Na~+-K~+-2Cl~-共转运蛋白在L6骨骼肌细胞调节性体积增加中的作用(英文)

在许多类型的哺乳动物细胞中,细胞体积对介导胰岛素发挥其生理功能起关键作用.细胞体积调节机制在胰岛素的主要靶组织骨骼肌中尤为重要.为了解该组织中细胞体积调节的机制,对Na+K+2Cl-共转运蛋白在大鼠L6骨骼肌细胞中的表达及其在调节性体积增加中的作用进行了研究.应用免疫印迹法,在L6肌管细胞中检测到分子量为170kD的钠钾氯共转运蛋白.K+(86Rb+)输入实验结果表明,54%的86Rb+是通过钠钾氯共转运蛋白输入细胞的,而其余86Rb+的输入是通过钠钾三磷酸腺苷酶和其他未知转运蛋白实现的.当L6细胞被置于高渗透压溶液(420mosmolL)中,导致细胞体积减少40%,同时钠钾氯共转运蛋白的活性迅速增加(高达2倍).细胞体积在60min内恢复至正常,但在钠钾氯共转运蛋白抑制剂bumetanide存在时,这种调节性体积增加的过程被阻断.这些结果表明,L6肌管细胞表达具有生理活性的钠钾氯共转运蛋白;此转运蛋白被高渗透压诱导造成的细胞体积缩小激活的现象表明,它是细胞调节性体积增加(RVI)机制中的关键元素,在L6骨骼肌细胞体积的调节中起重要作用.     

Effect of high-NaCl or high-KCl diet on hepatic Na+- and K+-receptor sensitivity and NKCC1 expression in rats

We previously reported that the bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter (NKCC1) is involved in the hepatic Na+ and K+ sensor mechanism. In the present study, we examined the effects of a high-NaCl or high-KCl diet on hepatic Na+ and K+ receptor sensitivity and NKCC1 expression in the liver of Sprague-Dawley rats. RT-PCR and Western blots were used to measure NKCC1 mRNA and protein expression, respectively. Infusion of hypertonic NaCl or isotonic KCl + NaCl solutions into the portal vein increased hepatic afferent nerve activity (HANA) in a Na+ or K+ dose-dependent manner. After 4 wk on a high-NaCl or high-KCl diet, HANA responses were attenuated compared with animals fed a normal diet, and NKCC1 expression was reduced. These results show that a high-NaCl or high-KCl diet decreases NKCC1 expression in the liver, and it might cause a reduction in hepatic Na(+)- and K(+)-receptor sensitivity.     

TNF-alpha down-regulates the Na+-K+ ATPase and the Na+-K+-2Cl-cotransporter in the rat colon via PGE2

TNF-alpha is believed to play a pivotal role in the pathogenesis of inflammatory bowel diseases which have diarrhea as one of their symptoms. This work studies the effect of the cytokine on electrolyte and water movements in the rat distal colon using an intestinal perfusion technique and attempts to determine its underlying mechanism of action. TNF-alpha inhibited net water and chloride absorption, down-regulated in both surface and crypt colonocytes the Na+-K+-2Cl- cotransporter, and reduced the protein expression and activity of the Na+-K+ ATPase. Indomethacin up-regulated the pump and the cotransporter in surface cells but not in crypt cells, and in its presence, TNF-alpha could not exert its effect, suggesting an involvement of PGE2 in the cytokine action. The effect of TNF-alpha on the pump and symporter was studied also in cultured Caco-2 cells in isolation of the effect of other cells and tissues, to test whether the cytokine acts directly on intestinal cells. In these cells, TNF-alpha and PGE2 had a similar effect on the pump expression and activity as that observed in crypt cells but were without any effect on the Na+-K+-2Cl- cotransporter. It was concluded that the effect of the cytokine on colonocytes is mediated via PGE2. By inhibiting the Na+-K+ ATPase, it reduces the Na+ gradient needed for NaCl absorption, and by down-regulating the expression of the Na+-K+-2Cl- symporter, it reduces basolateral Cl- entry and luminal Cl- secretion. The inhibitory effect on absorption is more significant than the inhibitory effect on secretion resulting in a decrease in net electrolyte uptake and consequently in more water retention in the lumen.     

Physiology and pathophysiology of Na+/H+ exchange and Na+ -K+ -2Cl- cotransport in the heart, brain, and blood

