Molecular Identity of the NKCC Cotransporters - Essay Example

www.academia-research.com Sumanta Sanyal d: 18/03/07 An Analytical Investigation into the Molecular Identity of the NKCC Cotransporters in Differentiated and Undifferentiated Murine Neuronal Cell Line CAD Abstract This paper introduces a research project that shall investigate the expression patterns of the NKCC (Sodium-Potassium-coupled Chloride) Cotransporters in a newly-developed murine neuronal cell line CAD (caspase-activated deoxyribonuclease) to try and establish molecular identity and other traits, such as genetic implications, of the two cotransporters in this subfamily - NKCC1 and NKCC2. The new cell line has been introduced hereafter and it is noted here that, significantly, it is one of the few thoroughly neuronal cell lines that can be induced to reversibly differentiate under culture conditions. This property is of special significance to the project as the emphasis of research is on the NKCC1 member, one that is known to have two isoforms - NKCC1a and NKCC1b. Of these NKCC1b is also known to be found in brain RNA (Gamba, 2005). It is noted here, though, that the two isoforms of the NKCC1 cotransporter is found only in the European eel (Anguilla anguilla) as per research of Cutler and Cramb, 2002. Nevertheless, there is ample evidence that NKCC1, in human and other mammalian species, is functionally implicated in CNS cells. It is observed by Gamba, 2005, that the NKCC1 cotransporter is activated by receptors and assists in neurotransmission by driving anions into the cell. It is also observed by Strange et al, 2000, that the work of the NKCC1 cotransporter complements that of the KCC2 one. The choice of the culture medium, the neuronal-specific CAD cell line, and the somewhat CNS-specific NKCC1 dovetails perfectly for a research attempt that seeks to establish new facts on the molecular identity and other expression patterns of these unique electroneutral cotransporters in cells of the central nervous system (CNS). G. Gamba's excellent 2005 review article on these cotransporters has been extensively used in this paper because it is the most comprehensive document prepared to date being inclusive of all aspects described so far. The Cation-Coupled Cotransport System The cation-coupled (Sodium and Potassium cations only) chloride cotransporters are a unique transmembranal transport system that is electrically neutral yet very effective in action (Gamba, 2005). These cotransporters constitute a secondary transcellular transport system that complements the primary cation transcellular transport system - the one mediated by the enzyme --ATPase. In the primary system the mover is an electrochemical gradient while in this chloride cotransporter system there is no such gradient, the reason why it has acquired the electroneutral label, and imbalances in chloride anion concentrations between intracellular and extracellular media constitute the prime mover of the system (Gamba, G., 2005). In absorptive and secretory epithelia there is need to transport ions and solutes in and out of the cells. Specific plasma membrane proteins mediate this transport system by either effecting sodium influx and potassium efflux with accompaniment of those ions and/or solutes that need to be transported (Gamba, G., 2005). Except in choroidal plexus, these cations move through the epithelial basolateral membrane mediated by the action of the enzyme --ATPase that creates an electrochemical gradient across the membrane. The plasma proteins mediate this transcellular transport that utilises this gradient to move target ions across the membrane and thus this system is called a secondary one while the enzyme-mediated cation movement is called the primary system. These two component systems together constitute the primary ion transport system across cellular membranes in human physiology (Gamba, G., 2005). On the other hand, the cation-coupled cotransport system is considered a secondary one and a very important one at that. The cations - and - are coupled with the anions in a 1:1 stoichiometry that works electroneutrally. These electroneutral cation-coupled chloride cotransporters themselves constitute the primary movers of the system (Gamba, G., 2005). Both epithelial and non-epithelial cells have this mechanism which has a net effect of chloride translocation across cell membranes. This is so because as soon as the cations are transported