Abstract
The K+–Cl− co-transporter KCC2 (SLC12A5) tunes the efficacy of GABAA receptor-mediated transmission by regulating the intraneuronal chloride concentration [Cl−]i. KCC2 undergoes activity-dependent regulation in both physiological and pathological conditions. The regulation of KCC2 by synaptic excitation is well documented; however, whether the transporter is regulated by synaptic inhibition is unknown. Here we report a mechanism of KCC2 regulation by GABAA receptor (GABAAR)-mediated transmission in mature hippocampal neurons. Enhancing GABAAR-mediated inhibition confines KCC2 to the plasma membrane, while antagonizing inhibition reduces KCC2 surface expression by increasing the lateral diffusion and endocytosis of the transporter. This mechanism utilizes Cl− as an intracellular secondary messenger and is dependent on phosphorylation of KCC2 at threonines 906 and 1007 by the Cl−-sensing kinase WNK1. We propose this mechanism contributes to the homeostasis of synaptic inhibition by rapidly adjusting neuronal [Cl−]i to GABAAR activity.
Highlights
The K+–Cl− co-transporter KCC2 (SLC12A5) tunes the efficacy of GABAA receptor-mediated transmission by regulating the intraneuronal chloride concentration [Cl−]i
We asked whether pharmacological modulation of GABAergic inhibition impacts the membrane dynamics of KCC2 using quantum dot-based single-particle tracking (QD-SPT) technique in cultures of hippocampal neurons (DIV 21–24)
Whereas KCC2 is known to be rapidly downregulated by enhanced neuronal activity and glutamatergic neurotransmission in mature neurons[10, 12, 14], only one study so far had tested the effect of GABA signaling on KCC2 post-translational regulation
Summary
The K+–Cl− co-transporter KCC2 (SLC12A5) tunes the efficacy of GABAA receptor-mediated transmission by regulating the intraneuronal chloride concentration [Cl−]i. Enhanced glutamatergic synaptic activity increases KCC2 membrane diffusion, leading to transporter escape from clusters located near excitatory and inhibitory synapses and endocytosis from the plasma membrane[10] This regulation involves the Ca2+-dependent, PP1mediated, dephosphorylation of KCC2 at serine (S) 940 and the Ca2+ activated, calpain-dependent cleavage of the KCC2 carboxyterminal domain (CTD)[11, 12]. Blocking GABAAR-dependent inhibition rapidly increases KCC2 membrane dynamics, reducing its membrane clustering, stability, and activity of the transporter We show that this mechanism is mediated by chloride ions via the Cl−-sensitive serine/threonine WNK1 kinase-dependent phosphorylation of KCC2 at threonines (T) 906 and 1007, two key regulatory sites of KCC2 activity during brain development[17, 18]. We speculate that antagonizing WNK1 kinase activity may be a promising strategy to restore inhibition by restoring Cl− homeostasis in diseases like epilepsy, schizophrenia, and neuropathic pain
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