WNK1 as an Osmolality Sensor: Mechanism and Structure-Function Studies

Background: The circumventricular organs (CVO’s) of the brain lack a blood-brain barrier. CVO neurons detect serum hyperosmolality to stimulate the production and release of arginine vasopressin (AVP). We recently reported that WNK1 in CVO neurons senses hyperosmolality and activates Kv3.1 channel to increase action potential to increase AVP release. Boyd-Shiwarski et al reported that WNK1 forms condensates during cellular dehydration and activates NKCC1 to restore cell volume. The C-terminal intrinsically disordered region (IDR) is important for formation of the condensates. Here, we studied the role of OSR1/SPAK for WNK1 to activate Kv3.1 and the role of IDR in this activation. Methods: Mice withCVO deletion ofSPAK and OSR1 were produced by stereotaxic injection of Cre-recombinase-carrying AAV virus into CVO of Spak-null and Osr1-floxed mice (Sapk-/-;Osr1flox/flox). Water homeostasis was examined in metabolic cage studies. AVP levels were measured. The Kv3.1 current was studied in HEK cells expressing recombinant WNK1 (or mutants), SPAK, and Kv3.1. Biochemical kinase activity of WNK1 was examined by western blot. Results: Hyperosmolality increased p-OSR1/SPAK in mouse CVOs. Mice with OSR1/SPAK deletion in CVOs exhibited polyuria with decreased urine osmolality that persisted in water restriction, indicating diabetes insipidus. Circulating levels of AVP and copeptin were lower in water restricted OSR/SPAK deletion mice vs controls. In whole-cell recording with 20 mM Cl- in the pipette (intracellular), exposure to physiological extracellular hyperosmolality (5 mM NaCl, “HTS”) increased Kv3.1 currents in HEK cells expressing full-length WNK1, SPAK, and Kv3.1. Increasing pipette Cl- to 100 mM blunted the activation of Kv3.1 by HTS. Importantly, cells expressing just the kinase domain of WNK1 (aa 1-491), SPAK, and Kv3.1 responded to HTS. Moreover, HTS induced autophosphorylation of WNK1 (1-491). Conclusion: The WNK1-mediated osmosensation in CVO neurons involves activation of Kv3.1 through SPAK/OSR1. WNK1 kinase domain can detect extracellular hyperosmolality independently of the C-terminal IDR region. The results support the structural data that water and chloride in the cavity of WNK kinase domain negatively regulate the kinase activity. How IDR coordinates with the kinase domain to regulate downstream effectors awaits future studies. Funding: NIDDK Support