across the membranes together with the anions the cations - and , either jointly or singly - are quickly compensated by the --ATPase-mediated cation transport system. This, in effect, leaves the translocated anions unattended and creates an imbalance in concentration of these anions in the intra- and extracellular media (Gamba, G., 2005). This secondary cotransport system is functionally effective in diverse mammalian physiological actions like transepithelial ion absorption and secretion, cell volume regulation and setting chloride ion concentrations at required levels in intra- and extracellular media. It shall be seen later that this chloride ion imbalance mechanism is highly effective in certain neurotransmssion activities in neuronal precincts. CAD (caspase-activated deoxyribonuclease) Cell Line CATH.a Cell Line: Since the research will be conducted in the CAD cell line culture it is necessary to introduce this newly developed cell line. Also, since the research will be conducted with primary emphasis being placed on the NKCC1 isoform, which is CNS-specific to some extent, it is necessary to point out that truly neuronal cell cultures that can be induced to differentiate reversibly are still not very easy to develop and very few, if any, are in existence. There are a few neuronal-specific cell lines that exhibit differentiated phenotypes such as PC12 cell lines and the P19 cell lines, among others (Wang and Oxford, 2000), but these are not developed from CNS sources and they do not exhibit fully differentiated phenotypic features. Thus, it became necessary in recent times to develop a cell line like the CAD line that is capable of exhibiting fully-differentiated neuronal features and is derived exclusively from CNS sources. There exists a catecholaminergic cell line CATH.a that is developed by driving tumourigenesis in target cells in transgenic mice. The tumours are induced by oncogenes driven by cell specific promoters and the tumours are tyrosine hydroxylase (TH) positive and, as such, they are expressed exclusively in neuronal cells that specifically express TH (Wang and Oxford, 2000). Actually, when the TH gene is transcriptionally regulated it promotes neoplastic growth (Wang and Oxford, 2000). The CATH.a cell line has some unique properties that have signified it as a source for developing a neuronal-specific cell line CAD. The CATH.a cell line synthesise dopamine and norepinephrine. They express, as stated earlier, the catecholaminergic enzymes tyrosine hydroxylase and dopamine hydroxylase (Wang and Oxford, 2000). Structurally, they include neuronal-specific molecular components like neurofilaments and synapophysin but not neurites (Wang and Oxford, 2000). They also exhibit voltage-dependent ion channels - the tetrodoxin-sensitive sodium channels, the L- and N-type calcium channels and the potassium channels, among possible others (Wang and Oxford, 2000). Nevertheless, the CATH.a cell line had to be improved upon because it does not express many neuronal markers such as the neurites and it cannot be made to differentiate morphologically (Wang and Oxford, 2000). CAD Cell Line: Subcloning of the CATH.a cells, with only cells with unusually short processes being selected, on a single cell basis was used to develop the CAD cell line. These new cells have an undifferentiated phenotype that can proliferate indefinitely but, upon deprivation of serum from its culture medium, it stops proliferating and begins to differentiate into neuronal-like phenotypes. The differentiation, as mentioned earlier, is reversible that is induced immediately by re-introduction of serum into the culture medium. Proliferation commences with reversion (Wang and Oxford, 2000). The cells are choice research medium because they can be electrophysically induced from a very low resting potential to become active with high potentiality with mild hyperpolarization. The cells can thus be made to start sodium and potassium currents across their membranes (Wang and Oxford, 2000). Before differentiation, sodium currents induced are tetrodoxin (TTX)-sensitive and are conventionally active and inactive. The potassium currents can be distinguished pharmacologically into two rectifier type channels. Though there are also calcium cation channels these are not mentioned here as they are of no relevance to this particular research project. When the cells are made to differentiate they demonstrate three distinct