Lim Wee Meng, Chin Eunice W M, Tang Bor Luen, Chen Tingting, Goh Eyleen L K
Neuroscience Academic Clinical Programme, Duke-NUS Medical School, Singapore, Singapore.
NUS Graduate School for Integrative Sciences and Engineering, Singapore, Singapore.
Front Mol Neurosci. 2021 Nov 10;14:762142. doi: 10.3389/fnmol.2021.762142. eCollection 2021.
The activation of chloride (Cl)permeable gamma (γ)-aminobutyric acid type A(GABA) 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 in mature neurons (DIV13-15) but had no effect on immature neurons (DIV6-8). This hyperpolarized 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 observed in WNK3-deficient neurons can be reversed by the KCC2 inhibitor, VU024055, thus indicating that WNK3 acts through KCC2 to influence . 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.
氯离子（Cl）通透的γ-氨基丁酸A型（GABA）受体的激活在成熟神经元中诱导突触抑制，而在未成熟神经元中诱导兴奋。这种由GABA极性控制的发育“开关”功能取决于出生后由阳离子-氯离子共转运体（CCC）成员KCC2介导的神经元内氯离子浓度的降低。丝氨酸-苏氨酸激酶WNK3（无赖氨酸[K]）是所有CCC的有效调节因子，在神经元中表达。在这里，我们表征了WNK3的功能及其在培养的胚胎第18天（E18）海马神经元GABA能信号传导中的作用。我们观察到随着神经元成熟，WNK3表达下降。敲低WNK3在成熟神经元（DIV13 - 15）中显著超极化，但对未成熟神经元（DIV6 - 8）没有影响。WNK3缺陷神经元中的这种超极化不是由于NKCC1和KCC2的总表达，其保持不变。然而，膜上磷酸化KCC2减少，表明KCC2氯化物输出活性增加。此外，在WNK3缺陷神经元中观察到的超极化可被KCC2抑制剂VU024055逆转，因此表明WNK3通过KCC2起作用来影响……值得注意的是，敲低WNK3导致成熟而非未成熟神经元的形态变化。WNK3缺陷成熟神经元的电生理特征显示电容降低，但内在兴奋性和突触兴奋性增加。因此，我们的研究表明WNK3维持神经元中的“成年”GABA能抑制基调，并在神经元的形态发育和兴奋性中起作用。
