The Molecular Identity of Cotransporter KCC in Mouse Neuronal Cell Line - Essay Example

www.academia-research.com Sumanta Sanyal d: 19/03/07 Molecular identity of co transporter KCC in differentiated and undifferentiated mouse neuronal cell line CAD. Introduction A Short Background: Seven electroneutral cation-coupled cotransporters have been found in the physiological systems of mammals to date (Delpire, E., 2000). These, together with two orphan members, belong to the SLC12 gene family (Gamba, G., 2005) and cloning of the cDNA encodes the different members. These are all sodium/potassium-coupled chloride cotransporters described succinctly below. They can be functionally classified broadly into four groups according to the cation coupling to the chloride ion, the stoichiometry of the transport process and the relative sensitivities to inhibitors. 1. The sodium-coupled benzothiaziadine (or thiazide) sensitive cotransporter (NCC) that is renal-specific in action; 2. the sodium-coupled sulfamoylbenzoic (or bumetanide) sensitive cotransporter that is also renal specific like its thiazide counterpart; 3. the two sodium/potassium-coupled sulfamoylbenzoic (or bumetanide) sensitive cotransporters (NKCC1 and NKCC2) that are loop diuretic sensitive; and 4. the four potassium-coupled dihydroindenyloxy-alkanoic acid (DIOA) (or furosemide) sensitive cotransporters (KCC1, 2, 3 and 4) (Delpire, E., 2000, Gamba, G., 2005). As mentioned earlier partially, the NCC and the NKCC2 cotransporters are renal-specific in action while the other five are more widely found and evident throughout the central nervous system (Delpire, E., 2000). The and solute carriers move through plasma membranes always accompanied by in equal proportions in a 1:1 stoichiometry (Gamba, G., 2005). Functionally, these membrane proteins are active in transepithelial ion absorption and secretion, cell volume regulation and setting intracellular anions below or above their electrochemical potential equilibriums (Gamba, G., 2005), among other possible functions. The last activity, specially appertaining to KCC2, is of special interest to this study. The last decade has seen much advance in their study and most of the knowledge that is available on them has become so within this short period. These family members are also extensively involved in the pharmacology and pathophysiology of cardiovascular and neuronal regions of mammalian anatomy. Loop diuretics and thiazide diuretics that are some of the most prescribed drugs in the world target some of the family members while genetic mutations that inactivate three specific members cause inherited diseases like Bartter's, Gitelman's and Anderman's (Gamba, G., 2005). It should be noted here that all the functional-structural characteristics of the above seven cotransporters have not been comprehensively discovered and described. This is amply evident from G. Gamba's extensive 2005 paper investigating comprehensively the hitherto unrevealed functional-structural characteristics of the seven. Thus, that deficiency lends purpose to the dissertation this paper is an introduction to again, specifically in relation to KCC2, the particular potassium-coupled chloride cotransporter this project is investigating. CAD Cells True cultured cell lines derived of purely neuronal origin are of immense value to biochemical and molecular study of neurons and their diverse functional implications together with other neurotransmission elements like the KCC cotransporters in this instance. This is so because primary neuronal cells thrive indifferently in cultures with low reproducibility and often are found mixed with other types of cells not of interest to the particular research at hand (Wang and Oxford, 2000). There are some specific neuronal differentiated phenotype exhibiting cell lines available to the researcher such as the PC12 cells, the P19 cells and the MN9D cells, among others, but these are not purely derived of the CNS and furthermore do not exhibit fully differentiated features. Nevertheless, in recent times (Wang and Oxford, 2000), a catecholaminergic cell line called CATH.a has been developed by the usage of oncogenes driven by cell specific promoters that, in turn, drive tumourigenesis in target cells in transgenic mice. The tumours are tyrosine hydroxylase (TH) positive and are exclusively expressed in neuronal populations that express the TH gene. The