Properties and Functions of Ca2+ Channels in Hair Cells

CA调节毛细胞的多种功能。我们的目标是了解多个电压门控钙通道是如何赋予这些钙依赖功能的。这项建议的总体主旨是部署创新的分子生物学、电生理学和成像技术-其中许多技术源于以前的钙通道研究-以发现毛细胞中钙通道生理学的基本和新的可访问领域。这一推力推动了三个目标，以解决毛细胞钙通道生理学的各个方面，每个方面都具有基本和治疗意义。总体假设是毛细胞表达Cav3.x(T型)钙通道，该通道可能赋予毛细胞特有的功能(例如，某些发育阶段的特征性爆裂活动)，并有助于毛细胞的成熟。其目的是：(1)阐明毛细胞中钙通道亚型及其功能的未解决的方面。尽管存在巨大的潜在生理影响，但非L型钙通道的特性仍不确定。利用生物物理和分子生物学技术，我们将鉴定毛细胞中的非L型通道为T型通道，并表明基因的选择性剪接产生不同变体的T型通道，这些通道是毛细胞特有的。(2)探讨钙通道亚型在毛细胞功能发育中的作用。我们预测毛细胞中VGCC的表达存在发育转换。我们认为不同的钙通道亚型参与了毛细胞的早期发育过程。(3)明确L钙通道和T型钙通道在毛细胞局部和整体钙结构域调控中的作用。要使钙离子在不同的细胞内过程中发挥信号作用，必须精确控制其局部浓度，以防止不同途径之间的串扰。我们假设，从单个到不同大小的簇，钙通道亚型的异质性表达赋予了不同的钙结构域和多种钙依赖功能。在这里，我们将通过进行全细胞和单通道记录以及使用高分辨率成像技术同时记录钙结构域来确定毛细胞中钙通道的基本特性。将确定毛细胞中钙处理的空间和时间特征，并将制定局部和全球钙处理的模型。总体而言，这项建议将回答毛细胞中钙通道生理学的基本未知问题。

Ca regulates multiple functions in hair cells. Our objectives are to understand how multiple voltage-gated Ca channels confer these Ca-dependent functions. The overall thrust of this proposal is to deploy innovative molecular biological, electrophysiological, and imaging techniques- many inspired from previous Ca channel studies- for the discovery of fundamental and newly accessible arenas of Ca channel physiology in hair cells. This thrust drives three aims that address various aspects of hair cell Ca channel physiology, each with fundamental and therapeutic implications. The overall hypothesis is that hair cells express Cav3.x (T-type) Ca channels that may confer hair cell-specific functions (e.g. characteristic bursting activity at certain stages in development) and contribute towards maturation of hair cells. The Aims are: (1) To clarify unresolved aspects of the subtypes of Ca channels in hair cells and their functions. The identity of non L-type Ca channels remains uncertain, despite large potential physiological ramifications. Using biophysical and molecular biological techniques, we will identify the non L-type channels in hair cells as T-type channels and show that alternative splicing of mRNA produces different variants of T-type channels that are specific to hair cells. (2) To determine the role of Ca-channel subtypes in functional development of hair cells. We predict that there is a developmental switch in the expression of VGCCs in hair cells. We expect that different Ca channel subtypes are involved in early developmental processes of hair cells. (3) To define the role of Ca entry through L- and T- type Ca channels in the control of local and global Ca domains in hair cells. For Ca ions to be effective as signals in different intracellular processes, their local concentration has to be precisely controlled to prevent crosstalk between different pathways. We hypothesize that heterogeneous expression of Ca channel subtypes from single to different-sized clusters confers varying Ca domains and multiple Ca dependent functions. Here, we will determine the elementary properties of Ca channels in hair cells by performing whole-cell and single-channel recordings and simultaneous recording of Ca domains using high resolution imaging techniques. The spatial and temporal characteristics of Ca handling in hair cells will be identified and a model of local and global Ca handling will be formulated. Overall, this proposal will answer fundamental unknowns of Ca channel physiology in hair cells.