Chloride potassium symporter 5 explained

Potassium-chloride transporter member 5 (aka: KCC2 and SLC12A5) is a neuron-specific chloride potassium symporter responsible for establishing the chloride ion gradient in neurons through the maintenance of low intracellular chloride concentrations.^[1] It is a critical mediator of synaptic inhibition, cellular protection against excitotoxicity^{[2] [3]} and may also act as a modulator of neuroplasticity.^{[4] [5] [6] [7]} Potassium-chloride transporter member 5 is also known by the names: KCC2 (potassium chloride cotransporter 2) for its ionic substrates, and SLC12A5 for its genetic origin from the SLC12A5 gene in humans.^[1]

Animals with reduced expression of this transporter exhibit severe motor deficits, epileptiform activity, and spasticity.^[4] KCC2 knockout animals, in which KCC2 is completely absent, die postnatally due to respiratory failure.^[4]

Location

KCC2 is a neuron-specific membrane protein expressed throughout the central nervous system, including the hippocampus, hypothalamus, brainstem, and motoneurons of the ventral spinal cord.^[6]

At the subcellular level, KCC2 has been found in membranes of the somata and dendrites of neurons,^[8] with no evidence of expression on axons.^[4] KCC2 has also been shown to colocalize with GABA_A receptors, which serve as ligand-gated ion channels to allow chloride ion movement across the cell membrane. Under normal conditions, the opening of GABA_A receptors permits the hyperpolarizing influx of chloride ions to inhibit postsynaptic neurons from firing.^[3]

Counterintuitively, KCC2 has also been shown to colocalize at excitatory synapses.^[2] One suggested explanation for such colocalization is a potential protective role of KCC2 against excitotoxicity.^{[2] [3]} Ion influx due to the excitatory synaptic stimulation of ion channels in the neuronal membrane causes osmotic swelling of cells as water is drawn in alongside the ions. KCC2 may help to eliminate excess ions from the cell in order to re-establish osmotic homeostasis.

Structure

KCC2 is a member of the cation-chloride cotransporter (CCC) superfamily of proteins.^[9]

As with all CCC proteins, KCC2 is an integral membrane protein with 12 transmembrane domains and both N- and C-terminal cytoplasmic domains. The terminal cytoplasmic domains can be phosphorylated by kinases within the neuron for rapid regulation.

Two Isoforms: KCC2a, KCC2b

There are two isoforms of KCC2: KCC2a and KCC2b.^{[4] [10]} The two isoforms arise from alternative promoters on the SLC12A5 gene and differential splicing of the first mRNA exon.^{[4] [10]} The isoforms differ in their N-termini, with the KCC2a form constituting the larger of the two splice variants.^[11]

KCC2a levels remain relatively constant during pre- and postnatal development.^[11]

KCC2b, on the other hand, is scarcely present during prenatal development and is strongly upregulated during postnatal development. The upregulation of KCC2b expression is thought to be responsible for the “developmental shift” observed in mammals from depolarizing postsynaptic effects of inhibitory synapses in early neural networks to hyperpolarizing effects in mature neural networks.^[4]

KCC2b knockout mice can survive up to postnatal day 17 (P17) due to the presence of functional KCC2a alone, but they exhibit low body weight, motor deficits and generalized seizures.^[4] Complete KCC2 knockouts (both KCC2a and KCC2b absent) die after birth due to respiratory failure.^[4]

Oligomerization

Both KCC2 isoforms can form homomultimers, or heteromultimers with other K-Cl symporters on the cell membrane to maintain chloride homeostasis in neurons. Dimers, trimers, and tetramers involving KCC2 have been identified in brainstem neurons.^[12] Oligomerization may play an important role in transporter function and activation, as it has been observed that the oligomer to monomer ratio increases in correlation to the development of the chloride ion gradient in neurons.^[12]

Developmental changes in expression

KCC2 levels are low during mammalian embryonic development, when neural networks are still being established and neurons are highly plastic (changeable). During this stage, intracellular chloride ion concentrations are high due to low KCC2 expression and high levels of a transporter known as NKCC1 (Na⁺/K⁺ chloride cotransporter 1), which moves chloride ions into cells.^[13] Thus, during embryonic development, the chloride gradient is such that stimulation of GABA_A receptors and glycine receptors at inhibitory synapses causes chloride ions to flow out of cells, making the internal neuronal environment less negative (i.e. more depolarized) than it would be at rest. At this stage, GABA_A receptors and glycine receptors act as excitatory rather than inhibitory effectors on postsynaptic neurons, resulting in depolarization and hyperexcitability of neural networks.^{[4] [6] [7]}

During postnatal development, KCC2 levels are strongly upregulated while NKCC1 levels are down regulated.^[13] This change in expression correlates to a developmental shift of the chloride ion concentration within neurons from high to low intracellular concentration. Effectively, as the chloride ion concentration is reduced, the chloride gradient across the cellular membrane is reversed such that GABA_A receptor and glycine receptor stimulation causes chloride ion influx, making the internal neuronal environment more negative (i.e. more hyperpolarized) than it would be at rest. This is the developmental shift of inhibitory synapses from the excitatory postsynaptic responses of the early neural development phase to the inhibitory postsynaptic responses observed throughout maturity.