transcriptional regulation of the TH gene promotes neoplastic growth (Wang and Oxford, 2000). CATH.a cells have certain unique properties that make them attractive as sources of other derivative clones such as the CAD culture line. This shall be disclosed subsequently. The CATH.a cells synthesise dopamine and norepinephrine and express catecholamine synthetic enzymes, TH and dopamine hydroxylase (Wang and Oxford, 2000). Structurally, CATH.a cells have neuron-specific molecules that structurally evolve into neurofilaments and synaptophysin. What is of great interest to this research project is that they also express voltage dependent cation channels - the tetrodoxin (TTX)-sensitive sodium channels, L- and N-type calcium channels and potassium channels (Wang and Oxford, 2000). Nevertheless, these cells cannot be used for the present research since they cannot be made to differentiate morphologically. Therefore, the research project has identified the murine (caspase-activated deoxyribonuclease (CAD) (Cao, G., et al, 2001) cell line to do the research work with. These cells are unique subclones of the murine CATH.a cell lines and preserve many characteristics of that line that are desirable to this research effort. Specifically, they are cloned singly from spontaneous variants of the parental line that have unusually short processes not found in most of the CATH.a cells. When cultured in serum and medium they remain undifferentiated and proliferate but as soon as they are deprived of serum they differentiate into a neuronal-like phenotype and stop proliferating (Wang and Oxford, 2000). What makes these cells so attractive to research efforts like this is that, while the undifferentiated cells have round or oval shaped cells with few neuronal-like processes the differentiated cells demonstrate CNS neuronal characteristics with spindle-shaped body and long processes (Wang and Oxford, 2000). These differentiated CAD cells have processes with ultrastructural components that promote maximal transmission parameters. They have parallel microtubules and intermediate filaments, varicosities with both large dense-core and smaller clear-core vesicles (Wang and Oxford, 2000). They express a number of neuron specific proteins like class 3 -tubulin, GAP-43, SNAP-25 and synaptotagmin (Wang and Oxford, 2000). They express enzymatically active tyrosine hydroxylase (TH) and accumulate L-Dopa (Wang and Oxford, 2000). Thus, they are ideal for this purpose because of their special neuronal morphology, their excitability properties that can be induced to fire to relatively strong action potentials when artificially hyperpolarized even from very low potentials and their distinctly apparent changes in channel function with differentiation (Wang and Oxford, 2000). The Co-Transporters (KCC1, 2, 3 and 4) The -coupled chloride cotransport system was first recorded in low potassium sheep's red blood cells with evidence of swelling and N-ethylmaleimide (NEM)-activated efflux pathway (Gamba, G, 2005). The and cotransport system is interdependent characterised by a 1:1 stoichiometry and low-affinity constants for both ions. Though the system was first discovered in red blood cells and has subsequently been established as being functionally and physiologically evident primarily in RBCs it has since been found manifest in many other cells and tissues including neurons, vascular smooth muscles, endothelium, epithelia, heart and even skeletal muscle. Evidence of functional capabilities at these diverse locations suggest that the system can be implicated in functional roles such as regulatory volume decrease, transepithelial salt exchange, renal generation, myocardial loss during ischemia and regulation of ion levels in neuronal precincts (Gamba, G, 2005). In all, four members of this subfamily of cotransporters have been discovered - KCC-1, -2, -3 and -4. The four genes that encode each have also been revealed by the fact that this subfamily is genetically homologous to the sodium-coupled chloride cotransporters. The encoding genes all belong to the SLC12 family and the ones that encode each isoform are as follows: SLC12A4 - KCC-1 SLCA5 - KCC-2 SLCA6 - KCC-3 SLCA7 - KCC-4 (Gamba, G., 2005) More specific details are available from Table 1, (Appendix). As becomes manifest after studying the table, not all the relevant details of the four isoforms have been completely described yet. Before proceeding