Cation-chloride cotransporters in neuronal development, plasticity and disease

Abstract

Electrical activity in neurons requires a seamless functional coupling between plasmalemmal ion channels and ion transporters. Although ion channels have been studied intensively for several decades, research on ion transporters is in its infancy. In recent years, it has become evident that one family of ion transporters, cation-chloride cotransporters (CCCs), and in particular K(+)-Cl(-) cotransporter 2 (KCC2), have seminal roles in shaping GABAergic signalling and neuronal connectivity. Studying the functions of these transporters may lead to major paradigm shifts in our understanding of the mechanisms underlying brain development and plasticity in health and disease.

Figure 1. Developmental expression profiles, functions and secondary structures of CCCs

a | Line plots showing the average exon array signal intensity of cation-chloride cotransporter (CCC) transcripts in the human neocortex from the early fetal period to late adulthood. The profiles are qualitatively similar for the hippocampus, amygdala and cerebellar cortex. Of the five CCCs expressed in the human neocortex (K⁺–Cl⁻ cotransporter 1 (KCC1), KCC2, KCC3, KCC4 and Na⁺ – K₊–2Cl⁻ cotransporter 1 (NKCC1)), only mRNA encoding KCC2 undergoes robust developmental upregulation. NKCC1 shows moderate postnatal upregulation, and KCC3 and NKCC1 are expressed at high levels throughout life while expression of KCC1 and KCC4 is low. The levels of NKCC2 and Na⁺–Cl⁻ cotransporter (NCC) are below the level (6 on the y axis) for a gene to be considered as expressed according to the criteria described in REF. . Only the expression of KCC2 is neuron-specific. Expression data are from the data bank described in REF. and accessible at the Human Brain Transcriptome website. b | NKCC1 and KCC2 control intracellular Cl⁻ concentration ([Cl⁻]_i) in many central neurons. In immature neurons, the Na⁺ gradient generated by the Na⁺/K⁺ ATPase drives cellular uptake of Cl⁻ via NKCC1 and KCC2 has a minor role. This generates a depolarizing Cl⁻ current across GABA_A receptors (GABA_ARs). During neuronal maturation, functional KCC2 attains a high level of expression and the Cl⁻ current becomes hyperpolarizing. Cl⁻ extrusion by KCC2 is fuelled by the Na⁺/K⁺ ATPase-dependent K⁺ gradient. NKCC1 and KCC2, like all CCCs, are electroneutral, in other words, they do not generate any current by themselves. c | Upregulation of KCC2 facilitates the structural and functional development of cortical dendritic spines in an ion-transport-independent manner, probably through effects on the actin–spectrin cytoskeleton. d | Secondary structures of human KCC2b and NKCC1b splice isoforms, highlighting residues critical for function. Dephosphorylation of KCC2 at threonine residues T906 and T1007 and phosphorylation at serine residue S940 are associated with functional activation. Phosphorylation by protein kinase C (PKC) at S940 promotes KCC2 membrane stability. Phosphorylation of NKCC1 by SPAK and OSR1 (oxidative stress responsive kinase 1) at the depicted N-terminal residues leads to functional activation. KCC2 transport function is inhibited by phosphorylation of T906 and T1007 and dephosphorylation at S940. Phosphorylation at T1007 and dephosphorylation at S940 are likely to be mediated by SPAK/OSR1 kinases, and protein phosphatase 1 (PP1), respectively. An arginine-to-histidine mutation at residue 952 (R952H), discovered in humans, results in loss of both transport activity and spinogenesis by KCC2. The approximate (~30 kDa) C-terminal fragment of KCC2 cleaved by calpain is shown in blue. Dephosphorylation of NKCC1 by PP1 at N-terminal threonine residues renders it transport-deficient. Putative glycosylation sites in KCC2 and NKCC1 are highlighted. AMPAR, AMPA receptor. Part d is adapted from 2-D models provided by B.Forbush, Yale University, New Haven, Connecticut, USA.

