Molecular mechanisms for olfactory signal transduction

采用全细胞膜片钳技术记录单个嗅觉感受器细胞(ORC)的放电。同时，在电压钳条件下，测量各通道的活动。电流注入引起重复放电。放电频率依赖于注入电流的大小，表现为饱和。当细胞受到气味刺激时，在电压钳条件下(保持电位=-60 mV)，54个细胞对桉叶油素产生内向电流。内向电流的幅度依赖于刺激周期，反映了刺激剂量的时间积分。剂量-效应关系可用Hill方程拟合。在电流钳制条件下，气味诱导了重复的尖峰。在这些细胞中，尖峰频率是剂量依赖的，表现为饱和，类似于电流注射的结果。由于转导通道和棘波产生步骤都表现出剂量依赖性的饱和，我们探索了是什么步骤(S)实际上决定了兽类动物信号过程的饱和。通过检查同一细胞中电流和尖峰电流的剂量-反应关系，发现饱和剂量在很大程度上依赖于转导步骤的剂量。这表明动态范围受到转导系统的强烈影响。从膜的非线性出发，建立了一个简单的模型，可以解释随着传导通道希尔系数的增大，去极化-浓度曲线的陡度变大。此外，据推测，动态范围的一个临界水平至少部分地被膜非线性所修正。此外，我们还在识别介导嗅觉转导的第二信使、交叉适应机制以及一些关于人类嗅觉的生理测量方面进行了实验。

Spike discharges of single olfactory receptor cells (ORCs) were recorded with the whole-cell patch clamp method applied to the slice preparation. In parallel, activities of transduction channels were measured under the voltage-clamp condition. Current injection induced repetitive discharges. The firing frequency was dependent on the amount of injected current, showing saturation. When cells ware stimulated by odorants, 54 out of 306 cells exhibited inward currents in response to cineole under the voltage-clamp condition (holding potential =-60 mV). The amplitude of the inward current was dependent on the stimulus period, reflecting the time-integration for the stimulus dose. The dose-response relation could be fitted by the Hill equation. Under the current-clamp condition, odorants induced repetitive spikes. In those cells spike frequency was dose-dependent, expressing saturation similar to the result obtained by current injection. Since both transduction channel and spike generative steps expressed saturation in their dose dependences, we explored what step(s) actually determines saturation in ORCs' signaling processes. By examining dose-response relations of both the current and spikes in the same cells, saturating dose was found to be dependent largely on that of the transduction step. This suggests that the dynamic range is strongly influenced by the transduction system. A simple model derived from the non-linearity of the membrane could explain that the steepness of the depolarization-concentration curve became bigger as Hill coefficient of the transduction channel was increased. In addition, it was speculated that a critical level of dynamic range was, at least in part, modified by the membrane non-linearity.In addition we made experiments on identifying second messenger mediating olfactory transduction, mechanisms for cross-adaptation and some physiological measurements from human subjects regarding olfactory perception.