with the individual cotransporters it is deemed necessary to explain in some detail the position of the electroneutral cotransporters in human physiological processes. The Electroneutral Cotransporters There are some specific plasma membrane proteins in absorptive and secretory epithelia that mediate the entrance and exit of transcellular ions into and out of individual cells. This transcellular ion transport is either mediated solely on influx and efflux or on transport mechanisms where these cations are accompanied by other ions or solutes. In epithelial basolateral membrane (except in choroidal plexus) these cations move through the action of the enzyme --ATPase, a system where a electrochemical gradient is produced across the cellular membranes to promote the passage of the cations. Thus, these mediating membrane proteins are known as secondary transporters because the ions or molecules are not moved by ATP hydrolysis but by the electrochemical gradients produced by the primary ions. This is the primary mode of transport of cations in human physiological systems (Gamba, G. 2005) There is also another means of transport - the secondary system - where the cations ( and ) are associated with negative ions in a 1:1 stoichiometry where no electrochemical gradient is produced. This is thus called an electroneutral transport system where the primary transporters are called the electroneutral cation-coupled chloride cotransporters (Gamba, G., 2005). This system is heavily implicated in ion absorptive and secretory mechanisms in both epithelial and non-epithelial cells and the net effect of this is transport of ions in and out of such cells. This is so because the accompanying cations ( and ) are quickly compensated, when transported, by the action of the --ATPase system. This net translocation system is implicated in both maintenance and regulation of cellular volume and in other activities such as the neurotransmission mechanisms in neuronal processes (Gamba, G., 2005). This last is of great interest to this research project because it deals primarily with KCC-2, a cotransporter known to be highly neuronal specific. It is also reported that KCC transport is activated by nitric oxide donors and inhibited by the cGMP pathway, protein phosphatases and tyrosine kinases in a concentration and time dependent manner. The cGMP/PKG (/protein kinase G) pathway regulates the passage of ions in this system (D Fulvio, M., et al, 2001). However, to be more comprehensive, since not all the regulatory mechanisms of the KCC cotransporters have been fully described yet, it is noted that KCC promoted ion exchange is regulated by multiple activators and inhibitors through their putative kinase-phosphatase cascades (a feature explained later) (Shen, MR, et al, 2004). Co-Transporter KCC-1 Genetic Detail: As mentioned earlier in the introductory portion of this section, the four cotransporter genes were revealed by what is called the silico-cloning technique. Unknown DNA sequences put in the Genebank were subsequently linked to their phenotypic counterparts in the relevant organisms. These expressed sequences were subsequently registered by their finders in the sequence tag database (dbEST) set up in 1992 and extended throughout the 1990s and after. There was one set of human-derived sequences among those registered at dbEST that were >50% identical to the sodium-coupled chloride cotransporter genes BSC1 (for NKCC-2) and BSC2 (for NKCC-1) (Gamba, G., 2005). This suggested that these close sequences belonged to a near member of the cotransporter family. Gillen et al, subsequently came upon these sequences and were successful in isolating full-length clones from human, rat and rabbit kidneys that encoded a membrane protein of 1,085 amino acid residues. This residue sequence was expressed as a 3.8 kb transcript in all the tested tissues (Gamba, G., 2005). This was confirmed to be the KCC-1 cDNA when it was taken from rabbit and (human embryonic kidney) HEK-293 cells were transfected with it. They exhibited known characteristics of the potassium-coupled chloride cotransporters with (retinoblastoma protein) uptake and efflux mechanisms (Gamba, G., 2005). KCC-1, in that first instance, induced transport that was Na free, Cl dependent, furosemide sensitive and activated by NEM or cell swelling (Gamba, G., 2005). Though KCC-1 is not the focus of this research, its genetic origins have