories of olfaction.     

Molecular biological research on olfactory chemoreception in fishes

This review describes recent molecular biological research on olfactory chemoreception in fishes. The recent rapid development of molecular biological techniques has provided new valuable information on the main and vomeronasal olfactory receptor (OR) genes, the axonal projection from ciliated, microvillous and crypt-olfactory receptor cells to the olfactory bulb, properties of odorant substances and olfactory imprinting and homing in salmon. Many important questions, however, remain unanswered on functional differences among OR genes, on ligand binding to each OR and on the molecular biological mechanisms underlying olfactory imprinting and homing in salmon. Olfactory chemoreception is believed to be the oldest sensory cue for both animal survival and adaptation to various different environments. Further intensive molecular biological research on olfactory memory formation and remembrance should be carried out to clarify the fundamental process of olfactory chemoreception in fishes.     

Morituri te salutant? Olfactory signal transduction and the role of phosphoinositides

During the past 150 years, researchers have investigated the cellular, physiological, and molecular mechanisms underlying the sense of smell. Based on these efforts, a conclusive model of olfactory signal transduction in the vertebrate's nose is now available, spanning from G-protein-mediated odorant receptors to ion channels, which are linked by a cyclic adenosine 3',5'-monophosphate-mediated signal transduction cascade. Here we review some historical milestones in the chronology of olfactory research, particularly emphasising the role of cyclic nucleotides and inositol trisphosphate as alternative second messengers in olfactory cells. We will describe the functional anatomy of the nose, outline the cellular composition of the olfactory epithelium, and describe the discovery of the molecular backbone of the olfactory signal transduction cascade. We then summarize our current model, in which cyclic adenosine monophosphate is the sole excitatory second messenger in olfactory sensory neurons. Finally, a possible significance of microvillous olfactory epithelial cells and inositol trisphosphate in olfaction will be discussed.     

Odorant-binding proteins in vertebrates and insects: similarities and possible common function

Among the various chemoreception systems, pheromone perceptionin insects provides perhaps the best biochemical model for comparingwith olfaction in vertebrates. The proteins recently identifiedin the two systems are reviewed and their characteristics arecompared with particular reference to the soluble odorant-bindingand pheromone-binding proteins that are present in large concentrationsin both systems. Hypotheses about the functions of these bindingproteins in modulating the olfactory signals are reported anddiscussed.     

Anatomical contributions to odorant sampling and representation in rodents: zoning in on sniffing behavior

Odorant sampling behaviors such as sniffing bring odorant molecules into contact with olfactory receptor neurons (ORNs) to initiate the sensory mechanisms of olfaction. In rodents, inspiratory airflow through the nose is structured and laminar; consequently, the spatial distribution of adsorbed odorant molecules during inspiration is predictable. Physicochemical properties such as water solubility and volatility, collectively called sorptiveness, interact with behaviorally regulable variables such as inspiratory flow rate to determine the pattern of odorant deposition along the inspiratory path. Populations of ORNs expressing the same odorant receptor are distributed in strictly delimited regions along this inspiratory path, enabling different deposition patterns of the same odorant to evoke different patterns of neuronal activation across the olfactory epithelium and in the olfactory bulb. We propose that both odorant sorptive properties and the regulation of sniffing behavior may contribute to rodents' olfactory capacities by this mechanism. In particular, we suggest that the motor regulation of sniffing behavior is substantially utilized for purposes of "zonation" or the direction of odorant molecules to defined intranasal regions and hence toward distinct populations of receptor neurons, pursuant to animals' sensory goals.     

The sense of smell: molecular basis of odorant recognition

Most animal species rely on odorant compounds to locate food, predators, or toxins. The sense of smell is also involved in animal communication, and revealing the underlying mechanisms will therefore facilitate a deeper understanding of animal behaviour. Since the 1940s different theories have speculated on the fundamental basis of olfaction. It was assumed that odorant molecules were recognized by selective protein receptors in the nose, triggering a nervous signal processed by the brain. The discovery of these receptors in the early 1990s allowed great progress in understanding the physiological and biochemical principles of olfaction. An overview of the different mechanisms involved in the coding of odour character as well as odour intensity is presented here, focusing on the biochemical basis of odorant recognition. Despite the enormous progress achieved in recent years, details of odorant-receptor interaction at the molecular level and the mechanisms of olfactory receptor activation are poorly understood. The likely role of metal ions in odorant recognition is discussed, and also the perireceptor events involved in odorant transport and biotransformation, with a view to providing a comprehensive overview of mammalian olfaction to guide future computational structural models and the design of functional experiments. Recent studies have analysed the olfactory genome of several species, providing information about the evolution of olfaction. The role of the olfactory system in animal communication is also described.     