channel differentiations. Two are of relevance to this research project. There is a 4-fold decrease in sodium current density and a corresponding 1.5-fold increase in potassium current density (Wang and Oxford, 2000). The fluctuation in current density with differentiation may be of interest to this research project. Uniquely, before differentiation, these cells are round or oval without any neuronal-like structures but, upon differentiation, they become spindle-shaped and begin to exhibit neuronal-like long processes (Wang and Oxford, 2000). This unique structural change upon differentiation can be fruitfully investigated in this research project as well. The differentiated CAD cells demonstrate long processes with ultrastructural components like parallel microtubules with intermediate filaments as well as varicosities with both large dense-bodied and small clear-bodied vesicles. This enables maximum space for neurotransmission (Wang and Oxford, 2000). The differentiated cells also express certain neuron-specific proteins like class 3 -tubulin, synaptotagmin, GAP-43 and SNAP-25 (Wang and Oxford, 2000). They also express active TH (tyrosine hydroxylase) and accumulate L-Dopa (Wang and Oxford, 2000). The CAD cell line, though it carries forward many essential features of its parent cell line CATH.a, does not, quite preferentially for this research purpose, express the SV40 T antigen that is found in its parent line (Wang and Oxford, 2000). The paper thus finds that the new CAD cell line, being highly neuronal in nature when induced to differentiate, seems suitable for research into possible expression patterns of the cotransporter sub-family . The Cation-Coupled Cotransporters Now that the cation-coupled chloride cotransport system and the new CAD cell line chosen for this research project has been spelled out it is time for the paper to move on to these unique cotransporters themselves. All the cotransporters, seven of them together with two orphan members, shall be expounded on very succinctly. Again, Gamba's paper has been chosen to guide in this as it is the most comprehensive document on these cotransporters as well as being the most up-to-date. All seven cotransporters belong to the SLC12 gene family (Table 1, Appendix). Though many of their genetic orientations have been described, to date, there are still some gaps in this. They can all be encoded by their individual cDNAs. There are four broad groups among them and the grouping has been done on the basis of three distinctions - the particular cation ( or or both) that is coupled to the ion, the stoichiometry involved in the transportation and the particular inhibitor the cotransporter group is sensitive to. The grouping is given as follows. i. The sodium-coupled cotransporter (NCC) makes one group. There is only one in this group and its renal-specific in action and sensitive to thiazide or benzothiaziadine. ii. Another sodium-coupled cotransporter (NCC) makes the second group. It is distinct from its counterpart in the first group by its sensitivity to sulfamoylbenzoic acid or bumetanide. It is renal-specific also. iii. The third group is constituted by two sodium/potassium-coupled cotransporters (NKCC1 and NKCC2). These are loop diuretic and also sensitive to sulfamoylbenzoic acid or bumetanide. iv. The fourth and last group is made up of the four potassium-coupled cotransporters (KCC1, KCC2, KCC3 and KCC4). These are dihydroindenyloxy-alkanoic acid (DIOA) (or furosemide) sensitive. (Adapted: Gamba, G., 2005) Stoichiometry-wise the cations and pass through cell membranes together with the chloride ions in a 1:1 ratio. The two NCC cotransporters together with the NKCC2 one are renal-specific in action while all the other cotransporters are widely expressed throughout mammalian bodies in various isoforms and encoded manifestations. All of them are functionally involved in transepithelial ion transfer in absorptive and secretory arenas, in cell volume regulation and electroneutral ion transfer to exclusively translocate the anions. Nevertheless, it must be noted here that not all of them are catholically involved in all these functions (Gamba, G., 2000). For example, KCC2 is brain-specific and assists in GABAergic inhibitory action by maintaining optimum electrochemical potential across neuronal membranes by regulating ion transfer. Thus, it is noteworthy that function often depends upon which specific body part and what type