been furnished here in such detail because the same process of discovery is applicable to KCC-2, KCC-3 and KCC-4, and this lengthy discussion will pre-empt later repetition. Structure: The molecular identity of KCC-1 is touched upon very lightly here. It has a central domain that is slightly hydrophobic flanked by short amino-terminal and long carboxy-terminal domains that are intracellular. These are all predictive. The central domain is comprised of 12 putative transmembrane segments (Gamba, G., 2005). The distinction between KCC-1 and the other KCCs and the sodium cotransporters is the difference in length and location of its extracellular loop. This loop is longer in KCC-1 and located between putative segments TM5 and TM6 with 4 potential N-linked glycosylation sites. This feature is conserved among rat, rabbit and human KCC-1s (Gamba, G., 2005). Function and Location: Studying the level of expression of KCC-1 mRNA suggests that it may be involved in erythroid maturation (Gamba, G., 2005). Since KCC1 is found expressed in many tissues it is also suggested that it is an isoform responsible for cell volume regulation (Strange, K., 2000). D Fulvio et al (2001) reports presence of KCC1/KCC3 mRNA in primary cultures of rat vascular smooth muscle cells (VSMCs) in the ratio 2:1 by specific semiquantitative reverse transcription PCR. This section on KCC-1 has been deliberately kept short as it is not the focus of this research. More material is certainly available. Co-Transporter KCC-3 Genetic Details: KCC3 is a more complex study than the other cation-coupled cotransporters since it has two isoforms - KCC3a and KCC3b. Three separate groups researched on EST records to find the gene (SLC12A6) that encodes the isoforms from human mRNA by two different strategies (Gamba, G., 2005). More details are not provided to conserve brevity. Hiki et al isolated a cDNA from human umbilical vein endothelial cells (HUVECs) after treating them with vascular endothelial growth factor (VEGF) that conformed 77% with KCC1 and 73% with KCC2. It encoded a protein of 1099 amino acid residues length identical in hydropathy profile to KCC1 and -2 (Gamba, G., 2005). The Hiki et al study in transfected HEK-293 cells showed that KCC3 performed as a furosemide-sensitive and NEM-activated cotransporter that showed slight but significant response to hypotonicity (Gamba, G., 2005). The gene was expressed in several tissues and located on human chromosome 15q13. Interestingly, Mount et al isolated KCC3 cDNA together with the KCC4 one from human muscle cDNA library to reveal by Northern blot analysis that the cDNA expressed variably by two 6-7-kb bands in several tissues suggesting alternate splicing. Race et al, almost simultaneously with Mount et al, used a similar technique to isolate the KCC3 cDNA from human placenta cDNA library (Gamba, G., 2005). Both these cDNA encoded a 1150 amino acid residue chain that differed from Hiki et al group's 1099 residue protein by length and sequence at the amino-terminal domain (Gamba, G., 2005). The SLC12A6 gene has two first exons that encode differently. They were named exon 1a for the KCC3a isoform and exon 1b for KCC3b. Exon 1a encodes 90 amino acids while the shorter exon 1b encodes 39 amino acids (Gamba, G., 2005). Thus, KCC3a encodes 1150 residues while KCC3b encodes 1099. It is notable that KCC3a, with its longer exon 1a chain, has more potential phosphorylation sites within the extra residues than the shorter KCC3b (Gamba, G., 2005). Location: KCC3 seems to be found widely in cells and tissues such as vascular endothelial cells, heart, kidney, brain, placenta, liver and lung (Shen et al, 2001). KCC3 functional details are available hereafter in the general features section. Co-Transporter KCC-4 Molecular Genetics with Structural Identity: Mount et al used the bdEST records to discover the fourth isoform of the potassium-coupled chloride cotransporters - KCC-4. They conducted polymerase chain reaction (PCR) strategy on mouse and human kidney mRNA to find that the KCC-4 cDNA encodes a long chain of 1083 amino acid residues with a hydropathic orthography similar to the other cotransporters of the potassium-coupled family (Gamba, G., 2005). There is varying degree of similarity between KCC-4 and the other KCC cotransporters on an individual basis. This is as follows: with KCC-1: 67%; with KCC-2: 72%; and with KCC-3: 67% (Gamba, G., 2005). KCC-4 cDNA