Figure 2. Mechanisms of ionic plasticity and their temporal domains

Cation-chloride cotransporters (CCCs) control ionic plasticity across various overlapping time scales that range from seconds to weeks, and beyond. a | Short-term ionic plasticity of GABAergic signalling is based on fast, activity-dependent transmembrane movements of Cl⁻ and HCO₃⁻, which alter the driving force and polarity of GABA-induced currents and thereby cause changes in the postsynaptic membrane potential (V_m). On the left, a single presynaptic event leads to a hyperpolarization of V_m. On the right, repetitive activation of GABAergic terminals evokes a biphasic V_m response. In biphasic V_m responses, the depolarizing HCO₃⁻ current leads to an increase in GABA_A receptor (GABA_AR)-mediated uptake of Cl⁻ and an increase in intracellular Cl⁻ concentration ([Cl⁻]_i). The consequent K⁺–Cl⁻ cotransporter 2 (KCC2)-mediated extrusion of K⁺ increases the extracellular K⁺ concentration ([K⁺]_o) and has a depolarizing or even functionally excitatory action on V_m. b | Fast functional regulation of CCCs on a time scale of minutes to hours is mediated by post-translational mechanisms, including (de)phosphorylation of key residues on the intracellular domains of KCC2 and Na⁺–K⁺–2Cl⁻ cotransporter 1 (NKCC1) (see also FIG. 1d) and calpain-mediated cleavage of KCC2. Constitutive membrane recycling (dashed arrows) of KCC2 is regulated by the phosphorylation state of the C-terminal serine residue S940. Phosphorylation of S940 by protein kinase C (PKC) limits clathrin-mediated endocytosis of KCC2. By contrast, protein phosphatase 1 (PP1)-dependent dephosphorylation of S940 leads to internalization of KCC2 and a reduction in neuronal Cl⁻ extrusion capacity following intense activation of NMDA receptors (NMDARs) and an increase in [Ca²⁺]. Under such conditions, KCC2 is also C-terminally cleaved by the Ca²⁺- and brain-derived neurotrophic factor (BDNF)-activated protease calpain, which results in irreversible inactivation of KCC2 (BOX 3). NKCC1 is kinetically regulated through a Cl⁻-sensing cascade involving WNK (with no lysine kinase) and the STE20-related kinases SPAK and OSR1 (oxidative stress responsive kinase 1; not shown). WNKs are allosterically modulated by Cl⁻, with a decrease in [Cl⁻]_i leading to activation of SPAK and OSR1 by WNKs. Consequent SPAK-mediated phosphorylation of key N-terminal threonine residues (for example, T212 and T217; see also FIG. 1d) of NKCC1 results in its activation and Cl⁻ accumulation. By contrast, dephosphorylation of these residues by PP1 inactivates NKCC1. Reciprocal regulation of transport activities of NKCC1 and KCC2 by the WNK SPAK and WNK OSR1 cascade has been demonstrated in heterologous expression systems and may also take place in neurons. At least one SPAK and OSR1 phosphorylation site (T1007) is found in KCC2b, the principal KCC2 splice variant found in neurons. However, it is not clear how phosphorylation of KCC2 by SPAK and OSR1 affects neuronal Cl⁻ extrusion capacity. c | Neuron-specific expression of KCC2 is ensured via multiple transcriptional mechanisms, including the actions of neuron-restrictive elements (NRSEs; also known as RE1), which silence SLC12A5 (encoding KCC2) in non-neuronal cells and neuron-enriched transcription factors (TFs). Neuron-enriched TFs, for example, members of the early growth response (EGR; not shown) family, are sensitive to signalling by neurotrophic factors, such as BDNF and its receptor tropomyosin-related kinase B (TRKB), which exert qualitatively different effects on SLC12A5 transcript expression in immature and mature neurons. The TRKB-mediated cascades may reverse during neuronal trauma, resulting in recapitulation of immature-like Cl⁻ homeostasis in diseased neurons (BOX 3). Long-term consolidation of changes in KCC2 following trauma or during epileptogenesis is likely to be mediated to an extent by the above transcriptional mechanisms.

Figure 3. Cation-chloride cotransporters in pain

There are two major theories concerning the role of cation-chloride cotransporters (CCCs) in chronic pain, and more specifically in touch-evoked pain (allodynia). Under normal conditions (part a), the activation of Aβ fibres, the myelinated fibres responsible for light touch sensation, leads to primary afferent depolarization in C fibres and consequent presynaptic shunting inhibition of pain-conducting C fibres by dorsal horn GABAergic interneurons. This requires the expression of Na⁺–K⁺–2Cl⁻ cotransporter 1 (NKCC1) in dorsal root ganglion (DRG) C fibre neurons. The inhibition blocks the signalling of the C fibres to lamina I projection neurons. Following injury, signalling cascades lead to phosphorylation and thereby kinetic activation of NKCC1 (FIG. 1d) in C fibre terminals. This may be linked to an increase in [Cl⁻]_i, potentially converting Aβ fibre-mediated inhibition into frank excitation of C fibres and leading to activation of pain signalling via lamina I projection neurons. This provides a neurophysiological explanation for allodynia. Very recent evidence suggests that nociceptive-specific (NS) neurons in the deep laminae of the dorsal horn (lamina V) lose KCC2 expression following peripheral nerve injury (PNI), thereby altering the response of these neurons to GABA and unmasking an Aβ fibre-mediated input to NS neurons, effectively converting them to wide dynamic range (WDR) neurons (part b). As in the scenario outlined in part a, this gives the Aβ fibre pathway access to pain signalling through feedforward activation of projection neurons in lamina I/II. Importantly, conversion of NS neurons to WDR neurons following PNI is reversed by positive modulation of KCC2 and this treatment also reduces touch-evoked pain in behavioural assays. GABA_AR, GABA_A receptor.