Osmosensing by WNK Kinases

Abstract

WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol 400 or osmolyte ethylene glycol, and that this activation is opposed by chloride. Small Angle X-ray Scattering of WNK3 in the presence and absence of PEG400, static light scattering in ethylene glycol, and crystallography of WNK1 were used to understand mechanism. Osmosensing in WNK3 and WNK1 appear to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.

LRRC8A-containing chloride channel is crucial for cell volume recovery and survival under hypertonic conditions

“…Under these conditions, no differences in WNK1 phosphorylation or RVI were observed between LZ and KO cells, thereby suggesting that the effect of LRRC8A-containing channels on the modulation of the WNK1–NKCC axis is through the modulation of intracellular Cl − concentrations. Actually, direct activation of the purified kinase domain of WNK by osmolytes is promoted by reducing [Cl − ] ( 27 ), in concordance with our findings that LRRC8A-mediated Cl − efflux prevents excessive accumulation of intracellular Cl − and promotes WNK1–NKCC activation in intact cells.…”

“…We propose a mechanism ( Fig. 4 E ) by which activation of the p38/MSK1 pathway phosphorylates and activates the LRRC8A chloride and promotes Cl − efflux, which in turn facilitates shifting the conformational equilibrium of WNK1 from the chloride-bound inhibitory state toward the less-hydrated, active state triggered by hypertonicity ( 27 ). Thus, the combination of both direct activation of WNK1 by hyperosmotic stress and reduction in Cl − -mediated autoinhibition may represent a mechanism to ensure optimal WNK1 activation and guarantee NKCC1-mediated transport to promote RVI and cell survival in response to hypertonic environments.…”

“…Therefore, most of the effectors participating in the regulation of cell volume are identified, but less is known about the mechanisms that initiate RVI. Recent evidence suggests direct osmosensing by the kinase domain of WNK due to a displacement of the equilibrium from a chloride binding conformation (inhibitory) to a low water availability (activating) conformation under high osmolarity conditions ( 27 ). In this model, reduction of the intracellular [Cl − ] may facilitate hyperosmotic-dependent activation of WNK and RVI, in line with reports from the early 1990s pointing to the link between low intracellular [Cl − ] and osmotically activated NKCC transport ( 12 , 15 ).…”

LRRC8A reduces intracellular chloride to permit WNK activation in response to hypertonic stress

“…Chloride binds and stabilizes an auto-inhibited dimeric configuration (14). Osmotic stress, mimicked in vitro with crowding agents (15), induces WNK1 and WNK3 to autophosphorylate (16). Crowding agents induce monomerization as measured by gel filtration and low-angle X-ray scattering.…”

WNK kinases sense molecular crowding and rescue cell volume via phase separation

“…These mechanisms likely serve permissive roles that mitigate WNK1 inactivation during shrinkage (Goldsmith and Huang, 2021). A third study found that the purified WNK1 and WNK3 kinase domains activate in vitro in the presence of molecular crowders (Akella et al, 2021). This effect was associated with dehydration of the kinase domain, suggesting that solvent effects participate in a direct osmosensing mechanism.…”