of cells the particular cotransporter is expressed in. All the cotransporters have interesting pharmacological and pathophysiological implications and their sensitivities to the various inhibitors can be specifically utilised to provide therapy and relief in many diseases. Also, mutated genes that encode some of these cotransporters are known to initiate such disorders like Bartter's Disease, Gitelman's Disease and Anderman's Disease (Gamba, G., 2000). Hereafter, the paper shall exclusively deal with the two NKCC cotransporters that have been signified as the object of research. The Cotransporters NKCC2 Genetic Identity: All the cotransporters belong to the SLC12 gene family, as mentioned earlier. The two bumetanide sensitive cotransporters are encoded by the SLC12A1 and SLC12A2 genes respectively. SLC12A1 encodes the NKCC2 isoform (Table 1, Appendix) that is renal-specific and is found expressed exclusively in the apical membrane of the thick ascending limb of Henle (Gamba, G., 2005). Payne and Forbush and Gamba, et al, simultaneously identified NKCC1 in mammalian kidney in 1994. The former group took cDNA encoding T84 human colonic basolateral cotransporter and constructed a -DNA random-primed probe and used it to screen rabbit cortical and medullary cDNA libraries inserted in ZAP (Gamba, G., 2005). They identified a clone 4750 bp in length from which they realised a 3297 bp bit that encoded a protein of 1099 amino acid residues. The presence of this protein was revealed by Northern Blot analysis to be expressed totally only in RNA from kidney. The duo conjectured that since this cotransporter protein was very identical with the basolateral isoform it was the apical one (Gamba, G., 2005). Payne and his group had earlier identified the basolateral NKCC1 isoform and, so, this one was named NKCC2. It is apically expressed and is renal-specific in action. Interestingly, in the same year 1994, Gamba et al also identified a 4546 bp clone in a cDNA library segmented from poly RNA taken from the inner stripe of the outer medulla of rat kidney (Gamba, G., 2005). They screened this with a -DNA random-primed probe taken from the coding region of a flTSC transporter and identified a 3285 bp section encoding a protein of 1095 amino acid residues. Again, Northern Blot analysis to identify tissue distribution revealed that the protein was expressed exclusively in total RNA from kidney (Gamba, G., 2005). Gamba's group had previously named the thiazide-sensitive cotransporter cDNA clone identified in flounder urinary bladder as flTSC. Thus, the bumetanide-sensitive apical cDNA clone Gamba and Co. had identified in rat renal outer medulla was named rBSC1. Thus, this apically expressed renal-specific bumetanide-sensitive cotransporter is either called BSC1, as per Gamba and Co., or NKCC2, as per Payne and Co. (Gamba, G., 2005). Since then, this sodium-potassium-coupled chloride cotransporter has been identified in both mouse and human kidney. Simon et al (Gamba, G., 2005) identified BSC1/NKCC2 in human kidney in their search for the genetic cause of Bartter's Disease and found this cotransporter implicated with mutation of the SLC12A1 gene (Gamba, G., 2005). They found that the human BSC1/NKCC2 is 95% identical to rabbit and 93% identical to mouse and rat BSC1/NKCC2. Molecular Identity: BSC1/NKCC2 has a central hydrophobic domain with a 475 residue bit with 12 putative transmembranic segments that is flanked by two predominantly hydrophilic domains. One of these hydrophilic domains is a short amino-terminal domain of 165 amino acids while the other is a carboxy-terminal domain of 450 residues (Gamba, G., 2005). Both these domains are thoroughly intra-cellular. Both these domains have several putative protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites. The central hydrophobic domain has a long hydrophilic loop located between the transmembanic segments 7 and 8. This loop contains two putative N-glycosylation sites (Gamba, G., 2005). Molecular Variation: Molecular variation in these electroneutral cotransporters has been widely reported with numerous isoforms for each member of the family. There are six such variants for the BSC1/NKCC2 cotransporter reported in mouse kidney (Gamba, G., 2005). They are all results of two alternate splicing mechanisms. One splicing mechanism is due to three mutually exclusive cassette exons (Gamba, 2005) of 96 bp which encode 32 residues in the second half