The strict regulation of intracellular and extracellular chloride concentrations is a vital requirement for the physiological functioning of cells and also influences neuronal excitability. Cation-chloride co-transporters (CCCs) transporter Cl- across the cell membrane. Na+-K+-2Cl- co-transporters (NKCCs) transporter Cl- into the cell. Of the two known isoforms NKCC1 is expressed in the nervous system and is responsible for the high intracellular Cl- concentrations present during neuronal development that make the actions of GABA and glycine hyperpolarising. K+-Cl- co-transporters (KCCs) transport Cl- out of the cell. Of the four known isoforms KCC2 is expressed only in the nervous system and is vital for the ontogenic switch in Cl- concentrations that renders GABA and glycine hyperpolarising. Other KCC isoforms present in the nervous system (KCC1, KCC3 and KCC4), anion exchangers and the enzyme carbonic anhydrase contribute to the actions of NKCC1 and KCC2. The extent of the role they play still needs to be discovered. The spatial and temporal expressions patterns of NKCC1 and KCC2 are lacking in clarity. However, both transporters are expressed throughout the mammalian brain and the consensus is that NKCC1 expression is high during neuronal development and decreases following maturation and KCC2 expression is low initially but is upregulated during neuronal maturation.
The regulation of intracellular and extracellular ions is important for controlling physiological processes such as cell volume, cell pH and establishing ionic gradients required for the operation of neuronal ion channels (Payne et al., 2003). Active transport of Na+ and K+ by the Na-K ATPase generates inward Na+ currents and outward K+ currents. These concentration gradients are utilised by cation-chloride co-transporters (CCCs) to move Cl- across the cell membrane in an electro-neutral mechanism to regulate the [Cl-]i necessary for the hyperpolarising actions of γ-aminobutyric acid (GABA) and glycine in the mature nervous system (Payne et al., 2003). Activation of glycine and GABAA receptors cause neuronal Cl- influx and neuronal hyperpolarisation by the inwardly directed Cl- electrochemical gradient generated by low [Cl-]i and high [Cl-]o maintained by CCCs (Balakrishnan et al., 2003).
Interestingly following birth and in early postnatal life GABAergic and glycinergic transmissions are depolarising and excitatory and switch to hyperpolarising and inhibitory between P7-P14 in the rat (Cherubini et al., 1991; Rivera et al., 2005). Depolarising responses lead to activation of voltage-gated calcium channels causing an increase in intracellular Ca2+. It is thought that this response is important for neuronal development and contributes to neuronal differentiation, proliferation, migration and maturation (Rivera et al., 1999; Payne et al., 2003). CCCs have been shown to play an important part in this developmental shift.
CCCs can be classified into three gene groups and consist of seven members (Payne et al., 2003). They are transport proteins that regulate intracellular ion concentrations. Na+-K+-2Cl- co-transporters (NKCCs) and Na+-Cl- co-transporters (NCCs) mediate Cl- uptake into the cell and K+-Cl- co-transporters (KCCs) mediate Cl- extrusion from the cell (Payne et al., 2003). This can be seen in Figure 1. NKCCs transport Na+, K+ and Cl- into the cell and KCCs extrude K+ and Cl- from the cell. High [Cl-]o is required for the hyperpolarising response to GABA in mature neurons.
Figure 1: Cation-Chloride Co-Transporters and the Ions they Transport. NKCC mediates Na+, K+ and Cl- uptake and KCC mediates K+ and Cl- extrusion. Upon opening GABAA receptors allow Cl- influx, along the electrochemical gradient created by the co-transporters, leading to hyperpolarisation of the cell in mature neurons. Arrow heads dictate the direction of ion movement.
There are two NKCC isoforms (NKCC1-2), four KCC isoforms (KCC1-4) and one NCC isoform (Payne et al., 2003). Structurally CCCs are comprised of a small intracellular N-terminus, twelve transmembrane spanning segments and a large intracellular C-terminus with high sequence homology between members of the same family (e.g. KCC1-2) and low sequence homology between different families (Payne et al., 1996; Payne et al., 2003). CCCs show developmental and tissue specific expression patterns and for the purpose of this literature review I will focus on the neuronal specific NKCC1 and KCC2.
KCC2 was first identified following a screen for CCCs displaying homology to NKCC1 (Payne et al., 1996). It was found to be a 124 kDa protein, 1116 amino acids in length sharing predicted structural homology with other CCCs. Unlike KCC1, which is ubiquitously expressed throughout rat tissue, KCC2 expression is restricted to neurons in the central nervous system (CNS) (Payne et al., 1996; Rivera et al., 1999; Kanaka et al., 2001). KCC2 mRNA has been detected in rat DRGs (Lu et al., 1999), however this method used antibody immunofluorescence rather than the standard blotting and electrophoretic techniques utilised by others. KCC2 mRNA is also absent in glial cells (Payne et al., 1996). GABAergic transmission in the immature rat brain is depolarising and it is thought that KCC2 mediates the ontogenic switch to hyperpolarising transmission. Using in situ hybridisation, Rivera et al., (1999), were able to show a developmental upregulation of KCC2 expression consistent with the change from depolarising to hyperpolarising transmission of GABAergic signalling. To show a direct relationship further studies utilised anti-sense oligonucleotides to knock-out gene expression in cultured hippocampal slices. Electrophysiological studies showed an almost complete abolition of the hyperpolarising actions of GABA in these slices (Rivera et al., 1999). However, complete knock-out of KCC2 in mice is lethal and leads to death following birth due to an inability to breathe (Hubner et al., 2001). Elsewhere in the CNS developmental KCC2 expression varies between different brain regions. In the thalamus KCC2 expression can be seen from embryonic day 20 (E20) whereas in the hippocampus KCC2 may not be seen up until postnatal day 16 (P16) (Rivera et al., 1999). Weak labelling of KCC2 mRNA can be visualised at birth in the cortex and becomes stronger at P5 (Rivera et al., 1999). In the rat cerebellum KCC2 mRNA can be detected weakly at P0 with signal intensity increasing up to P14 and adult levels reached at P21 (Mikawa et al., 2002). In the rat auditory brainstem KCC2 mRNA expression is abundant during development (P0-P16) (Balakrishnan et al., 2003). This is an intriguing result as during P0-P6 glycinergic transmission in the brainstem is depolarising, even in the presence of KCC2. Western blot analysis showed KCC2 protein levels were constant during P0-P16, however the protein was not inserted into the plasma membrane until later on in development, at P6-P16, when glycinergic transmission becomes hyperpolarising (Balakrishnan et al., 2003). This shows different developmental processing of KCC2 can exist between different brain areas.