Function

Current literature suggests that KCC2 serves three primary roles within neurons:

1. Establishing the chloride ion gradient necessary for postsynaptic inhibition
2. Protecting neuronal networks against stimulation-induced excitotoxicity
3. Contributing to dendritic spine morphogenesis and glutamatergic synaptic function

Postsynaptic inhibition

KCC2 is a potassium (K⁺)/chloride (Cl⁻) symporter that maintains chloride homeostasis in neurons. The electrochemical chloride gradient established by KCC2 activity is crucial for classical postsynaptic inhibition through GABA_A receptors and glycine receptors in the central nervous system. KCC2 utilizes the potassium gradient generated by the Na⁺/K⁺ pump to drive chloride extrusion from neurons.^[4] In fact, any disruption of the neuronal K⁺ gradient would indirectly affect KCC2 activity.

Loss of KCC2 following neuronal damage (i.e. ischemia, spinal cord damage, physical trauma to the central nervous system) results in the loss of inhibitory regulation and the subsequent development of neuronal hyperexcitability, motor spasticity, and seizure-like activity^[6] as GABA_A receptors and glycine receptors revert from hyperpolarizing to depolarizing postsynaptic effects.

Cellular protection

High levels of stimulation and subsequent ionic influx through activated ion channels can result in cellular swelling as osmotically-obliged water is drawn into neurons along with ionic solutes. This phenomenon is known as excitotoxicity.^[2] KCC2 has been shown to be activated by cell-swelling, and may therefore play a role in eliminating excess ions following periods of high stimulation in order to maintain steady-state neuronal volume and prevent cells from bursting.^[2]

This role may also account for the fact that KCC2 has been known to colocalize near excitatory synapses, even though its primary role is to establish the chloride gradient for postsynaptic inhibition.^{[2] [3]}

Morphogenesis and function of glutamatergic synapses

In addition to controlling the efficacy of GABAergic synapses through chloride homeostasis, KCC2 play a critical role in the morphogenesis and function of glutamatergic synapses within the central nervous system. Studies on hippocampal tissue in KCC2 knockout animals showed that neurons lacking KCC2 have stunted dendritic growth and malformed dendritic spines. Recent studies demonstrate that KCC2 plays a critical role in the structure and function of dendritic spines^[5] which host most excitatory synapses in cortical neurons. Through an interaction with actin cytoskeleton, KCC2 forms a molecular barrier to the diffusion of transmembrane proteins within dendritic spines, thereby regulating the local confinement of AMPA receptors and synaptic potency.^[5]

It has been proposed that the downregulation of KCC2 observed following neuronal trauma, and the consequent depolarizing shift of GABA_A-mediated synapses, may be an aspect of neuronal de-differentiation. De-differentiation of damaged portions of the nervous system would allow for neuronal networks to return to higher levels of plasticity in order to rewire surviving neurons to compensate for damage in the network.^{[4] [6] [7]} In addition, reduced glutamatergic transmission upon KCC2 downregulation may serve as a homeostatic process to compensate for the reduced GABA transmission due to altered chloride extrusion.^[5]

Oncogenesis

Mutations in SLC12A5 are associated with colon cancer.^[14]

Regulation

Transcriptional regulation: TrkB receptor signalling

KCC2 is transcriptionally downregulated following central nervous system injury by the TrkB receptor signalling transduction cascade (activated by BDNF and NT-4/5).^{[15] [16] [17]}

Post-translational regulation: phosphorylation

It is conventionally thought that phosphorylation inactivates or downregulates KCC2, however there is recent evidence to suggest that phosphorylation at different sites on the KCC2 protein determines different regulational outcomes:

• Wnk1/Wnk3 and tyrosine kinase (i.e. TrkB) phosphorylation downregulates KCC2 activity.^{[15] [16] [17] [18]}
• PKC phosphorylation of the C-terminus Ser940 residue of the KCC2 protein upregulates KCC2 activity by increasing surface stability.^[4] Conversely, Ser940 dephosphorylation leads to enhanced membrane diffusion and endocytosis of KCC2.^[19]

KCC2 has an extremely high rate of turnover at the plasmalemma (minutes),^[4] suggesting that phosphorylation serves as the primary mechanism for rapid regulation.

