WNK-SPAK/OSR1 signaling pathway facilitates ictal activity via reduced neuronal chloride extrusion rate

Seizures upregulate Na ⁺ -K ⁺ -2Cl ^- (NKCC1)-mediated Cl ^- influx and downregulate K ⁺ -Cl ^- (KCC2)-mediated Cl ^- efflux via the WNK-SPAK/OSR1 kinases, leading to cytoplasmic chloride ([Cl ^- ] _i ) accumulation, reduced GABAergic inhibition and anticonvulsant failure. Early studies found that inhibiting WNK-kinase reduced baseline [Cl ^- ] _i (E _Cl ) and seizures via increased KCC2 activity. However, increased KCC2 activity alone should not affect E _Cl whose determinants are more complex. We determined the net effects of WNK-SPAK/OSR1 pathway inhibitor WNK463 on E _Cl and [Cl ^- ] _i transients during spontaneous ictal-like discharges (ILDs). We found that WNK463 reduced interictal [Cl ^- ] _i but did not change baseline [Cl ^- ] _i measured in the presence of TTX. WNK463 enhanced neuronal Cl ^- extrusion during and after ILDs, before abolishing ILDs. Pharmacological inhibition and targeted siRNA silencing demonstrated that the anti-ictal effects of WNK463 involved both NKCC1 and KCC2. Thus, mutual NKCC1 inhibition and KCC2 activation via the WNK-SPAK/OSR1 pathway exert powerful anti-ictal effects by facilitating [Cl ^- ] _i extrusion during ILDs.