Neto2 is a KCC2 interacting protein required for neuronal Cl ⁻ regulation in hippocampal neurons

Abstract

KCC2 is a neuron-specific K + -Cl − cotransporter that is essential for Cl − homeostasis and fast inhibitory synaptic transmission in the mature CNS. Despite the critical role of KCC2 in neurons, the mechanisms regulating its function are not understood. Here, we show that KCC2 is critically regulated by the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2). Neto2 is required to maintain the normal abundance of KCC2 and specifically associates with the active oligomeric form of the transporter. Loss of the Neto2:KCC2 interaction reduced KCC2-mediated Cl − extrusion, resulting in decreased synaptic inhibition in hippocampal neurons. that makes fast GABA A Rmediated synaptic inhibition possible is maintained by the neuronspecific K + -Cl − cotransporter KCC2 (4), a member of the cationchloride cotransporter SLC12 gene family (5). KCC2 is essential for survival, as KCC2 knockout mice die immediately at birth due to respiratory failure (6). In the adult nervous system, decreased KCC2 expression correlates with neuropathic pain, spasticity following spinal cord injury, and epileptic seizures (5, 7-11).Based on the critical importance of KCC2 in the brain, an understanding of the mechanisms that promote and maintain KCC2 expression and efficacy is essential for designing therapeutic strategies to treat neurological disorders characterized by KCC2 dysfunction and the subsequent loss of inhibitory synaptic transmission. A major limitation in the development of these strategies is a lack of understanding regarding the cellular mechanisms regulating KCC2. In particular, KCC2 interacting proteins required for KCC2 transport and function in the mature CNS have not been identified, which represents a large gap in our fundamental knowledge regarding neuronal Cl − regulation and inhibitory synaptic transmission.Here, we identify neuropilin and tolloid like-2 (Neto2) as a KCC2 interacting protein in vivo and demonstrate that this interaction is required for normal neuronal Cl − homeostasis. Neto2 is a complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing transmembrane protein abundantly expressed in neurons (12), which is important for proper neurological function (13). CUB domains are evolutionarily conserved protein domains (14) that participate in protein-protein interactions (15, 16). In the present study, we performed an unbiased proteomic screen and discovered that Neto2 interacts with KCC2. We elucidated the role of this association and characterized its importance in the normal neurophysiological function of KCC2. Because Neto proteins were previously identified as auxiliary subunits of ionotropic glutamate receptors, including kainate and NMDA receptors (17-21), this study further extends the role of the Neto proteins to inhibitory synapses. ResultsNeto2 and KCC2 Interact in Vivo. Recent reports have demonstrated that Neto2 and its homologous protein Neto1 have important functions at excitatory synapses. Neto1 interacts with both the N-methyl D-aspartate receptor (NMDAR) and kainate receptor comp...

Regulation of neuronal chloride homeostasis by neuromodulators

“…2004) and other members of the excitatory synapse (Ivakine et al . 2013; Mahadevan et al . 2014) are physically coupled with KCC2.…”

Exciting Times: New Advances Towards Understanding the Regulation and Roles of Kainate Receptors

“…KCC2 is crucial because it establishes the electrochemical chloride gradient for postsynaptic inhibition through GABA A Rs [121,122] and its dysregulation is implicated in autism spectrum disorder (ASD; [123]) and epilepsy [124]. KCC2 interacts with both Neto2 [125] and GluK2 [126], which increase the total abundance and enhance surface expression of KCC2 [14]. KCC2 and GluK2 form macromolecular assemblies that traffic together, and this complex regulates intraneuronal chloride homeostasis to support GABA A R-mediated transmission [126].…”

A Novel Regulatory Locus of Phosphorylation in the C Terminus of the Potassium Chloride Cotransporter KCC2 That Interferes with N-Ethylmaleimide or Staurosporine-mediated Activation*♦

“…On the cellular level, location in membrane rafts was shown to modify KCC2 transport activity (23,24). On the molecular level, interaction partners such as the ATPase subunit ␣2 (25), CIP1 (26), Neto2 (27), and several protein kinases, including brain-specific creatine kinase (28), SPAK (29), OSR1 (30), and WNKs (31) were identified. The regulatory role of phosphorylation is in line with previous pharmacological studies.…”