CELLULAR BASIS OF TUNING IN THE COCHLEA

拟议研究的目标是表征细胞 有助于海龟耳蜗毛细胞调谐的机制。 通过了解这些细胞机制如何有助于 频率选择性，我们将能够评估其局限性 以及对包括人类在内的高等脊椎动物听觉的适用性。 全细胞膜片钳和单通道膜片钳的结合 技术将用于从鉴定的细胞中分离的孤立细胞， 海龟耳蜗的区域。 第一个目的是分析 毛细胞纤毛束的机械反应，以及 确定这种反应在整个转导中的作用 机制 的来源、强度和电压敏感性 将确定纤毛运动。 传感器的能力 将确定检测该运动的通道，并且任何 尺寸和电压灵敏度随特性的变化 频率将被评估。 第二个目标是描述 此外，基底外侧毛细胞膜中的电导 与电共振有关 尺寸的变化和 已知的细胞中的膜电流的动力学 特征频率将使用细胞内 灌注。 特定的协议解决了内部隔离问题 电流由于顶端换能器和基底侧钙 电流，表征可能的产电转运 将钙排出细胞，并将钙与细胞分离 钙激活的钾电流。 第三 目的是分析钙激活的 钾离子通道和表征钙离子和电压 灵敏度 由于这种电流的动力学行为是高度 与毛细胞的特征频率相关 被调谐，它被认为在频率上起主要作用。 选择性 同时细胞附着的单通道和整个- 细胞记录将被用来比较行为的单一 通道和宏观钙激活钾 电流 拟议的研究将分析这些离子电流，以评估 是否毛细胞调谐由于基底外侧离子电导 同时存在，或通过，机制，通过 睫状体束中的转导过程。 离子流 在电压钳下测量的电压将用于重建 在电流钳中观察到膜共振。

The goal of the proposed research is to characterize the cellular mechanisms that contribute to tuning in turtle cochlear hair cells. By understanding how these cellular mechanisms contribute to frequency selectivity, we will be able to assess their limitations and applicability to hearing in higher vertebrates including man. A combination of whole-cell and single-channel patch-clamp techniques will be used on solitary cells isolated from identified regions of the turtle cochlea. The first aim is to analyze the mechanical response of the hair-cell ciliary bundle, and to determine the role of this response in the overall transduction mechanism. The source, strength and voltage-sensitivity of the ciliary motion will be determined. The ability of the transducer channel to detect this motion will be ascertained, and any variation in the size and voltage-sensitivity with characteristic frequency will be assessed. The second aim is to characterize further the conductances in the basolateral hair-cell membrane implicated in electrical resonance. Changes in the size and kinetics of the membrane currents in cells of known characteristic frequency will be analyzed using intracellular perfusion. Specific protocols address the isolation inward currents due to the apical transducer and the basolateral calcium current, the characterization of possible electrogenic transport of calcium out of the cell, and a separation of the calcium from the calcium-activated potassium currents in a single cell. The third aim is to analyze the kinetic behavior of the calcium-activated potassium channel and to characterize the calcium- and voltage- sensitivity. Since the kinetic behavior of this current is highly correlated with the characteristic frequency to which a hair cell is tuned, it is presumed to play a major role in frequency selectivity. Simultaneous cell-attached single-channel and whole- cell recording will be used to compare the behavior of the single channel and the macroscopic calcium-activated potassium current. The proposed research will analyze these ionic currents to assess whether hair-cell tuning due to basolateral ionic conductances coexists with, or is enhanced by, mechanisms acting via the transduction process in the ciliary bundle. The ionic currents measured under voltage-clamp will be used to reconstruct the membrane resonance seen in current-clamp.