The human NKCC1 homologue was identified as a 1212 amino acid protein with a molecular weight of 170 kDa (Payne et al., 1995). NKCC1 mRNA was found to be ubiquitously expressed in many tissues including the brain (Payne et al., 1995). Unlike KCC2 which acts to extrude Cl- from the cell NKCC1 transports Cl- into the cell (Yamada et al., 2004). NKCC1 mRNA has been shown to be expressed highly in developing neurons, where responses of the neurons to GABA were depolarising (Yamada et al., 2004). However, following neuronal maturation and the switch of GABA to hyperpolarising, NKCC1 mRNA was not detected (Yamada et al., 2004). Using in situ hybridisation NKCC1 mRNA has been detected in the olfactory bulb, the hippocampus, the trigeminal nucleus, the cerebellum, the spinal cord and DRGs (Kanaka et al., 2001). However no information was given on the maturity of the rats used in these studies. In the auditory brainstem NKCC1 expression is absent in the presence of immature depolarising glycinergic transmission suggesting other inwardly directed chloride transporters may play a role in establishing the high [Cl-]i required for depolarisations here (Balakrishnan et al., 2003). NKCC1 mRNA was expressed however following maturity of the cells in the auditory brainstem. A NKCC1 knock-out mouse model showed that knock-out of the gene encoding NKCC1 affected spontaneous and depolarising activity in pyramidal neurons and delayed the maturation of glutamate and GABA synapses (Pfeffer et al., 2009). However, in a different knock-out mouse model disruption of NKCC1 had no effect on the generation of spontaneous network activity and KCC2 expression was unaltered (Sipila et al., 2009). This suggests that in the absence of GABAergic depolarisations homeostatic mechanisms take place that regulate neuronal activity. This is in contrast to Pfeffer et al., (2009) where disruption of NKCC1 resulted in loss of spontaneous and depolarising network activity. The significance of the results from the complete knock-out of a gene is difficult to determine and the significance of both of these studies are debatable (Wright et al., 2009).
Other Important Transporters and Enzymes
KCC1, KCC3 and KCC4 have all been found to be expressed in the nervous system and play an important part in maintaining [Cl-] gradients (Payne et al., 2003). KCC1 mRNA has been shown to be upregulated during post-natal maturation in the developing rat cerebellum (Mikawa et al., 2002). KCC1 expression is low in the developing mouse brain and begins to be upregulated following birth (Li et al., 2002). KCC3 is weakly detected in the developing mouse brain and KCC4 expression was shown to be high during neuronal development but decreased following birth (Li et al., 2002).
Other important transporters include sodium dependent and sodium independent anion exchangers which mediate the uptake of Cl- in exchange for bicarbonate ions (HCO3-) (Payne et al., 2003).
The enzyme carbonic anhydrase (isoform VII - CAVII) is also important in the control of intracellular HCO3- levels and consequently [Cl-]i and GABAergic transmission (Rivera et al., 2005). CAVII converts CO2 and water to HCO3- which is able to pass through GABAA receptors resulting in a depolarising current (Rivera et al., 2005). This depolarisation causes an influx of Cl- raising its intracellular concentration. Subsequent depolarisations then occur due to an efflux of Cl- to lower [Cl-]i, whether this efflux is by a co-transporter (e.g. KCC2) or another mechanism is unknown.
CCCs, in particular KCC2 and NKCC1, are important in controlling [Cl-]i. It would appear that NKCC1 is important for controlling the developmentally high levels of [Cl-]i necessary for the depolarising actions of GABAergic and glycinergic transmission. This initial phase of depolarisation during development is important for the development of the nervous system. KCC2 on the other hand appears to be upregulated during development and is responsible for the switch in Cl- concentrations that renders GABA and glycine hyperpolarising. Although a lot of work has been carried out on the expression patterns of CCCs in the mammalian brain there are conflicting reports and a lack of clarity. Also, different animal models are used which may confuse the results further. Rats are born with neuronally immature brains which continue to develop during the first post natal weeks. Mice are born relatively neuronally mature and this can make comparisons between the two models difficult. More work is needed to clarify the temporal and spatial expression profiles of CCCs to try to determine the extent to which they control neuronal excitability in the developing and mature nervous system. Finally the role and expression patterns of other transporters and enzymes involved in Cl- regulation need to be addressed.