isolated by Velazquez and Silva from rabbit kidney has been found to be variant from that of mouse and human. It encodes a 1,105 amino acid residue with a 23 residue extra section that is positioned on the amino-terminal domain as two separate segments of 11 and 12 residues each (Gamba, G., 2005). Function and Location: KCC-4 has been found to be expressed in many tissues including with high levels in heart and kidney and low levels in brain (Gamba, G., 2005). In the CNS KCC-4 is primarily located in the cranial nerves. Functionally, KCC-4 can be expressed actively by hypotonic solutions and by exposure to NEM (N-ethylmaleimide). This brevity and utilisation solely of Gamba's paper for KCC-4 has the same logic as that given for KCC-1. Co-Transporters: General Features Certain general features pertaining to KCC1, 3 and 4 are placed here on a pointwise basis to enable easy comprehension. 1. KCC mRNA transcripts for KCC1, 3 and 4 have been found to be important modulators for cervical cancer cell proliferation and invasiveness. This may be specifically through the KCC activation potentials of growth factors such as insulin-like growth factor 1 (IGF1) (Shen MR, et al, 2004). Shen MR, et al, 2001, report that KCC3 seems to be specifically involved in cell cycle progression, being most influenced by the various growth factors though it did not seem to be involved in cell volume regulation as there was no evidence of it being osmolality-sensitive (Shen et al, 2001). 2. Shen MR et al, 2001, report that KCC1 and KCC4 are involved in cell volume regulation and their activity is osmolality-sensitive. Co-Transporter KCC-2 Phylogenetics: Payne et al used two records from dbEST taken from the human brain that showed 35% identity with the human BSC2/NKCC1. They used a 286-bp cDNA from rat brain to amplify it by PCR to subsequently use the result as a template for a -DNA random primed probe under low stringency conditions into the cDNA of rat brain. They thus produced 19 clones out of which 7 were KCC-1 and the rest were the closely related cotransporter KCC-2. The 5566-bp full clone encodes a 1116 amino acid residue chain with a predicted molecular mass of 123 kDA and that is 67% identical to KCC-1 (Gamba, G., 2005, Payne, JA, et al, 1996). A similar cDNA was isolated and sequenced from mouse by the US government "Mammalian Gene Collection Program Team" (Gamba, G., 2005). KCC-2 cDNA was first isolated from human neuronal-derived cell lines by Song et al using a PCR based homology approach (Gamba, G., 2005). The 5907-bp cDNA encodes 1116 amino acid residues that are 99% identical to the one that encodes rat KCC-2 (Gamba, G., 2005). Differentiated NT2-N cells with retinoic acid express KCC-2 leading to the conclusion that the cotransporter is neuronal-based. Structural and Locational Implications: Hydrophobic studies indicate that KCC-2 has the same hydrophobic centre as the other potassium-coupled chloride cotransporters with 12 putative transmembrane segments with a topology similar to KCC-1 with the extracellular loop located between the 5 and 6 segments (Gamba, G., 2005, Payne, JA, et al, 1996). Northern Blot analysis revealed that a 5.6 bp transcript was expressed only in polyRNA from the central nervous system. This made the gene brain-specific (Gamba, G., 2005, Payne, JA, et al, 1996). This was amply corroborated when analysis of segments of rat nervous system cell lines such as the primary astrocytes, glioma cell lines, pheochromocytoma cell lines proved positive for KCC-1 but negative for KCC-2 (Gamba, G., 2005, Payne, JA, et al, 1996). In situ hybridisation analysis revealed that KCC-2 is present in all layers of the cortex, all areas of the hippocampus and in the granular layer of the cerebellum while white matter elicited no signal indicating that KCC-2 is present only in grey matter, evidently in neurons (Gamba, G., 2005, Payne, JA, et al, 1996). Structural and Functional Implications: John Payne and his group of researchers, when they observed that KCC2 was exclusively expressed in neuronal precincts, realised that that it was the specific potassium-coupled isoform that was responsible for regulating levels in neurons. In many neurons the chloride dependent synaptic inhibition is reliant upon maintenance of low intracellular chloride [levels and a continuous inwardly fluxed electrochemical gradient favouring chloride influx (Payne, JA, et al, 1996). Inhibitory