No Evidence for Ionotropic Pheromone Transduction in the Hawkmoth Manduca sexta

Insect odorant receptors (ORs) are 7-transmembrane receptors with inverse membrane topology. They associate with the conserved ion channel Orco. As chaperon, Orco maintains ORs in cilia and, as pacemaker channel, Orco controls spontaneous activity in olfactory receptor neurons. Odorant binding to ORs opens OR-Orco receptor ion channel complexes in heterologous expression systems. It is unknown, whether this also occurs in vivo. As an alternative to this ionotropic transduction, experimental evidence is accumulating for metabotropic odor transduction, implicating that insect ORs couple to G-proteins. Resulting second messengers gate various ion channels. They generate the sensillum potential that elicits phasic-tonic action potentials (APs) followed by late, long-lasting pheromone responses. Because it is still unclear how and when Orco opens after odor-OR-binding, we used tip recordings to examine in vivo the effects of the Orco antagonist OLC15 and the amilorides MIA and HMA on bombykal transduction in the hawkmoth Manduca sexta. In contrast to OLC15 both amilorides decreased the pheromone-dependent sensillum potential amplitude and the frequency of the phasic AP response. Instead, OLC15 decreased spontaneous activity, increased latencies of phasic-, and decreased frequencies of late, long-lasting pheromone responses Zeitgebertime-dependently. Our results suggest no involvement for Orco in the primary transduction events, in contrast to amiloride-sensitive channels. Instead of an odor-gated ionotropic receptor, Orco rather acts as a voltage- and apparently second messenger-gated pacemaker channel controlling the membrane potential and hence threshold and kinetics of the pheromone response.

昆虫气味受体（odorant receptors, ORs）是一类具有反向膜拓扑结构（inverse membrane topology）的7次跨膜受体（7-transmembrane receptors），它们可与保守离子通道Orco（conserved ion channel Orco）形成功能复合体。作为分子伴侣（chaperon），Orco可维持ORs在纤毛（cilia）中的定位；同时作为起搏通道（pacemaker channel），Orco能够调控嗅觉受体神经元（olfactory receptor neurons）的自发活动。在异源表达系统（heterologous expression systems）中，气味分子与ORs结合可激活OR-Orco受体离子通道复合体，但目前尚不清楚该过程是否同样存在于活体（in vivo）中。作为离子型转导（ionotropic transduction）的替代途径，越来越多的实验证据支持代谢型气味转导（metabotropic odor transduction）机制，即昆虫ORs可与G蛋白（G-proteins）偶联。由此产生的第二信使（second messengers）可门控多种离子通道，进而产生感器电位（sensillum potential），最终诱发相位-紧张型动作电位（action potentials, APs）以及后续持续时间较长的晚期信息素响应。由于目前仍不明确OR与气味分子结合后，Orco通道的激活机制与具体时机，我们采用尖端记录技术（tip recordings），在活体状态下检测了Orco拮抗剂OLC15（Orco antagonist OLC15）以及阿米洛利类化合物（amilorides）MIA和HMA对烟草天蛾（Manduca sexta）体内bombykal转导过程的影响。与OLC15不同，两种阿米洛利类化合物均降低了信息素依赖的感器电位幅值，以及相位型AP响应的频率。与之相反，OLC15可降低神经元自发活动，延长相位型响应的潜伏期，并呈授时因子时间依赖性（Zeitgebertime-dependently）地降低晚期持续型信息素响应的频率。我们的研究结果表明，与阿米洛利敏感通道不同，Orco并未参与初级转导事件。相较于气味门控的离子型受体，Orco更倾向于作为电压门控且可被第二信使调控的起搏通道，通过控制膜电位进而调控信息素响应的阈值与动力学特性。