second coiled-coil domain of WNK4 mediates the downregulation of TRPV4. WNK4 and calcium-binding protein 39 (Cab39) act together to activate transporters NKCC1 and NKCC2. Role in pseudohypoaldosteronism type 2 Dysregulation of WNK4 kinase activity In 2001, four missense mutations in the WNK4 gene were identified in patients with pseudohypoaldosteronism type 2 (PHAII) (Fig. 1). Three of these mutations (E562K, D564A, and Q565E) are charge-changing substitutions in the acidic motif of WNK4, which are conserved among all members of the WNK family in human and rodent species. The fourth substitution (R1185C) is located in the calmodulin-binding domain near the second coiled-coil domain. Few other PHAII mutations in WNK4 have also been reported. Examples of these mutations include E560G, P561L, and D564H, all of which are located close to or in the acidic motif; and K1169E which is located between the coiled-coil 2 and the calmodulin-binding domain. The PHAII mutations appear to disrupt the mechanisms underlying Ca2+-sensitivity of WNK4 kinase. Two mechanisms are important in this regard. First, the PHAII-causing mutations in the acidic motif make the kinase domain insensitive to Ca2+ concentration. The acidic motif of WNK4 potentially acts as a Ca2+ sensor, and WNK4 kinase activity rises when Ca2+ concentration is elevated. This has been demonstrated using isolated WNK4 kinase domain truncated to contain the acidic motif. The kinase activity is elevated when a PHAII-causing mutation is present in the acidic motif, similar to what is observed in a Ca2+-binding state (Fig. 3). Second, the WNK4 C-terminal region containing the calmodulin-binding domain and multiple SGK1 phosphorylation sites inhibits the WNK4 activity at the resting state. However, when Ca2+ levels are elevated, Ca2+/calmodulin complex binds to the C-terminal region, derepressing WNK4 kinase activity. Additionally, the RFXV motif is believed to interact with the autoinhibitory domain and subsequently triggers a conformational change that brings the C-terminal and kinase domain close for the inhibitory effect to take place. Angiotensin II increases the SPAK phosphorylation and activates NCC through a WNK-dependent mechanism. The activation of SPAK and NCC by angiotensin II is abrogated by WNK4 knockdown. Activation of angiotensin II receptor AT1 couples to Gq/11 to activate phospholipase C and to increase the intracellular Ca2+ concentration. An increase in Ca2+ concentration then elevates WNK4 activity through mechanisms described above (Fig. 3, left panel). The PHAII-causing mutations in the acidic motif and the R1185C mutation in the calmodulin-binding domain constitutively activate the WNK4 kinase domain