Activity-dependent downregulation

KCC2 is downregulated by excitatory glutamate activity on NMDA receptor activity and Ca²⁺ influx.^{[7] [18]} This process involves rapid dephosphorylation on Ser940 and calpain protease cleavage of KCC2, leading to enhanced membrane diffusion and endocytosis of the transporter,^[19] as demonstrated in experiments using single particle tracking.

Glutamate release occurs not only at excitatory synapses, but is also known to occur after neuronal damage or ischemic insult.^[7] Thus, activity-dependent downregulation may be the underlying mechanism by which KCC2 downregulation occurs following central nervous system injury.

See also

• Solute carrier family

Further reading

• Chevy Q, Heubl M, Goutierre M, Backer S, Moutkine I, Eugène E, Bloch-Gallego E, Lévi S, Poncer JC . KCC2 Gates Activity-Driven AMPA Receptor Traffic through Cofilin Phosphorylation. . Journal of Neuroscience . 6 . 48 . 15772–86 . 2013 . 26631461 . 10.1523/JNEUROSCI.1735-15.2015 . 6605455 . free .
• Chamma I, Chevy Q, Poncer JC, Levi S . Role of the neuronal K-Cl co-transporter KCC2 in inhibitory and excitatory neurotransmission. . Front Cell Neurosci . 35 . 15772–86 . 2015 . 22363264 . 10.3389/fncel.2012.00005 . 3282916. free .
• Hebert SC, Mount DB, Gamba G . Molecular physiology of cation-coupled Cl⁻ cotransport: the SLC12 family . Pflügers Arch. . 447 . 5 . 580–93 . 2004 . 12739168 . 10.1007/s00424-003-1066-3 . 21998913 .
• Rivera C, Voipio J, Kaila K . Two developmental switches in GABAergic signalling: the K⁺-Cl⁻ cotransporter KCC2 and carbonic anhydrase CAVII . J. Physiol. . 562 . Pt 1 . 27–36 . 2005 . 15528236 . 10.1113/jphysiol.2004.077495 . 1665491 .
• Andersson B . A "double adaptor" method for improved shotgun library construction . Anal. Biochem. . 236 . 1 . 107–13 . 1996 . 8619474 . 10.1006/abio.1996.0138 . vanc. Wentland MA . Ricafrente JY . 3 . Liu . W . Gibbs . RA .
• Yu W, Andersson B, Worley KC, Muzny DM, Ding Y, Liu W, Ricafrente JY, Wentland MA, Lennon G, Gibbs RA . Large-scale concatenation cDNA sequencing . Genome Res. . 7 . 4 . 353–8 . April 1997 . 9110174 . 139146 . 10.1101/gr.7.4.353 .
• Hirosawa M, Nagase T, Ishikawa K, Kikuno R, Nomura N, Ohara O . Characterization of cDNA clones selected by the GeneMark analysis from size-fractionated cDNA libraries from human brain . DNA Res. . 6 . 5 . 329–36 . October 1999 . 10574461 . 10.1093/dnares/6.5.329 . free .
• Hübner CA, Stein V, Hermans-Borgmeyer I, Meyer T, Ballanyi K, Jentsch TJ . Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition . Neuron . 30 . 2 . 515–24 . May 2001 . 11395011 . 10.1016/S0896-6273(01)00297-5 . 10.1.1.476.2965 . 4971832 .
• Sallinen R, Tornberg J, Putkiranta M, Horelli-Kuitunen N, Airaksinen MS, Wessman M . Chromosomal localization of SLC12A5/Slc12a5, the human and mouse genes for the neuron-specific K(+)-Cl(-) cotransporter (KCC2) defines a new region of conserved homology . Cytogenet. Cell Genet. . 94 . 1–2 . 67–70 . 2001 . 11701957 . 10.1159/000048785 . 33299788 .
• Song L, Mercado A, Vázquez N, Xie Q, Desai R, George AL, Gamba G, Mount DB . Molecular, functional, and genomic characterization of human KCC2, the neuronal K-Cl cotransporter . Brain Res. Mol. Brain Res. . 103 . 1–2 . 91–105 . June 2002 . 12106695 . 10.1016/S0169-328X(02)00190-0 .
• Bräuer M, Frei E, Claes L, Grissmer S, Jäger H . Influence of K-Cl cotransporter activity on activation of volume-sensitive Cl- channels in human osteoblasts . Am. J. Physiol., Cell Physiol. . 285 . 1 . C22–30 . July 2003 . 12637262 . 10.1152/ajpcell.00289.2002 .
• Lee H, Chen CX, Liu YJ, Aizenman E, Kandler K . KCC2 expression in immature rat cortical neurons is sufficient to switch the polarity of GABA responses . Eur. J. Neurosci. . 21 . 9 . 2593–9 . May 2005 . 15932617 . 2945502 . 10.1111/j.1460-9568.2005.04084.x .
• Mercado A, Broumand V, Zandi-Nejad K, Enck AH, Mount DB . A C-terminal domain in KCC2 confers constitutive K+-Cl- cotransport . J. Biol. Chem. . 281 . 2 . 1016–26 . January 2006 . 16291749 . 10.1074/jbc.M509972200 . free .
• Vanhatalo S, Palva JM, Andersson S, Rivera C, Voipio J, Kaila K . Slow endogenous activity transients and developmental expression of K+-Cl- cotransporter 2 in the immature human cortex . Eur. J. Neurosci. . 22 . 11 . 2799–804 . December 2005 . 16324114 . 10.1111/j.1460-9568.2005.04459.x . 37264065 .
• Lee HH, Walker JA, Williams JR, Goodier RJ, Payne JA, Moss SJ . Direct protein kinase C-dependent phosphorylation regulates the cell surface stability and activity of the potassium chloride cotransporter KCC2 . J. Biol. Chem. . 282 . 41 . 29777–84 . October 2007 . 17693402 . 10.1074/jbc.M705053200 . free .
• Uvarov P, Ludwig A, Markkanen M, Pruunsild P, Kaila K, Delpire E, Timmusk T, Rivera C, Airaksinen MS . A novel N-terminal isoform of the neuron-specific K-Cl cotransporter KCC2 . J. Biol. Chem. . 282 . 42 . 30570–6 . October 2007 . 17715129 . 10.1074/jbc.M705095200 . free .

