Structural and functional roles of KCC2 in developing cortex

Cation chloride cotransporters (CCCs) are critical for controlling intracellular chloride homeostasis. The CCC family is composed of four isoforms of K-Cl cotransporters (KCC1-4), two isoforms of Na-K-2Cl cotransporters (NKCC1-2), one Na-Cl cotransporter (NCC) and two the structurally related proteins with unknown function, CCC8 also known as cation-chloride cotransporter interaction protein, CIP, and CCC9. KCC2 is a neuron-specific isoform, which plays a prominent role in controlling the intracellular Cl- concentration in neurons and is responsible for producing the negative shift of GABAA responses from depolarizing to hyperpolarizing during neuronal maturation. In the present studies we first used in situ hybridization to examine the developmental expression patterns of the cation-chloride cotransporters KCC1-4 and NKCC1. We found that they display complementary expression patterns during embryonic brain development. Most interestingly, KCC2 expression in the embryonic central nervous system strictly follows neuronal maturation. In vitro data obtained from primary and organotypic neuronal cultures support this finding and revealed a temporal correlation between the expression of KCC2 and synaptogenesis. We found that KCC2 is highly expressed in filopodia and mature spines as well as dendritic shaft and investigated the role of KCC2 in spine formation by analyzing KCC2-/- neurons in vitro. Our studies revealed that KCC2 is a key factor in the maturation of dendritic spines. Interestingly, the effect of KCC2 in spine formation is not due to Cl- transport activity, but mediated through the interaction between KCC2 C-terminal and intracellular protein associated with cytoskeleton. The interacting protein we found is protein 4.1N by immunoprecipitation. Our results indicate a structural role for KCC2 in the development of functional glutamatergic synapses and suggest KCC2 as a synchronizer for the functional development of glutamatergic and GABAergic synapses in neuronal network. Studies on the regulatory mechanisms of KCC2 expression during development and plasticity revealed that synaptic activity of both the glutamatergic and GABAergic system is not required for up-regulation of KCC2 during development, whereas in acute mature hippocampal slices which undergo continuous synchronous activity induced by the absence of Mg2+ solution, KCC2 mRNA and protein expression were down-regulated in CA1 pyramidal neurons subsequently leading to a reduced capacity for neuronal Cl- extrusion. This effect is mediated by endogenous BDNF-TrkB down-stream cascades involving both Shc/FRS-2 and PLCγ-CREB signaling. BDNF mediated changes in KCC2 expression indicate that KCC2 is significantly involved in the complex mechanisms of neuronal plasticity during development and pathophysiological conditions.

Cellular ionic concentrations have to be maintained for correct physiological functions of the cell. Ionic concentrations are regulated by membrane expressed channels and cotransporters. In this study, we focused on chloride regulatory mechanisms in central nervous system by studying the functional role of a cation-chloride cotransporter (CCC) protein family. The cation-chloride cotransporter family contains four isoforms of K-Cl cotransporters (KCC1-4), two isoforms of Na-K-2Cl cotransporters (NKCC1-2), one Na-Cl cotransporter (NCC) and two the structurally related proteins with unknown function, CCC8 also known as cation-chloride cotransporter interaction protein, CIP, and CCC9. We first analyzed the developmental expression pattern of the cation-chloride cotransporters KCC1-4 and NKCC1. We found that they display complementary expression patterns during embryonic brain development indicating chloride regulatory mechanisms are critically involved in neuronal development. Most interestingly, KCC2 expression in the embryonic central nervous system strictly follows neuronal maturation and there is a temporal correlation between the expression of KCC2 and synaptogenesis. Furthermore, we found that KCC2 is highly expressed in filopodia and mature spines as well as dendritic shaft. Thus, we investigated the role of KCC2 in spine formation by analyzing the phenotype of KCC2 knock out neurons. The study revealed that KCC2 is a key factor in the maturation of excitatory synapses. Mechanistic study demonstrated that a structural interaction between KCC2 C-terminal and cytoskeleton associated protein 41.N underlies the effect of KCC2 in excitatory synapse formation. These results suggest KCC2 acts as a molecular synchronizer in the functional maturation of excitatory and inhibitory synapses. During development, the expression of KCC2 is up-regulated. We demonstrated that the up-regulation of KCC2 do not require the synaptic activity of both the glutamatergic and GABAergic system. However, KCC2 mRNA and protein expression were down-regulated in CA1 pyramidal neurons in acute mature hippocampal slices which undergo continuous synchronous activity induced by the absence of Mg2+ solution. This effect is mediated by endogenous BDNF-TrkB down-stream cascades involving both Shc/FRS-2 and PLCγ-CREB signaling. In general, our results indicate that KCC2 is significantly involved in the complex mechanisms of developmental neuronal disorders such as autism and schizophrenia.