Molecular mechanisms for olfactory signal transduction

用全细胞膜片钳技术记录单个嗅感受器细胞的棘波放电。同时，在电压钳条件下测量转导通道的活性。电流注入诱导重复放电。发射频率取决于注入电流的量，表现出饱和。在电压钳条件下（保持电位=-60 mV），306个细胞中有54个细胞对桉叶素产生内向电流。内向电流的幅度依赖于刺激时间，反映了刺激剂量的时间积分。剂量-反应关系可用Hill方程拟合。在电流钳条件下，气味诱导重复尖峰。在这些细胞中，尖峰频率是剂量依赖性的，表示饱和类似于通过电流注入获得的结果。由于转导通道和峰电位生成步骤都以其剂量依赖性表达饱和，因此我们探索了在ORC的信号传导过程中哪些步骤实际上决定饱和。通过研究同一细胞中电流和尖峰的剂量-反应关系，发现饱和剂量在很大程度上取决于转导步骤。这表明动态范围受到转导系统的强烈影响。由膜的非线性导出的一个简单模型可以解释随着转导通道的希尔系数的增大，去极化-浓度曲线的陡度变大。此外，我们还对嗅觉信号转导的第二信使、交叉适应的机制以及嗅觉感知的一些生理测量进行了实验研究。

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.