Abstracts

Rationale: Approximately 30% of epilepsy patients display pharmacoresistance to current treatments, highlighting a critical need for novel therapeutic strategies. The neuron-specific K+/ Cl– transporter KCC2 maintains low intracellular Cl– ([Cl–]i), which is a prerequisite for inhibitory gamma-amino butyric acid (GABA) transmission. Importantly, deficits in KCC2 and GABAergic inhibition are implicated in epilepsy. KCC2 phosphorylation plays a key role in regulating transporter activity, stability, and GABAergic inhibition. Notably, transgenic mice with KCC2 threonine-to-alanine substitutions, which prevent phosphorylation, at residues 906 and 1007 (KCC2-T906A/T1007A), show strong resistance to in vitro models of seizures (Moore et al., 2018). These findings implicate KCC2 as a novel target for restoration of GABAergic deficits and seizure control. The with-no-lysine kinase (WNK) and STE20/SPS1-related proline/alanine-rich kinase (SPAK) pathway is highly conserved across cell types and lies directly upstream of KCC2-T1007. While the WNK-SPAK pathway has been studied for its role in cell-volume regulation and blood pressure, it has not been tested for its role in KCC2-mediated Cl– homeostasis in the brain. Here, we test the hypothesis that pharmacological inhibition of the WNK-SPAK pathway will enhance KCC2 activity, and increase the efficacy of GABAergic inhibition. Methods: Electrophysiology. Gramicidin-perforated and whole-cell patch methods were used for in vitro assays. Electroneutral KCC2 activity cannot be directly measured, however, the Cl– reversal potential (approximated as the GABAA reversal potential (EGABA)), can be measured via voltage ramp protocols. Thus EGABA is the gold-standard measure of KCC2 activity. [Cl–]i values were then derived from measured EGABA values using the Nernst equation. Biochemistry. Western blot analysis and immunoprecipitations were employed. Pharmacological WNK inhibition. Our work takes advantage of the highly selective pan-WNK kinase inhibitor WNK463 (Yamada et al., 2016). Results: First we examined SPAK and KCC2-T1007 phosphorylation following WNK inhibition in vitro. Neurons treated with WNK463 exhibited a significant reduction in SPAK and KCC2-T1007 phosphorylation (n=3; p<0.001), while total levels of SPAK and KCC2 protein were unchanged (n=3; p=0.128). KCC2-T1007 phosphorylation is also significantly reduced in brain slices when exposed to WNK463 (n=3 p<0.0001). Next, we found that WNK inhibition via WNK463 induces a negative shift in EGABA, coupled with a significant decrease in [Cl–]i in vitro (n=10, p=0.0024). We also report that neurons pre-incubated with WNK463 had faster rates of KCC2-mediated Cl– extrusion and resistance to Cl– loading (n=8, p<0.0001). Conclusions: We have shown that pharmacological WNK inhibition enhances GABAergic inhibition in vitro and that these effects coincide with a significant decrease in KCC2-T1007 and SPAK kinase phosphorylation. We have also shown that neurons pre-treated with WNK463 are better equipped to combat robust Cl– loading, and can maintain inhibitory GABAergic transmission under such challenges. These findings provide solid evidence for the WNK-SPAK pathway as a novel target to enhance KCC2 activity and restore deficits in GABAergic inhibition.ReferencesMoore YE, Deeb TZ, Chadchankar H, Brandon NJ, Moss SJ (2018) Potentiating KCC2 activity is sufficient to limit the onset and severity of seizures. Proc Natl Acad Sci USA 115:10166-10171.Yamada K, Park HM, Rigel DF, DiPetrillo K, et al. (2016) Small-molecule WNK inhibition regulates cardiovascular and renal function. Nat Chem Biol 12:896-898. Funding: T32 Training Grant