WNK3 Maintains the GABAergic Inhibitory Tone, Synaptic Excitation and Neuronal Excitability via Regulation of KCC2 Cotransporter in Mature Neurons.

Wee Meng Lim,Tingting Chen,Eunice W M Chin,Eyleen L K Goh,Bor Luen Tang

doi:10.3389/fnmol.2021.762142

Abstract

The activation of chloride (Cl−)permeable gamma (γ)-aminobutyric acid type A(GABAA) receptors induces synaptic inhibition in mature and excitation in immature neurons. This developmental “switch” in GABA function controlled by its polarity depends on the postnatal decrease in intraneuronal Cl− concentration mediated by KCC2, a member of cation-chloride cotransporters (CCCs). The serine-threonine kinase WNK3 (With No Lysine [K]), is a potent regulator of all CCCs and is expressed in neurons. Here, we characterized the functions of WNK3 and its role in GABAergic signaling in cultured embryonic day 18 (E18) hippocampal neurons. We observed a decrease in WNK3 expression as neurons mature. Knocking down of WNK3 significantly hyperpolarized EGABA in mature neurons (DIV13–15) but had no effect on immature neurons (DIV6–8). This hyperpolarized EGABA in WNK3-deficient neurons was not due to the total expression of NKCC1 and KCC2, that remained unchanged. However, there was a reduction in phosphorylated KCC2 at the membrane, suggesting an increase in KCC2 chloride export activity. Furthermore, hyperpolarized EGABA observed in WNK3-deficient neurons can be reversed by the KCC2 inhibitor, VU024055, thus indicating that WNK3 acts through KCC2 to influence EGABA. Notably, WNK3 knockdown resulted in morphological changes in mature but not immature neurons. Electrophysiological characterization of WNK3-deficient mature neurons revealed reduced capacitances but increased intrinsic excitability and synaptic excitation. Hence, our study demonstrates that WNK3 maintains the “adult” GABAergic inhibitory tone in neurons and plays a role in the morphological development of neurons and excitability.

Highlights

GABA is a major inhibitory neurotransmitter of the mature central nervous system (CNS) that plays a crucial role in controlling neuronal excitability in the brain (Ben-Ari et al, 2012)
WNK3 and K+-Cl- cotransporter 2 (KCC2) are expressed in most CNS regions albeit at varying levels in the adult mice (2 months), except that WNK3 is absent in the spinal cord (Figure 1A)
We used cultured hippocampal neurons to determine if the developmental expression of WNK3 in brain tissues can be recapitulated in these neurons in vitro (Figure 1C)

Summary

Introduction

GABA is a major inhibitory neurotransmitter of the mature central nervous system (CNS) that plays a crucial role in controlling neuronal excitability in the brain (Ben-Ari et al, 2012). The direction of GABA currents through ionotropic GABA receptors reverses from depolarizing to hyperpolarizing as the brain develops (Ben-Ari, 2014; Peerboom and Wierenga, 2021) This developmental ‘‘switch’’ in GABA function is controlled by its polarity of transmission, which can be reflected by the equilibrium potential of GABA (EGABA; Ouardouz and Sastry, 2005). In the adult brain, [Cl-]i is maintained at low levels, resulting in a hyperpolarizing effect of GABAergic signaling In this way, the EGABA in neonatal neurons is set at a relatively depolarized level, but subsequently shows a negative shift in the adult brain neurons (Yang et al, 2010). Decreases in input resistance and membrane time constant when GABAA receptors open, act by decreasing the temporal and spatial summation of excitatory inputs, regardless of the direction of Cl- flux (Blaesse et al, 2009)

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Conclusion