The current project """"""""Molecular studies of osmotic neural sensing"""""""" is based on the recent cloning by the applicant of the vanilloid receptor related osmotically activated ion channel (VR-OAC) and its identification as a possible osmolarity sensor in the hypothalamus of the central nervous system. In this proposal, the applicant outlines a series of experiments to further explore the functions of this novel molecule and its possible role in regulating salt balance in-vivo. Homeostasis of osmotic pressure is a vital prerequisite for all living cells. In vertebrate organisms including humans, systemic osmotic pressure is the most aggressively defended set-point value of all homeostatically regulated systems. Sensing of osmotic pressure as well as the adaptive response - drinking behavior and secretion of antidiuretic hormone - all localize anatomically to the hypothalamus. The recent cloning of VR-OAC and its localization to the hypothalamus opened the door to an understanding of this homeostatic circuit at the molecular level. The current application describes a set of experiments that will characterize the Vroac gene further. Most importantly, Vroac-/- mice will be generated by gene-targeting. A general knockout and a tissue-specific knockout for the central nervous system are proposed. These knockout mouse lines will be examined for drinking behavior, osmotic regulation, kidney function and fluid homeostasis including the regulation of blood pressure, axonal projections of VR-OAC expressing neurons and inner ear function. The potential of VR-OAC to restore genetically defective osmotic sensing in-vivo will be examined in a mutant strain of the roundworm, Caenorhabditis elegans, that has defects in osmotic sensing. In these studies, osm-9 mutant worms with such sensing defects will be crossed to transgenic worms that overexpress rat VR-OAC. This experimental set-up will allow for a rapid evaluation of structure-function relationships of VR-OAC in-vivo. By means of the proposed deletion of the Vroac gene and analysis ot Vroac-/- mice as well as the mutant worms, the applicant intends to pursue a critical question and develop a firm grasp of the immensely powerful tools of molecular genetics and in particular, mouse gene-targeting. Endowed with these techniques and the experience of functional genomics of the Vroac gene, the applicant intends to advance his career and become an independent junior investigator.
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