of the transmembranic domain TM2 and the contiguous intracellular loop placed between TM2 and TM3. The three cassette exons have been termed A, B, and F. Correspondingly, three splice variants of the BSC1/NKCC2 cotransporter has been reported in many tissues that are mutually exclusive through exclusion of the 32 amino acids encoded by the A, B, and F cassette exons. There is also report of an isoform containing both A and F by Yang et al (Gamba, G., 2005) though no corresponding protein has been found yet. This also may be of interest to this research project. Functional Identity: The primary function of the apical NKCC2 is to construct the major salt transport pathway in mammalian TALH (thick ascending limb of the loop of Henle). TALH is heavily involved in reabsorbing 16-20% of glomerular filtrate (Gamba, G., 2005). It has already been spelled out in a previous section how electroneutral ion exchange takes place and how the net effect is translocation across cellular membranes. In this case as well the NKCC2 cotransporter effects salt transfer from the lumen to the inner cells through this process and assists in the reabsorption work of the kidney. It is noted here that this is its most important function since it, like the TSC transporter, is present in apical membranes (Gamba, G. 2005). Thus, it can be targeted by the loop diuretics like bumetanide. The mechanism is quite complex and research papers, even original ones, are easily available but, since the more necessary target of research is the NKCC1 cotransporter, the NKCC2 cotransporter function is placed here in a compendium. NKCC1 Genetic Identity: As stated earlier, the NKCC1 cotransporter is basolaterally expressed and also bumetanide sensitive like its isoform NKCC2. It corresponds to the SLC12A2 gene (Table 1, Appendix). It is found expressed in both epithelial and non-epithelial cells. In epithelial cells it is expressed in basolateral membrane in organs and tissues like gills, trachea intestine and renal collecting duct (Gamba, G., 2005). Interestingly, it is expressed in the apical membrane of the choroids plexus (Gamba, G., 2005). Two groups also identified NKCC1 cDNA simultaneously in 1994. Xu et al (Gamba, G., 2005) utilised monoclonal antibodies J3 and J7 to probe shark rectal gland cDNA library. The antibodies were successful because they can recognise epitopes in the carboxy terminal domain of the cotransporter. A 5260 bp cDNA clone that encoded a protein of 1191 amino acid residues was revealed and subsequently transfected into HEK (human embryonic kidney)-293 cells and a bumetanide-sensitive cotransporter was identified that demonstrated functional characteristics similar to that already exhibited in the shark rectal gland. The cDNA was nominated NKCC1 (Gmaba, G., 2005). Northern Blot analysis for tissue distribution revealed that NKCC1 is expressed in many tissues. The mammalian ortholog was also identified by the same team in human colon derived T84 cell line when its cDNA library was probed using shark cDNA (Gamba, G., 2005). The identified 3036 bp bit revealed that human NKCC1 is made up of 1212 amino acid residues and it has a molecular weight of 132 kDa. Northern Blot analysis revealed that this human cotransporter is also expressed in many tissues. The second research group, Delfire et al, probed mouse inner medullary collecting duct derived cell line mIMCD-3 with degenerative primers designed over highly homologous regions of putative transmembranic domains 1 and 10 of TSC and BSC1/NKCC2 (Gamba, G., 2005). For the same reason as applied to BSC1/NKCC2 the authors denominated this basolateral cotransporter BSC2/NKCC1 (Gamba, G., 2005). Molecular Identity: The BSC2/NKCC1 topology is highly identical to that of its isoform BSC1/NKCC2. Diagram 1, Appendix, posits the NKCC1 structure against that of the KCC2 cotransporter, both being functionally neuronal. There is the central hydrophobic domain with a long hydrophilic loop moving extra-cellularly and located between the 7th and 8th transmembranal segments with two putative N-glycosylation sites (Gamba, G., 2005). The TSC transporter, though, it is noted here, has the same topology but with only one N-glycosylation site (Gamba, G., 2005). It is also emphatically noted here for the purpose of the research that the BSC2/NKCC1 topology has been experimentally proved while that of the BSC1/NKCC2 cotransporter has not. This is extremely important for the