(A) WNK1 exists in conformational equilibrium between chloride-bound autoinhibited dimer and chloride-free activation-competent monomer. Hyperosmolality extracts water from the cell and from the catalytic core of WNK1, which facilitates chloride unbinding, allowing autophosphorylation at S382 and be activated (38–40). WNK1 may activate Kv3.1 directly or indirectly through other intermediaries such as OSR1/SPAK. (B) Kv3.1 is a high-threshold voltage-gated K⁺ channel activated by membrane depolarization to –20 mV or above (24, 25). Activation of Kv3.1 shortens action potential duration, increases after hyperpolarization (AHP), and thus increases firing frequency (illustrated by red trace). Conversely, inhibition of Kv3.1 decreases firing frequency (blue trace). In support of this notion, we have found that TEA increased the action potential half-width (data not shown). (C) Exponential curvilinear relationship between AVP release and plasma osmolality begins at the threshold of approximately 280 mOsm/kg. WNK1 activation by cellular dehydration (Excitatory pathway; thick green line) plays an important role in AVP release by hyperosmolality. Additional mechanism(s) may be involved, at least for secretion at the basal state, which may include tonic inhibition of osmosensory neurons (Inhibitory pathway; thick solid red line). Loss of hypotonicity-mediated inhibitory pathway (thick dotted red line) may also contribute to hyperosmolality-induced AVP release. Compensation by the additional pathways may account for apparent similar AVP release defects in OVLT-selective deletion of WNK1 (by direct shRNA injection) versus neuronal deletion of WNK1. Extracellular hypertonicity may also activate WNK1 signaling cascade through molecular crowding of the protein (ref. 46) (data not shown).