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Key Event Title
Down Regulation, K-Cl co-transporter 2 (KCC2)
Key Event Components
Key Event Overview
AOPs Including This Key Event
Key Event Description
Biological state: K-Cl co-transporter 2 (KCC2) is a neuron-specific glucoprotein detectable mainly within the central nervous system and it is encoded by the SLC12 genes family. It is an active electro-neutral transporter that transfers K- and Cl- (1:1) through the neuronal membrane, the direction of which is dependent on the inter-membrane ion concentration (Payne, 1997). When it is close to the ionic equilibrium even slight changes in the concentration of any of the ions could lead to subsequent change of the flux direction, a property which is broadly used to characterize and measure KCC2 activity (Medina et al., 2014). In total, there are four genes encoding highly homologous K-Cl co-transporters (Payne et al., 1996), but only KCC2 has been found to exhibit basal transport activity in the CNS and to have a developmentally regulated profile (Song et al., 2002). This developmental increase of KCC2 reaches a pick during the second postnatal week in rodents and it has been suggested to be regulated by the neuronal activity itself (Gangulu et al., 2001), plausibly through the expression and function of different trophic factors (Kelsch et al., 2001; Ludwig et al., 2011 a, b). However, this hypothesis is still under investigation due to the controversial studies supporting that chronically blocked GABAA receptor did not negatively affected KCC2 up-regulation nor the neuronal hyperpolarizing activity (Ludwig et al., 2003; Titz et al., 2003).
Biological compartments: KCC2 has been suggested to be exclusively expressed in the central nervous system (CNS) (Blaesse et al., 2009), but more recent evidence indicate that KCC2 expression and function is present in other tissues as well, such as human fetal lens epithelial cells, chicken cardiomyocytes and cancer cells (Lauf et al., 2012; Antrobus et al., 2012; Wei et al., 2011). KCC2 is located in the plasma membrane of somata and dendrites in various brain regions, including the cortical neurons (Szabadics et al., 2006). This observation supports the notion that this co-transporter is implicated in the formation and function of GABAergic synapses, as it is well established that there is a preferential localization of GABAergic synapses to somata and dendritic shafts.
General role in biology: KCC2 serves as an active Cl- efflux mechanism responsible for the establishment of a Cl- gradient essential for the functional plasticity of GABAA and glycine receptors (Rivera et al., 1999). Specifically, lowering intracellular Cl− concentration enhances inhibition of GABA neurotransmission, whereas raising Cl− facilitates spontaneous neuronal activity (Farrant et al., 2007). The actual role of KCC2 is the maintenance of the Cl- reversal potential (ECl) less than the membrane potential (Em) which is a critical milestone for the regulation of postsynaptic inhibition by the GABAA and glycine receptors (Williams et al., 1999). Several studies have shown that impaired KCC2 activity and the subsequent raise of intracellular chloride concentration are evident in multiple neurological disorders, such as temporal lobe epilepsies, focal cortical dysplasia, ischemia and spasticity following spinal cord injury (Blaesse et al., 2009; Barmashenko et al., 2011; Talos et al., 2012; Jaenisch et al., 2010; Boulenguez et al., 2010). The common characteristic of these disorders is the dysregulation of GABAergic inhibition, suggesting that the plausible reason for this outcome is the depolarizing action of neurons, triggered by the raised Cl- levels which in turn lead to the enhanced neuronal network activity (Ben-Ari et al., 2012). However, no mutations or other structural impairments of KCC2 have been associated with a human disease up to date. KCC2 exhibits low levels of expression in hippocampal, cortical and retinal neurons in rat neonates, but shows a steady increase in expression until the end of the second postnatal week, which makes it a good candidate for the developmental decrease in neuronal Cl- concentration (Rivera et al., 1999) and the subsequent regulation of the GABAAR responses during the early neurodevelopmental stages and in many pathological conditions (Fiumelli et al., 2005). Thus, it has been also associated with the developmental switch of the GABAA neurotransmission in the brain (Rivera et al., 1999). The GABAergic action in the rat neonate is depolarizing, an effect which is rapidly reversed approximately at the end of the first postnatal week. It is now well documented that KCC2 is the key regulator of Cl- homeostasis during the early neurodevelopmental stages, ensuring the strictly programmed gradual decrease of intracellular Cl- , which in turn defines the above mentioned time point that the qualitative shift in GABAergic responses occurs (Rivera et al., 2005). KCC2 expression during late development is also important for synapse maturation, as it has been shown that in KCC2 knockout mice the neurons develop reduced number of synapses and elongated dendritic spines (Li et al., 2007). However, the physiological role of KCC2 is not well characterized, partly because there are no KCC2-specific pharmacological tools available (Payne et al., 2003).
