Project Summary Maintaining internal environment constancy is essential for life. The circumventricular organs (CVO’s) of the brain including organum vasculosum of the lamina terminalis (OVLT) and the subfornical organ (SFO) lack a blood-brain-barrier, function as central sensors to provide feedback regulation for maintaining osmotic equilibrium. Neurons in CVO’s detect changes in osmolality and transduce the signals into action potentials (AP’s) travelling down the axon projecting to magnocellular neurons in the paraventricular (PVN) and supraoptic nuclei (SON). Upon activation by AP’s, magnocellular neurons synthesize antidiuretic hormones (ADH), which is transported via axon process to the posterior pituitary gland and released into blood circulation herein. ADH acts on kidney to effect free water reabsorption. While hyperosmolality also stimulates thirst and drinking, separate neuronal networks are involved. Together, renal free water reclamation and drinking restore serum osmolality in response to water deprivation. The molecular identity of the osmolality sensor(s) in the OVLT/SFO neurons remains elusive. With-no-lysine [K] kinases WNK1-4 are protein kinases in which gain-of-function mutations of WNK1 and 4 in humans cause familial hypertensive and hyperkalemic disease called pseudohypoaldosteronism type II (PHA2). Our preliminary results strongly support the central hypothesis that WNK1 functions as a central osmosensor for osmolality regulation of ADH release. Mice with neuronal conditional knockout (cKO) of Wnk1 exhibit phenotypes of partial central diabetes insipidus (DI). WNK1 activate downstream oxidative-stress responsive-1 kinase (OSR1) and related SPAK (Ste20-related proline/alanine-rich kinase). WNK1-OSR1/SPAK kinase cascade regulates many ion channels and transporters. To support the hypothesis, Specific Aim-1 will test the hypothesis that OSR1 and/or SPAK acts downstream of WNK1 to regulate osmolality-induced ADH release. Control and mice with genetically altered WNK1 kinase cascade will be studied in metabolic cage under free water access and water restriction. Urine volume, urine and serum electrolyte, osmolality, ADH and copeptin levels will be measured. Specific Aim-2 will test the hypothesis that activation of Kv3.1b voltage-gated K+ channels by WNK1 increases AP firing in OVLT/SFO neurons leading to stimulation of ADH release by hyperosmolality. Electrophysiological recording of freshly isolated individual OVLT/SFO neurons, native neurons in situ in acute brain slice, and HEK cells expressing recombinant channels and WNK1 cascades will be performed. The proposed studies will reveal novel findings that an intracellular protein functions as a sensor for extracellular osmolality and provide fresh insights into how body maintains osmotic equilibrium in health and into disease processes that affect total body water homeostasis.