Properties and Functions of Ca2+ Channels in Hair Cells

钙调节毛细胞的多种功能。我们的目标是了解多个电压门控Ca通道如何赋予这些Ca依赖性功能。该提案的总体目标是部署创新的分子生物学，电生理学和成像技术-许多灵感来自以前的钙通道研究-发现的基本和新的毛细胞钙通道生理学领域。这一推力驱动三个目标，解决毛细胞钙通道生理学的各个方面，每个基本和治疗的影响。总体假设是毛细胞表达Cav3.x（T型）Ca通道，其可赋予毛细胞特异性功能（例如，在发育的某些阶段的特征性爆发活性）并有助于毛细胞的成熟。目的：（1）阐明毛细胞钙通道的亚型及其功能。非L型钙通道的身份仍然不确定，尽管大的潜在生理后果。使用生物物理学和分子生物学技术，我们将确定非L型通道的毛细胞T型通道，并表明，选择性剪接的mRNA产生不同的变体的T型通道是特定的毛细胞。(2)探讨钙离子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的表达存在发育开关。我们预期不同的钙通道亚型参与毛细胞的早期发育过程。(3)目的：明确L型和T型钙通道钙离子内流在毛细胞局部和整体钙结构域调控中的作用。为了使Ca离子在不同的细胞内过程中有效地作为信号，必须精确地控制它们的局部浓度以防止不同途径之间的串扰。我们推测，从单一到不同大小的簇的钙通道亚型的异质性表达赋予不同的钙结构域和多种钙依赖性功能。在这里，我们将确定在毛细胞中的钙通道的基本特性，通过执行全细胞和单通道记录和同时记录的钙域使用高分辨率成像技术。钙处理在毛细胞的空间和时间特性将被确定和局部和全局钙处理的模型将制定。总的来说，这个建议将回答毛细胞中钙通道生理学的基本未知数。

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.