WNK3 kinase maintains neuronal excitability by reducing inwardly rectifying K+ conductance in layer V pyramidal neurons of mouse medial prefrontal cortex

Abstract

The with-no-lysine (WNK) family of serine-threonine kinases and its downstream kinases of STE20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) may regulate intracellular Cl− homeostasis through phosphorylation of cation-Cl− co-transporters. WNK3 is expressed in fetal and postnatal brains, and its expression level increases during development. Its roles in neurons, however, remain uncertain. Using WNK3 knockout (KO) mice, we investigated the role of WNK3 in the regulation of the intracellular Cl− concentration ([Cl−]i) and the excitability of layer V pyramidal neurons in the medial prefrontal cortex (mPFC). Gramicidin-perforated patch-clamp recordings in neurons from acute slice preparation at the postnatal day 21 indicated a significantly depolarized reversal potential for GABAA receptor-mediated currents by 6 mV, corresponding to the higher [Cl−]i level by ~4 mM in KO mice than in wild-type littermates. However, phosphorylation levels of SPAK and OSR1 and those of neuronal Na+-K+-2Cl− co-transporter NKCC1 and K+-Cl− co-transporter KCC2 did not significantly differ between KO and wild-type mice. Meanwhile, the resting membrane potential of neurons was more hyperpolarized by 7 mV, and the minimum stimulus current necessary for firing induction was increased in KO mice. These were due to an increased inwardly rectifying K+ (IRK) conductance, mediated by classical inwardly rectifying (Kir) channels, in KO neurons. The introduction of an active form of WNK3 into the recording neurons reversed these changes. The potential role of KCC2 function in the observed changes of KO neurons was investigated by applying a selective KCC2 activator, CLP290. This reversed the enhanced IRK conductance in KO neurons, indicating that both WNK3 and KCC2 are intimately linked in the regulation of resting K+ conductance. Evaluation of synaptic properties revealed that the frequency of miniature excitatory postsynaptic currents (mEPSCs) was reduced, whereas that of inhibitory currents (mIPSCs) was slightly increased in KO neurons. Together, the impact of these developmental changes on the membrane and synaptic properties was manifested as behavioral deficits in pre-pulse inhibition, a measure of sensorimotor gating involving multiple brain regions including the mPFC, in KO mice. Thus, the basal function of WNK3 would be the maintenance and/or development of both intrinsic and synaptic excitabilities.

Delaying the GABA shift indirectly affects membrane properties in the developing hippocampus

“…It remains unclear how blocking KCC2 from DIV2 to DIV8 can alter membrane properties of neurons two weeks later. Our study adds to an increasing number of studies that demonstrate that intracellular chloride levels can modify ion channels, and therefore membrane excitability, in often unpredictable ways (Huang et al, 2012; Seja et al, 2012; Goutierre et al, 2019; Sinha et al, 2022). Most notably, it was shown that membrane levels of specific potassium channels are regulated via KCC2 (Goutierre et al, 2019) and the chloride-dependent kinase WNK3 (Sinha et al, 2022), and it will be important to further examine the role of various chloride channels in membrane excitability (Jentsch, 2016; Jentsch and Pusch, 2018; Akita and Fukuda, 2020).…”

“…Our study adds to an increasing number of studies that demonstrate that intracellular chloride levels can modify ion channels, and therefore membrane excitability, in often unpredictable ways (Huang et al, 2012; Seja et al, 2012; Goutierre et al, 2019; Sinha et al, 2022). Most notably, it was shown that membrane levels of specific potassium channels are regulated via KCC2 (Goutierre et al, 2019) and the chloride-dependent kinase WNK3 (Sinha et al, 2022), and it will be important to further examine the role of various chloride channels in membrane excitability (Jentsch, 2016; Jentsch and Pusch, 2018; Akita and Fukuda, 2020). Interestingly, effects seem to strongly depend on cell type (Seja et al, 2012) and timing of the chloride manipulation (Lim et al, 2021; Sinha et al, 2022).…”

“…Most notably, it was shown that membrane levels of specific potassium channels are regulated via KCC2 (Goutierre et al, 2019) and the chloride-dependent kinase WNK3 (Sinha et al, 2022), and it will be important to further examine the role of various chloride channels in membrane excitability (Jentsch, 2016; Jentsch and Pusch, 2018; Akita and Fukuda, 2020). Interestingly, effects seem to strongly depend on cell type (Seja et al, 2012) and timing of the chloride manipulation (Lim et al, 2021; Sinha et al, 2022). The changes in excitability that we observed at DIV21 may therefore reflect a complex sequence of subtle adaptations of ion channel distribution that is specific per cell type.…”

Treatment with furosemide indirectly increases inhibitory transmission in the developing hippocampus

“…However, the mechanism seems different as mIPSC frequency and synapse numbers were not affected in VU-treated slices (Peerboom et al, 2023). In interpreting our results and that of previous studies, it is hard to disentangle the versatile pharmacological profile of furosemide and the many roles for neuronal chloride in regulating cell volume (Jentsch and Pusch, 2018) and possibly cellular excitability (Huang et al, 2012; Seja et al, 2012; Jentsch and Pusch, 2018; Goutierre et al, 2019; Sinha et al, 2022; Peerboom et al, 2023). Our study further underscores the need for a beter understanding of the role of chloride as an intracellular messenger in developing neurons.…”

“…Our results suggest that one week treatment with furosemide indirectly affect ac�vity-induced swelling of neurons, but the mechanism remains unresolved. In interpre�ng our results and that of previous studies, it is hard to disentangle the versa�le pharmacological profile of furosemide and the many roles for neuronal chloride in regula�ng cell volume (Jentsch and Pusch, 2018) and possibly cellular excitability (Huang et al, 2012;Seja et al, 2012;Jentsch and Pusch, 2018;Gou�erre et al, 2019;Sinha et al, 2022;Peerboom et al, 2023).…”