Gephyrin Interacts with the K-Cl Cotransporter KCC2 to Regulate Its Surface Expression and Function in Cortical Neurons

Abstract

The K 1 -Clcotransporter KCC2, encoded by the Slc12a5 gene, is a neuron-specific chloride extruder that tunes the strength and polarity of GABA A receptor-mediated transmission. In addition to its canonical ion transport function, KCC2 also regulates spinogenesis and excitatory synaptic function through interaction with a variety of molecular partners. KCC2 is enriched in the vicinity of both glutamatergic and GABAergic synapses, the activity of which in turn regulates its membrane stability and function. KCC2 interaction with the submembrane actin cytoskeleton via 4.1N is known to control its anchoring near glutamatergic synapses on dendritic spines. However, the molecular determinants of KCC2 clustering near GABAergic synapses remain unknown. Here, we used proteomics to identify novel KCC2 interacting proteins in the adult rat neocortex. We identified both known and novel candidate KCC2 partners, including some involved in neuronal development and synaptic transmission. These include gephyrin, the main scaffolding molecule at GABAergic synapses. Gephyrin interaction with endogenous KCC2 was confirmed by immunoprecipitation from rat neocortical extracts. We showed that gephyrin stabilizes plasmalemmal KCC2 and promotes its clustering in hippocampal neurons, mostly but not exclusively near GABAergic synapses, thereby controlling KCC2-mediated chloride extrusion. This study identifies gephyrin as a novel KCC2 anchoring molecule that regulates its membrane expression and function in cortical neurons.

Loss of KCC2 in GABAergic Neurons Causes Seizures and an Imbalance of Cortical Interneurons

“…Premature overexpression of KCC2 disrupted brain development through a transport-independent interaction with the cytoskeleton-associated protein 4.1N ( Horn et al, 2010 ), while loss of KCC2 leads to excessive perinatal apoptosis ( Mavrovic et al, 2020 ), and KCC2 overexpression and pharmacological block lead to apoptosis in mature neurons ( Kontou et al, 2021 ). Studies of protein/protein interactions and work using transporter-deficient KCC2 mutants have revealed a previously unsuspected array of activity through interactions through membrane and cytoskeletal proteins ( Llano et al, 2020 ), such as kainate receptors ( Mahadevan et al, 2014 ), β-Pix ( Llano et al, 2015 ), KCNK9 channels ( Goutierre et al, 2019 ), gephyrin ( Al Awabdh et al, 2021 ), GABA B receptors ( Wright et al, 2017 ), and Neto2 ( Ivakine et al, 2013 ). Some of these interactions have effects independent of GABAergic signaling, such as increasing neuronal excitability through downregulating KCNK9 expression ( Goutierre et al, 2019 ).…”

“…However, KCC2 may still be involved in formation and plasticity of glutamatergic synapses in INs, which a follow-up study might investigate. On the other hand, GABA has been shown to promote neurite arborization and synaptogenesis of GABAergic synapses in multiple neuronal types ( Gascon et al, 2006 ; Ge et al, 2006 ; Bouzigues et al, 2007 ; Cancedda et al, 2007 ; Chattopadhyaya et al, 2007 ; Oh et al, 2016 ; Brady et al, 2018 ) and recently, an interaction between KCC2 and GABAergic synapse scaffolding protein gephyrin has been demonstrated ( Al Awabdh et al, 2021 ). Nonetheless, we found unremarkable expression of synaptic proteins and sIPSCs in layer 5 pyramidal neurons, suggesting normal development of GABAergic synapses in Dlx5 KCC2 cKO cortex.…”

The Yin and Yang of GABAergic and Glutamatergic Synaptic Plasticity: Opposites in Balance by Crosstalking Mechanisms

“…Gephyrin scaffolding interactions with non-receptor proteins provides an additional mechanism of synaptic regulation. For example, gephyrin directly interacts with and regulates the surface expression of the neuronal chloride extruder K + -Cl – cotransporter KCC2 ( Al Awabdh et al, 2022 ). KCC2 is expressed near both inhibitory and excitatory synapses ( Gulyás et al, 2001 ; Chamma et al, 2013 ) and is critical for generating the hyperpolarizing chloride gradient that allows for GABA A R inhibition in mature neurons.…”

Glioma‑neuronal interactions in tumor progression: Mechanism, therapeutic strategies and perspectives (Review)

“…This may result from a low expression of KCC2 and a high expression of NKCC1 ( 85 ). Another study demonstrated that KCC2 expression was downregulated by glioma via increasing the intracellular Zn 2+ concentration in neurons, eliciting GABA-dependent depolarization of co-cultured neurons ( 86 ). These data implied that glioma may contribute towards epilepsy by influencing both glutamatergic and GABAergic signaling in neurons.…”