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.
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