neurotransmitters, specifically -aminobutyric acid (GABA), and glycine elicit the inhibitory postsynaptic potential (IPSP) that, in turn, is dependent upon the conduction of anions, specifically ions in this case, through a ligand-gated channel in membranes. Specifically in the case of KCC2, the (A Type GABA) receptor functions as an ion channel that is highly selective of ions (Payne, JA, et al, 1996). The IPSP is set up when the ions enter through the ligand gate and hyperpolarizes the membrane and drives the membrane potential much above its threshold value. This much misbalances the electrochemical balance across the neuronal membrane that, if totally disrupted by continuous increase in potential, would cause the failure of action of neurotransmitters like GABA and glycine, that depend upon an optimum potential that is high but not too much so to set up the IPSP to conduct their inhibitory actions. To preserve this optimum potential, the secondary transportation system that KCC2 belongs to induce an active ion extrusion mechanism for the neurons that is reverse to the one set up by the neurotransmitter systems (Payne, JA, et al, 1996). This is the specific action KCC2 does in neurons and it is very important to the purpose of this paper. It is noted here, as per Diagram 1, (Appendix), the action of KCC2 is complemented by that of the sodium-potassium-coupled chloride cotransporter NKCC1 (Delfire, E., 2000). While KCC2 induces efflux in reverse response to ergic action, NKCC1 does the opposite. Subsequent to this, Nabekura et al, 2002, disclose the A Type GABA receptor mediated response in intact and injured neurons (in the team's particular case axotomised dorsal motor neurons of the vagus nerve) is initiated by ion efflux that, in effect, depolarises the membrane. Reversal potential of this type of receptor is equal to the equilibrium potential of ions across the membrane. In injured neurons, presumably, the intracellular ion concentration drove the anion efflux (Nabekura et al, 2002). The team found that in both control and injured (after axotomy) neurons regulation in ions was disturbed by furosemide and bumetanide and by altering the cation balance across the membrane. This suggested that the KCCs, being furosemide sensitive, became functionally altered (inhibited by the furosemide generic drug) and increased the ion balance through inaction after neuronal injury (Nabekura et al, 2002). This was proved correct when in situ hybridisation showed that the KCC2 mRNA was significantly reduced in the dorsal motor neurons after axotomy (Nabekura et al, 2002). Specific Neuronal Location: Williams et al, 1999, developed polyclonal antibodies to a KCC2 fusion protein and used these antibodies to localise and characterise KCC2 in the rat cerebellum. The protein is a 140-kDa glycoprotein that the antibodies recognised specifically only in the CNS. The protein was evenly distributed across primary cultured retinal amacrine cells that are known to exclusively form ergic synapses in cultures. The team found that the protein was expressed primarily in postsynaptic processes like neuronal somata and dendrites but were significantly absent from the axons and glia (Williams et al, 1999). The protein showed distinct co-localisation with subunits of receptor at the plasma membrane of granule cell somata and cerebellar glomeruli. It was more lightly evident at plasma membrane of Purkenji cell somata and it was molecularly evident at the dendritic processes signifying preference for inhibitory processes (Williams et al, 1999). Regulatory Action: Strange et al, 2000, reports that for KCC2 action there is a marked dependence on a tyrosine residue on the conserved carboxy terminal domain. Their experiments with Xenopus oocytes injected with KCC2 cRNA induced them to conclude that there is a distinct tyrosine phosphorylation site between R1081-Y1087 on the KCC2 carboxy terminal loop. This was also evident for KCC4 but not for the other two isoforms. They also concluded from additional pharmacological studies that this phosphorylation of KCC2 tyrosine residues had no regulatory role. They found that when the tyrosine residue at Y1087 was substituted with aspartate and arginine there was significantly reduced uptake under both isotonic and hypotonic conditions. Activity was normal or near normal when the Y1087 tyrosine residue was mutated to phenylalanine, alanine or