How It Is Measured or Detected
No OECD methods are available to measure KCC2 protein and mRNA levels. KCC2 protein levels can be measured by commercial available antibody sandwich ELISA kits, Western blotting, immunohistochemistry and immunofluorescence. KCC2 primers for different exons are available to determine mRNA levels by RT-PCR. The human SLC12A5 gene encompasses 24 coding exons, while the exons that are unique for KCC2 are 21 and 22 (Song et al., 2002).
Domain of Applicability
KCC2 co-transporter is present in the mouse and rat brain (Payne et al., 1996), as well as in human brain (Song et al., 2002). In both cases KCC2 exhibit significant constitutive K+–Cl- co-transport activity under isotonic conditions (Payne et al., 1997; Song et al., 2002). It has been shown that KCC2 null mice die at birth due to severe respiratory failure (Hubner et al., 2001), while targeted deletion of the KCC2 gene in mice results in seizure induction and neonatal lethality (Delpire and Lovinger, 2000). More recent studies revealed also KCC2 expression and function in chicken cardiomyocytes (Antrobus et al., 2012). C. elegans is the lowest organism from which a K-Cl co-transporter, or any member of the cation-chloride cotransporter family, has been characterized and it has been shown that it exhibits a higher level of conservation with the KCC2 than with all the other KCCs (co-transporters) in humans (Tanis et al., 2009) Kazachoc (kcc) gene in Drosophila is homologous to the human gene and it has been shown that it encodes a K/Cl co-transporter closely related to the mammalian KCC2 (Filippov et al., 2003). In both cases the KCC2-like genes in worms (Tanis et al., 2009; Bellemer et al., 2011) and flies (Hekmat-Scafe et al., 2006; Hekmat-Scafe et al., 2010) produces severe changes of the neuronal network properties leading to appearance of seizure activity, increased neuron hyperexcitability and modified formation of synaptic connections.
Antrobus SP, Lytle C, Payne JA. (2012). K+/Cl- co-transporter 2 KCC2 in chicken cardiomyocytes. Am J Physiol Cell Physiol. 303:C1180–C1191.
Barmashenko G, Hefft S, Aertsen A, Kirschstein T, Köhling R. (2011). Positive shifts of the GABAA receptor reversal potential due to altered chloride homeostasis is widespread after status epilepticus Epilepsia 52:1570–1578.
Bellemer A, Hirata T, Romero MF, Koelle MR. (2011). Two types of chloride transporters are required for GABA(A)receptor-mediated inhibition in C.elegans. EMBO J 30:1852–1863.
Ben-Ari Y, Khalilov I, Kahle KT, Cherubini E. (2012). The GABA excitatory/inhibitory shift in brain maturation and neurological disorders. Neuroscientist 18:467–486.
Blaesse P, Airaksinen MS, Rivera C, Kaila K. (2009). Cation chloride co-transporters and neuronal function. Neuron 61:820–838.
Boulenguez P, Liabeuf S, Bos R, Bras H, Jean-xavier C, Brocard C, Stil A, Darbon P, Cattaert D, Delpire E, Marsala M, Vinay L. (2010). Down-regulation of the potassium-chloride co-transporter KCC2 contributes to spasticity after spinal cord injury. Nature Med 16:302–307.
Delpire E, Lovinger DM. (2000). Frequent seizures and early lethality associated with disruption of the mouse KCC2 gene. J. Neurosci., 26:1148.
Farrant M, Kaila K. (2007). The cellular, molecular and ionic basis of GABAA receptor signalling. Prog Brain Res 160:59–87.
Filippov V, Aimanova K, Gill SS. (2003). Expression of an Aedes aegypti cation-chloride cotransporter and its Drosophila homologues. Insect Mol Biol. 12:319-331.
Fiumelli H, Cancedda L, Poo M. (2005). Modulation of GABAergic transmission by activity via postsynaptic Ca-dependent regulation of KCC2 function. Neuron 48:773–786.
Ganguly K, Schinder AF, Wong ST, Poo M. (2001). GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell 105:521–532.
Hekmat-Scafe DS, Lundy MY, Ranga R, Tanouye MA. (2006). Mutations in the K+/Cl- co-transporter gene kazachoc (kcc) increase seizure susceptibility in Drosophila. J Neurosci 26:8943–8954.
Hekmat-Scafe DS, Mercado A, Fajilan AA, Lee AW, Hsu R, Mount DB, Tanouye MA. (2010). Seizure sensitivity is ameliorated by targeted expression of K+/Cl- co-transporter function in the mushroom body of the Drosophila brain. Genetics 184:171–183.
Hubner CA, Stein V, Hermans-Borgmeyer I, Meyer T, Ballanyi K, Jentsch TJ. (2001). Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition. Neuron 30:515–524.