research effort as a direction can to set to conduct the research study in such a manner that BSC1/NKCC2 topology, that has not to date been experimentally resolved, can be studied in expression patterns in suitable cell cultures. The BSC2/NKCC1 topology is demonstrated in Diagram 1, Appendix. Gerelsaikhan and Turner (Gamba, G., 2005) used an in vitro translation system that studied the membrane-penetrating properties of the putative transmembranic helices. They fused each of these helices with a carboxy terminal with its reported multiple N-glycosylation sites. They discovered that NKCC1 indeed has 12 transmembranic segments and the first eight are classically 20 residues while the last four are of 36 residues length. They proposed from this that, together, with the variant lengths, the segments took up a hairpin-like shape across the membrane or were, at least, non-helical or partially helical in shape (Gamba, G., 2005). They further reported that the presence of asparagines and proline between the 9th and 10th and the 11th and 12th segments suggested that there was evidence of hairpin-like helices (Gamba, G., 2005). To date, as per Gamba, the BSC2/NKCC1 cotransporter has been reported in three other species - rat, Bos taurus and Anguilla anguilla (eel). The eel expressions are interesting because two isoforms NKCC1a and NKCC1b have been discovered (Gamba, G., 2005). The NKCC1b cotransporter isoform is expressed exclusively in eel brain RNA. Cutler and Cramb (Gamba, G., 2005) identified these two isoforms and found that variation is possible through the difference detected between NKCC1a and NKCC1b in the first 80-90 residues in the amino-terminal domain where degree of similarity is not >35%. The rest of the sequence has a 85%, making for an overall similarity of 80% (Gamba, G., 2005). NKCC1a of 13 kb was found expressed in all tissues while the NKCC1b isoform of 6 kb was found expressed in only brain RNA (Gamba, G., 2005). This isoform expression is explained by the duo as per the prevalent conjecture that the teleost genome, in ancient times, underwent duplication (Gamba, G., 2005). Molecular Variation: Spliced variants of the SLC12A2 gene encoding variants of the NKCC1 cotransporter has been reported in many organisms. Randall et al (Gamba, G., 2005) deliberately searched for such variants in mouse with PCR (polymerase chain reaction) amplifying segments containing two or three exons. They found a spliced variant in mouse brain RNA that lacked the entire 48 bp that makes up the 21st exon. This implies that a 16 residue transcript in the carboxy-terminal is missing. Tissue distribution analyses revealed that this variant without exon 21 is widely evident in the brain tissues except in the choroids plexus where one with the exon 21 is present (Gamba, G., 2005). This particular exon 21 lacking variant has also been reported in human ocular-trabecular meshwork cells and in other tissues (Gamba, G., 2005). The variant in 14 tested tissues evinced 68-fold variation in isoform (Gamba, G., 2005). This fact may be of great interest to the research project especially since the culture cell line CAD is murine in origin and of neuronal basis. It is also noted that this spliced variant without exon 21 is reported in most brain tissues and that its physiological significance remains unknown. The absence of the exon 21 removes the only putative PKA site in the whole SLC12A2 sequence (Gamba, G., 2005). Functional Identity: Again, it is noted here that NKCC1 also effects net ion translocation across cellular membranes to do all its work. Since it is present in basolateral membranes and is also ubiquitously expressed, it is heavily involved in cell volume regulation. When there are osmolarity differences between intracellular and extracellular media this cotransporter, being present in the basolateral membrane, effects , and transfer into or out of cell to either increase or decrease cell volume. When osmolarity is high extracellularly, there is net water outgo from the cell and it shrinks. To counter too much shrinkage, NKCC1 inducts ions into the cell to increase intracellular osmolarity and prevent water outgo. The opposite is done, ion outgo, when the osmolarity is high within the cell and water is drawn and the cell swells. The outgo of ions through NKCC1 cotransport decreases osmolarity within the cell and the swelling stops as water goes out (Gamba, G., 2005). NKCC1 is also heavily involved