isoleucine. The cotransporter activity was also reduced for KCC1 when the Y1087 congener was mutated to aspartate. Strange et al, 2000, thus concluded that the KCC cotransporters had Y1087 congeners that sustained conformational status of the carboxy terminus of the cotransporters that was essential for normal activity. Any mutative replacement of these Y1087 congeners with charged residues disrupted the conformational status of the carboxy terminus and reduced normal activity (Strange et al, 2000). Strange et al, 2000, also concluded that, though pharmacological studies failed to prove so, there was some regulatory activity associated with the Y1087 congeners in protein-protein interactions (Strange et al, 2000). The KCC2 gene regulatory region is believed to be position upstream of exon 1 (Karadsheh and Delfire, 2001). Neuron-Specificity: Northern Blot Analysis, in situ hybridisation and immunofluorescence studies on the KCC2 gene have revealed that KCC2 is expressed only in neurons. This neuron-specificity is due to a 21-bp motif downstream of exon 1, where the regulatory region is upstream of this exon, which resembles the consensus sequence of the neuronal-restrictive silencing element (NRSE) (Karadsheh and Delfire, 2001). The KCC2 element binds to nuclear proteins and inhibits transcription of a reporter gene in most nonneuronal cells suppressing KCC2 expression (Karadsheh and Delfire, 2001). KCC2 is known to be down-regulated by continuous synchronous neuronal activity via the BDNF (Brain-Derived Neurotopic Factor)-TrkB (tyrosine receptor Kinase B) signalling that recruits Shc (src homology 2 domain containing transforming protein) and PLC -coupled intracellular pathways (PLC- phospholipase C protein). It is reported that KCC2 turnover is high in neuronal cells that its expression and downregulation is mediated by a large number of elements and factors including the cAMP response element-binding protein (CREBP) (Rivera et al, 2004). Too much transcription and down-regulation details are not included here as it is envisioned that the research project will consult these but will nevertheless try to follow a course of its own. Post- and PreNatal Concentrations: Another observation made on the KCC2 and the complementary NKCC1 cotransporters is that in immature brains where GABA and glycine produce excitatory responses, whereas in adult brains they produce inhibitory responses, intracellular ion concentration in immature neurons is higher in a passive distribution than in normal adult brains. Thus, on activation of the GABA receptors there is outward flow of ions (excitatory response- depolarisation). Significantly, thus, in immature neurons there is greater expression of the NKCC1 cotransporter which assists in this outward ion efflux. With postnatal development, soon after birth, the intracellular concentration decreases below equilibrium levels and GABA responses become inhibitory (hyperpolarisation) with ion influx being more standard. This requires the KCC2 cotransporter and the NKCC1 one is down-regulated (Karadsheh and Delfire, 2001). Immunohistochemical Analysis The research project will undertake an immunohistochemistry analysis wherewith the expressed KCC2 protein in murine CAD cells, differentiated or undifferentiated, will be localised using the principle that antibodies bind specifically to antigens in biological tissues (IHC World, 2007). There are two manners in which this antibody-antigen reaction can be visualised. In the first method, the antibody is conjugated to an enzyme, such as a peroxidase, that catalyses a colour-producing reaction (also called immunoblot staining (IHC World, 2007). In the second method the antibody is tagged to a fluorospore, such as FITC and rhodamine, producing a reaction that induces fluorescence. This is also called immunofluorescence analysis (IHC World, 2007). Immunofluorescence is a much more sensitive process with possibilities of multiple protein interactions. Specific methods such as Western Blot analysis use electrophoresis to separate the test proteins, KCC2 protein here, on polyacrylamide gels and electroblot these proteins on a protein binding membrane. The membrane captures the proteins and immobilises them. Specific antibodies, like rabbit anti-KCC2 polyclonal antibodies, tagged with fluorescent spores like FITC-conjugated goat anti-rabbit IgG and the fluorescence induced by the reactions are