Jaenisch N, Witte OW, Frahm C. (2010). Downregulation of potassium- chloride co-transporter KCC2 after transient focal cerebral ischemia. Stroke 41:e151–e159.
Kelsch W, Hormuzdi S, Straube E, Lewen A, Monyer H, Misgeld U. (2001). Insulin-like growth factor 1 and a cytosolic tyrosine kinase activate chloride outward transport during maturation of hippocampal neurons. J Neurosci 21:8339–8347.
Lauf PK, DiFulvio M, Srivastava V, Sharma N, Adragna NC. (2012). KCC2a expression in a human fetal lens epithelial cell line. Cell Physiol Biochem 29:303–312.
Li H, Khirug S, Cai C, Ludwig A, Blaesse P, Kolikova J, Afzalov R, Coleman SK, Lauri S, Airaksinen MS, Keinänen K, Khiroug L, Saarma M, Kaila K, Rivera C. (2007). KCC2 interacts with the dendritic cytoskeleton to promote spine development. Neuron 56:1019–1033.
Ludwig A, Uvarov P, Soni S, Thomas-Crusells J, Airaksinen MS, Rivera C. (2011b). Early growth response 4 mediates BDNF induction of potassium chloride co-transporter 2 transcription. J Neurosci 31:644-649.
Ludwig A, Uvarov P, Pellegrino C, Thomas-Crusells J, Schuchmann S, Saarma M, Airaksinen MS, Rivera C. (2011a). Neurturin evokes MAPK dependent up-regulation of Egr4 and KCC2 in developing neurons. Neural Plast 1-8.
Ludwig A, Li H, Saarma M, Kaila K, Rivera C. (2003). Developmental up-regulation of KCC2 in the absence of GABAergic and glutamatergic transmission. Eur J Neurosci 18:3199–3206.
Medina I, Friedel P, Rivera C, Kahle KT, Kourdougli N, Uvarov P, Pellegrino C. (2014) Current view on the functional regulation of the neuronal K+/Cl− cotransporter KCC2. Front Cell Neurosci 8: 27.
Payne JA, Stevenson TJ, Donaldson LF. (1996). Molecular characterization of a putative K-Cl co-transporter in rat brain. A neuronal-specific isoform. J Biol Chem 271:16245-16252.
Payne JA. (1997). Functional characterization of the neuronal-specific K-Cl co-transporter: implications for [K+] coregulation. Am J Phys 273:C1516–C1525.
Payne JA, Rivera C, Voipio J, Kaila K. (2003). Cation-chloride co-transporters in neuronal communication, development and trauma. Trends Neurosci. 26:199-206.
Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, and Kaila K. (1999). The K/Cl co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397:251–255.
Rivera C, Voipio J, Kaila K. (2005). Two developmental switches in GABAergic signalling: the K+-Cl- cotransporter KCC2 and carbonic anhydrase CAVII. J Physiol 562:27-36.
Song L, Mercado A, Vázquez N, Xie Q, Desai R, George AL Jr, Gamba G, Mount DB. (2002). Molecular, functional, and genomic characterization of human KCC2, the neuronal K-Cl cotransporter. Brain Res Mol Brain Res. 103:91-105.
Szabadics J, Varga C, Molnar G, Olah S, Barzo P, Tamas G. (2006). Excitatory effect of GABAergic axo-axonic cells in cortical microcircuits. Science 311:233–235.
Talos DM, Sun H, Kosaras B, Joseph A, Folkerth RD, Poduri A, Madsen JR, Black PM, Jensen FE. (2012). Altered inhibition in tuberous sclerosis and type IIb cortical dysplasia. Ann Neurol 71:539–551.
Tanis JE, Bellemer A, Moresco JJ, Forbush B, Koelle MR. (2009). The potassium chloride co-transporter KCC-2 coordinates development of inhibitory neurotransmission and synapse structure in Caenorhabditis elegans. J Neurosci 29:9943–9954.
Titz S, Hans M, Kelsch W, Lewen A, Swandulla D, Misgeld U.(2003). Hyperpolarizing inhibition develops without trophic support by GABA in cultured rat midbrain neurons. J Physiol 550:719–730.
Wei WC, Akerman CJ, Newey SE, Pan J, Clinch NWV, Jacob Y, Shen MR, Wilkins RJ, Ellory JC. (2011). The potassium-chloride co-transporter 2 promotes cervical cancer cell migration and invasion by anion transport-independent mechanism. J Physiol 589:5349–5359.
Williams JR, Sharp JW, Kumari VG, Wilson M, Payne JA. (1999). The neuron-specific K–Cl cotransporter, KCC2. Antibody development and initial characterization of the protein. J Biol Chem 274:12656–12664.