in providing anions secreted in the apical membrane to cells through the cystic fibrosis transmembrane conductance regulator (CFTR)-related mechanism (Gamba, G., 2005). NKCC1 activity is tightly controlled by hormones induced by receptors such as isoproterenol and vasopressin (Gamba, G., 2005). The receptors phosphorylate the cotransporter protein that increases its activity. The converse is true for dephosphorylation when the cotransporter activity is diminished (Gamba, G., 2005). This GABA-ergic effect on the cotransporter is significant for GABA-inhibitory neurotransmission. The GABA-ergic action is induced by translocating ions into the cell through transmembranic ligand gates (Payne et al, 1996). This specific action is conducted through the activation of NKCC1 by receptors that do so by phosphorylating the cotransporter protein (Gamba, G., 2005). Thus, NKCC1 activity promotes excitatory GABA action. Several stimuli processes phosphorylate the cotransporter protein increasing activity. Dephosphorylation decreases it and inhibits NKCC1 action. For example, protein phosphatase 1 inhibitor calyculin A prevents dephosphorylation of the protein and sustains NKCC1 activity (Gamba, G, 2005). It is also noted that the cotransporter does not seem to be activated by a PKA mechanism. Recent structure-function studies have revealed that, aside from the cotransporter being activated by intracellular levels, especially decreases, the cotransporter regulation by phosphorylation/dephosphorylation is also mediated by threonine residues in the amino-terminal as well as other protein motifs on the terminal associated with binding of kinases and phosphatases (Gamba, G, 2005). This induces an inhibitory postsynaptic potential (IPSP) across cell membranes that hyperpolarizes the membrane and drives ions into the cell (Payne et al, 1996). An interesting study by Strange et al, 2000, as well as DeFazio, et al, 2002, revealed that NKCC1 activity is opposite to that of the potassium-coupled chloride cotransporter KCC2. KCC2 works to drive ions out of the cell (though this may not always be so, Strange, et al, 2000) when -action drives the intracellular electrochemical gradient up through the IPSP (inhibitory postsynaptic potential) it sets up (Payne, et al, 1996). KCC2 action regulates cell concentration to keep it at optimum levels so as not to totally disrupt the inhibitory action (Payne, et al, 1996). It is noted here that NKCC1 does the opposite through observed receptor action and draws in ions. It is also noted here that NKCC1 is expressed at high levels in the early stages of human life (up to postnatal for intraneuronal regulation) while this expression diminishes with age and KCC2 expression increases (Gamba, G, 2005). This fact may be explained by GABA excitatory action (NKCC1) prenatally and inhibitory action (KCC2) postnatally (Gamba, G, 2005). This too may of interest to the research. Immunohistochemical Analysis A immunohistochemical study will be conducted for NKCC1 protein expression in the differentiated and undifferentiated CAD cells after suitable cotransporter expression has been initiated by primers selected through methods like RT-PCR analysis. The immunoihistochemical study such as Western Blot analysis utilises the affinity antibodies have for antigens. There are two principal methods of analysis. The differences in methodology between these two depend more on how the antibody-antigen reactions can be visualised for easy optical assessment, nowadays with special software. The first method utilises antibodies conjugated with enzymes like peroxidases that produce colourful reactions (IHC World, 2007). This is usually called immunoblot analysis. The second method utilises antibodies tagged with fluorescent spores such as FITC and rhodamine (radioactive). During reaction with antigens the fluorescence produced is assessed with special microscopes, both light and electron, and quantified. For radioactive tags, autoradiography is conducted to quantify protein content. This is usually called immunofluorescence analysis (IHC World, 2007). Actually, analytical methods like Western blot analysis use electrophoresis to separate the proteins, the NKCC1 here, from the RT-PCR products onto polyacrylamide gels and electroblot them onto protein binding membranes. The membranes take hold of the proteins and transfix them (IHC World, 2007). They are ready for reaction with antibodies like rabbit