focused on by special high-calibre microscopes and quantified by special software like TINA (Rivera, 2004). The proteins may be expressed first with suitable mutated primer sections using methods of identification and purification such as reverse transcriptase polymer chain reaction (RT-PCR) analysis (Rivera, 2004). More details of the process is not essential as the research report will provide a more comprehensive account. Conclusion The principal aim of this paper is to introduce a research project that will investigate the molecular identity and expression patterns of the potassium-coupled chloride cotransporter KCC2 in cultured murine (caspase-activated deoxyribonuclease) CAD cell lines. The CAD cell line is specifically neuronal, a characteristic that is very suitable for KCC2 study as the cotransporter too is neuronal specific. Specific association between the culture cell line and the cotransporter has not been made in this paper. That has been left to the dissertation that shall elucidate in full the results of the research experiments. The paper, instead, expounds upon the seven cation-coupled chloride cotransporters that have been described to date with specific emphasis placed on KCC2. The cotransporter KCC2 is evident in neuronal processes with specific preference for inhibitory ones where ergic action is relevant. KCC2 action precedes the ergic one as the KCC2 action brings down the intracellular chloride concentration to optimum levels to make it possible for ergic action. The phylogenetic, molecular and structural-functional details of the cotransporters have also been provided with special emphasis on KCC2. The CAD cell line, new in studies like this, has been introduced. The cell line exhibits catecholaminergic voltage dependent currents across membranes. Specific experimentation details have not been provided. After providing the genetic, molecular and structural-functional details of the potassium-coupled chloride cotransporters the paper notes that, though there seem to be no controversy over what has been described so far of them, there are large gaps in information on many specific areas. Information on accurate chromosomal location, including that of KCC2, is not available. There is also no specific alignment between the pharmacological and physiological basis for the regulatory action of the cotransporters, specifically KCC2. This may be researched upon as a pharmacological association may allow development of new drugs with which the cotransporters, including KCC2, may be regulated in patients who may be suffering from malfunctioning regulatory action of these cotransporters. Of particular interest in this aspect is the prospect of KCC2 participating in human epilepsy where KCC2 deficiency may lead to the GABA inhibitory action being transferred to an excitatory one. Gamba, 2005, speculates that KCC2 position on human chromosome 20 may be investigated further in this aspect to investigate if KCC2 genetics plays any role in pathological conditions like epilepsy. Other possible areas of research would be the cell growth regulation roles of some of the isomers. The paper believes it has introduced the research project sufficiently. Reference 1. Cao, Guodong, et al, Caspase-Activated DNase/DNA Fragmentation Factor 40 Mediates Apoptotic DNA Fragmentation in Transient Cerebral Ischemia and in Neuronal Cultures, The Journal of Neuroscience, July 1, 2001, 21(13): 4678-4690. Extracted on 19th December, 2006, from: http://www.jneurosci.org/cgi/content/full/21/13/4678#Top 2. Delfire, E., Cation-Chloride Cotransporters in Neuronal Communication, News Physiol. 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Characteristics of SLC12 genes and their promoters Gene Cotransporter Chromosome Size, kb Number of Exons Promoter, bp Start Site, bp Reference Nos. SLC12A1 BSC1/NKCC2 Human: 15 80 26 2,255 -280 188, 330, 375, 423 Rat: 3 Mouse: 8 SLC12A2 BSC2/NKCC1 Human: 5 75 28 2,063 -270 80, 314, 332 Mouse: 18 SLC12A3 TSC Human: 16 55 26 1,019 -18 257, 308, 37, 400 Rat: 19 Mouse: 8 SLC12A4 KCC1 Human: 16 23 24 1,938 -121 180, 231, 392 Mouse: 5 SLC12A5 KCC2 Human: 20 30 24 NA NA 354, 380 Mouse: 8 SLC12A6 KCC3 Human: 15 NA NA NA NA 178, 292 SLC12A7 KCC4 Human: 5 NA NA NA NA 292 SLC12A8 CCC9 Human: 3 NA NA NA NA NA SLC12A9 CIP Human: 7 NA NA NA NA 49 NA, information not available. (Source: Gamba, G., 2005; Table 3. Posited without modification) Read More