Zonal organization of the mammalian main and accessory olfactory systems

Zonal organization is one of the characteristic features observed in both main and accessory olfactory systems. In the main olfactory system, most of the odorant receptors are classified into four groups according to their zonal expression patterns in the olfactory epithelium. Each group of odorant receptors is expressed by sensory neurons distributed within one of four circumscribed zones. Olfactory sensory neurons in a given zone of the epithelium project their axons to the glomeruli in a corresponding zone of the main olfactory bulb. Glomeruli in the same zone tend to represent similar odorant receptors having similar tuning specificity to odorants. Vomeronasal receptors (or pheromone receptors) are classified into two groups in the accessory olfactory system. Each group of receptors is expressed by vomeronasal sensory neurons in either the apical or basal zone of the vomeronasal epithelium. Sensory neurons in the apical zone project their axons to the rostral zone of the accessory olfactory bulb and form synaptic connections with mitral tufted cells belonging to the rostral zone. Signals originated from basal zone sensory neurons are sent to mitral tufted cells in the caudal zone of the accessory olfactory bulb. We discuss functional implications of the zonal organization in both main and accessory olfactory systems.     

Characteristics of odorant elicited calcium fluxes in acutely-isolated chick olfactory neurons

To understand avian olfaction, it is important to characterize the peripheral olfactory system of a representative bird species. This study determined the functional properties of olfactory receptor neurons of the chicken olfactory epithelium. Individual neurons were acutely isolated from embryonic day-18 to newborn chicks by dissection and enzymatic dissociation. We tested single olfactory neurons with behaviorally relevant odorant mixtures and measured their responses using ratiometric calcium imaging; techniques used in this study were identical to those used in other studies of olfaction in other vertebrate species. Chick olfactory neurons displayed properties similar to those found in other vertebrates: they responded to odorant stimuli with either decreases or increases in intracellular calcium, calcium increases were mediated by a calcium influx, and responses were reversibly inhibited by 100 M L–cis–diltiazem, 1 mM Neomycin, and 20 M U73122, which are biochemical inhibitors of second messenger signaling. In addition, some cells showed a complex pattern of responses, with different odorant mixtures eliciting increases or decreases in calcium in the same cell. It appears that there are common features of odorant signaling shared by a variety of vertebrate species, as well as features that may be peculiar to chickens.     

Evolutionary grass roots for odor recognition

Considerable evidence supports the idea that odorant recognition depends on specific sequence variations in olfactory receptor (OR) proteins. Much of this emerges from in vitro screens in heterogenous expression systems. However, the ultimate proof should arise from measurements of odorant thresholds in human individuals harboring different OR genetic variants, a research vein that has so far been only scantly explored. The study of McRae et al., published in this issue of Chemical Senses, shows how the recognition of a grassy odorant depends on specific OR interindividual sequence changes. It provides a clear relevant example for the impact of genetics on olfaction and is an excellent portrayal of the power of human genomics to decipher olfactory perception.     

The Odorant Receptor Co-Receptor from the Bed Bug,Cimex lectularius L

Recently, the bed bug, Cimex lectularius L. has re-emerged as a serious and growing problem in many parts of the world. Presence of resistant bed bugs and the difficulty to eliminate them has renewed interest in alternative control tactics. Similar to other haematophagous arthropods, bed bugs rely on their olfactory system to detect semiochemicals in the environment. Previous studies have morphologically characterized olfactory organs of bed bugs’ antenna and have physiologically evaluated the responses of olfactory receptor neurons (ORNs) to host-derived chemicals. To date, odorant binding proteins (OBPs) and odorant receptors (ORs) associated with these olfaction processes have not been studied in bed bugs. Chemoreception in insects requires formation of heteromeric complexes of ORs and a universal OR coreceptor (Orco). Orco is the constant chain of every odorant receptor in insects and is critical for insect olfaction but does not directly bind to odorants. Orco agonists and antagonists have been suggested as high-value targets for the development of novel insect repellents. In this study, we have performed RNAseq of bed bug sensory organs and identified several odorant receptors as well as Orco. We characterized Orco expression and investigated the effect of chemicals targeting Orco on bed bug behavior and reproduction. We have identified partial cDNAs of six C. lectularius OBPs and 16 ORs. Full length bed bug Orco was cloned and sequenced. Orco is widely expressed in different parts of the bed bug including OR neurons and spermatozoa. Treatment of bed bugs with the agonist VUAA1 changed bed bug pheromone-induced aggregation behavior and inactivated spermatozoa. We have described and characterized for the first time OBPs, ORs and Orco in bed bugs. Given the importance of these molecules in chemoreception of this insect they are interesting targets for the development of novel insect behavior modifiers.     

Improving the odorant sensitivity of olfactory receptor-expressing yeast with accessory proteins

Olfaction depends on the selectivity and sensitivity of olfactory receptors. Previous attempts at constructing a mammalian olfactory receptor-based artificial odorant sensing system in the budding yeast Saccharomyces cerevisiae suffered from low sensitivity and activity. This result may be at least in part due to poor functional expression of olfactory receptors and/or limited solubility of some odorants in the medium. In this study, we examined the effects of two types of accessory proteins, receptor transporting protein 1 short and odorant binding proteins, in improving odor-mediated activation of olfactory receptors expressed in yeast. We found that receptor transporting protein 1 short enhanced the membrane expression and ligand-induced responses of some olfactory receptors. Coexpression of odorant binding proteins of the silkworm moth Bombyx mori enhanced the sensitivity of a mouse olfactory receptor. Our results suggest that different classes of accessory proteins can confer sensitive and robust responses of olfactory receptors expressed in yeast. Inclusion of accessory proteins may be essential in the future development of practical olfactory receptor-based odorant sensors.