anti-NKCC1 antibodies (Flemmer et al, 2002). These antibodies may be conjugated with enzymes, as per the first method, or tagged with fluorescent material, as per the second method, and the visualised reactions quantified through microscopy and suitable software for procurement of the results. It is also noted that NKCC1 has certain phospho-specific antibodies that directly relate to phosphorylation of the NKCC1 N terminus during functional activation of the cotransporter (Flemmer et al, 2002). Though there are more general antibodies these special ones are also significant. This is a barebone treatment of the analytical methods with only the bare principles being stated. A more elaborate description of the methodology will be available in the report to the research experiments. Conclusion It is concluded here that the paper is considered quite successful in introducing the NKCC cotransporters for the dissertation that will project the research study. It is noted that the paper, as an introduction, has avoided too much specificity in favour of a comprehensive approach. This is why Gamba's paper has been utilised so extensively instead of specific research papers that are inclined to favour only a few aspects of the cotransporters while Gamba, in review, has been more comprehensive. 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 22nd December, 2006, from: http://www.jneurosci.org/cgi/content/full/21/13/4678#Top 2. Cutler, Christopher P., Cramb, Gordon, Two Isoforms of the cotransporter are expressed in the European eel (Anguilla anguilla), Biochimica et Biophysica Acta, 1566, (2002), 92-103. Extracted on 22nd December, 2006, from: http://www.medscape.com/medline/abstract/12421541queryText=eels 3. DeFazio, R. Anthony, et al, Activation of A Type -Aminobutyric Acid Receptors Excites Gonadotropin-Releasing Hormone Neurons, Molecular Endocrinology 16(12): 2872-2891, 2002. Extracted on 17th December, 2006, from: http://mend.endojournals.org/cgi/content/full/16/12/2872#top 4. Delfire, E., Cation-Chloride Cotransporters in Neuronal Communication, News Physiol. Sci., 15: 309-312; 2000. Extracted on 22nd December, 2006, from: http://physiologyonline.physiology.org/cgi/content/full/15/6/309#top 5. Flemmer, Andreas W., et al, Activation of the N-K-Cl Cotransporter NKCC1 Detected with a Phospho-Specific Antibody, J. Biol. Chem., Vol. 277, Issue 40, 37551-37558, Oct. 4, 2002. Extracted on 18th March, 2007, from: http://www.jbc.org/cgi/content/full/277/40/37551 6. Gamba, Gerardo, Molecular Physiology and Pathophysiology of Electroneutral Cation-Chloride Cotransporters, Physiol. Rev., 85: 423-493, 2005. Extracted on 24th December, 2006, from: http://physrev.physiology.org/cgi/content/full/85/2/423#ABSTRACT 7. Immunohistochemistry, IHC World, 2007. Extracted on 15th March, 2007, from: http://www.ihcworld.com/introduction.htm#top 8. Payne, John A., et al, Molecular Characterization of a Putative K-Cl Cotransporter in Rat Brain: A Neuronal Specific Isoform, Journal of Lipid Research, Vol. 271, No. 27, July 5, 1996, 16245-16252. Extracted on 22nd December, 2006, from: http://www.jbc.org/cgi/content/full/271/27/16245#abs 9. Wang, Haibin, and Oxford, Gerry S., Voltage-Dependent Ion-Channels in CAD Cells: A Catecholaminergic Neuronal Line That Exhibits Inducible Differentiation, The Journal of Neurophysiology, 84: 2888-2895, 2000. Extracted on 24th December, 2006, from: http://jn.physiology.org/cgi/content/full/84/6/2888#Top Appendix Diagram 1: Relative positions and activity characteristics of NKCC1 and KCC2 (Source: Delfire, E., 2000) TABLE 1. 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) Bibliography Isenring, Paul, et al, Comparison of Na-K-Cl Cotransporters NKCC1, NKCC2, and the HEK Cell Na-K-Cl - , J Biol Chem, Vol. 273, Issue 18, 11295-11301, May 1, 1998. Extracted on 24th December, 2006, from: http://www.jbc.org/cgi/content/full/273/18/11295#Top Qi, Yanping, et al, Characterisation of a CNS Cell Line, CAD, in which Morphological Differentiation is Initiated by Serum Deprivation, The Journal of Neuroscience, Vol. 17, No. 4, pp: 1217-1225, February, 1997. Extracted on 22nd December, 2006, from: http://www.jneurosci.org/cgi/content/full/17/4/1217#top Russell, John M., Sodium-Potassium-Chloride Cotransport, Physiological Reviews, Vol. 80, No. 1, January 2000, pp. 211-276. Extracted on 22nd December, 2006, from: http://physrev.physiology.org/cgi/content/full/80/1/211#SEC1 Read More