External links

• How Brain Injury Leads To Seizures, Memory Problems - medical news, 20 Oct 2006.

Notes and References

1. Web site: Entrez Gene: SLC12A5 solute carrier family 12, (potassium-chloride transporter) member 5.
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5. Gauvain G, Chamma I, Chevy Q, Cabezas C, Irinopoulou T, Bodrug N, Carnaud M, Lévi S, Poncer JC . The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines . Proc. Natl. Acad. Sci. U.S.A. . 108 . 37 . 15474–9 . September 2011 . 21878564 . 3174661 . 10.1073/pnas.1107893108 . 2011PNAS..10815474G . free .
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8. Báldi R, Varga C, Tamás G . Differential distribution of KCC2 along the axo-somato-dendritic axis of hippocampal principal cells . Eur. J. Neurosci. . 32 . 8 . 1319–25 . October 2010 . 20880357 . 10.1111/j.1460-9568.2010.07361.x . 11676308 .
9. Lee HH, Walker JA, Williams JR, Goodier RJ, Payne JA, Moss SJ . Direct protein kinase C-dependent phosphorylation regulates the cell surface stability and activity of the potassium chloride cotransporter KCC2 . J. Biol. Chem. . 282 . 41 . 29777–84 . October 2007 . 17693402 . 10.1074/jbc.M705053200 . free .
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13. Stil A, Liabeuf S, Jean-Xavier C, Brocard C, Viemari JC, Vinay L . Developmental up-regulation of the potassium-chloride cotransporter type 2 in the rat lumbar spinal cord . Neuroscience . 164 . 2 . 809–21 . December 2009 . 19699273 . 10.1016/j.neuroscience.2009.08.035 . 41738829 .
14. 24699064. 2014. Yu. C. Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing. Cell Research. Yu. J. Yao. X. Wu. W. K.. Lu. Y. Tang. S. Li. X. Bao. L. Li. X. Hou. Y. Wu. R. Jian. M. Chen. R. Zhang. F. Xu. L. Fan. F. He. J. Liang. Q. Wang. H. Hu. X. He. M. Zhang. X. Zheng. H. Li. Q. Wu. H. Chen. Y. Yang. X. Zhu. S. Xu. X. Yang. H. 29. 10.1038/cr.2014.43 . 24 . 6 . 701–12. 4042168.
15. Rivera C, Li H, Thomas-Crusells J, Lahtinen H, Viitanen T, Nanobashvili A, Kokaia Z, Airaksinen MS, Voipio J, Kaila K, Saarma M . BDNF-induced TrkB activation down-regulates the K⁺-Cl⁻ cotransporter KCC2 and impairs neuronal Cl⁻ extrusion . J. Cell Biol. . 159 . 5 . 747–52 . December 2002 . 12473684 . 2173387 . 10.1083/jcb.200209011 .
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19. Chamma I, Heubl M, Chevy Q, Renner M, Moutkine I, Eugène E, Poncer JC, Lévi S . Activity-dependent regulation of the K/Cl transporter KCC2 membrane diffusion, clustering, and function in hippocampal neurons . J. Neurosci. . 33 . 39 . 15488–503 . September 2013 . 24068817 . 10.1523/JNEUROSCI.5889-12.2013 . 6618451 . free .