Maintenance of a stable cell volume and intracellular pH is critical for normal cell function. Arguably, two of the most important ion transporters involved in these processes are the Na+/H+ exchanger isoform 1 (NHE1) and Na+ -K+ -2Cl- cotransporter isoform 1 (NKCC1). Both NHE1 and NKCC1 are stimulated by cell shrinkage and by numerous other stimuli, including a wide range of hormones and growth factors, and for NHE1, intracellular acidification. Both transporters can be important regulators of cell volume, yet their activity also, directly or indirectly, affects the intracellular concentrations of Na+, Ca2+, Cl-, K+, and H+. Conversely, when either transporter responds to a stimulus other than cell shrinkage and when the driving force is directed to promote Na+ entry, one consequence may be cell swelling. Thus stimulation of NHE1 and/or NKCC1 by a deviation from homeostasis of a given parameter may regulate that parameter at the expense of compromising others, a coupling that may contribute to irreversible cell damage in a number of pathophysiological conditions. This review addresses the roles of NHE1 and NKCC1 in the cellular responses to physiological and pathophysiological stress. The aim is to provide a comprehensive overview of the mechanisms and consequences of stress-induced stimulation of these transporters with focus on the heart, brain, and blood. The physiological stressors reviewed are metabolic/exercise stress, osmotic stress, and mechanical stress, conditions in which NHE1 and NKCC1 play important physiological roles. With respect to pathophysiology, the focus is on ischemia and severe hypoxia where the roles of NHE1 and NKCC1 have been widely studied yet remain controversial and incompletely elucidated.     

Tumor necrosis factor alpha down-regulates the Na+-K+ ATPase and the Na+-K+2Cl- cotransporter in the kidney cortex and medulla

The effect of TNF-alpha on the renal Na+-K+ pump and the Na+-K+2Cl- cotransporter was investigated in the rat. Animals were injected with the cytokine, and 4h later, a homogenate from the cortical and medullary tissues was prepared and used to assay the activity of the Na+-K+ ATPase and the protein expression of the pump and symporter. TNF-alpha reduced the activity and expression of the pump in both cortex and medulla, and its effect disappeared when animals were pre-treated with indomethacin, suggesting that TNF-alpha acts via PGE2. Higher levels of PGE2 were detected by enzyme immunoassay, in kidney tissues isolated from rats treated with PGE2, thus confirming this hypothesis. The cytokine also down-regulated the Na+-K+2Cl- cotransporter but this effect was not abrogated by indomethacin. PGE2, injected into animals, exerted a dose-dependent effect. Low doses did not have any effect on the two transporters in the cortex while high doses inhibited and down-regulated the pump and up-regulated the cotransporter. In the medulla low doses increased the activity and expression of the pump but down-regulated the cotransporter while high doses exerted an exactly opposite effect on the two transporters. It was concluded that the effect of TNF-alpha on the pump is mediated via PGE2 which is released at relatively high doses. The effect of the cytokine on the cotransporter is, however, independent of PGE2.     

Na+-K+-2Cl-共转运蛋白在L6骨骼肌细胞调节性体积增加中的作用

在许多类型的哺乳动物细胞中,细胞体积对介导胰岛素发挥其生理功能起关键作用.细胞体积调节机制在胰岛素的主要靶组织骨骼肌中尤为重要.为了解该组织中细胞体积调节的机制,对Na K 2Cl-共转运蛋白在大鼠L6骨骼肌细胞中的表达及其在调节性体积增加中的作用进行了研究.应用免疫印迹法,在L6肌管细胞中检测到分子量为170kD的钠钾氯共转运蛋白.K (86Rb )输入实验结果表明,54%的86Rb 是通过钠钾氯共转运蛋白输入细胞的,而其余86Rb 的输入是通过钠钾三磷酸腺苷酶和其他未知转运蛋白实现的.当L6细胞被置于高渗透压溶液(420mosmolL)中,导致细胞体积减少40%,同时钠钾氯共转运蛋白的活性迅速增加(高达2倍).细胞体积在60min内恢复至正常,但在钠钾氯共转运蛋白抑制剂bumetanide存在时,这种调节性体积增加的过程被阻断.这些结果表明,L6肌管细胞表达具有生理活性的钠钾氯共转运蛋白;此转运蛋白被高渗透压诱导造成的细胞体积缩小激活的现象表明,它是细胞调节性体积增加(RVI)机制中的关键元素,在L6骨骼肌细胞体积的调节中起重要作用.     

Na+-K+-2Cl- cotransporters and Cl- channels regulate citric acid cough in guinea pigs.

Loop diuretics have been shown to inhibit cough and other airway defensive reflexes via poorly defined mechanisms. We test the hypothesis that the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC1) is expressed by sensory nerve fibers innervating the airways where it plays an important role in regulating sensory neural activity. NKCC1 immunoreactivity was present on the cell membranes of most nodose and jugular ganglia neurons projecting to the trachea, and it was present on the peripheral terminals of putative mechanosensory nerve fibers in the airways. In urethane-anesthetized, spontaneously breathing guinea pigs, bolus application of citric acid (1 mM to 2 M) to an isolated and perfused segment of the tracheal mucosa evoked coughing and respiratory slowing. Removal of Cl- from the tracheal perfusate evoked spontaneous coughing and significantly potentiated cough and respiratory slowing reflexes evoked by citric acid. The NKCC1 inhibitor furosemide (10-100 microM) significantly reduced both the number of coughs evoked by citric acid and the degree of acid-evoked respiratory slowing (P < 0.05). Localized tracheal pretreatment with the Cl- channel inhibitors DIDS or niflumic acid (100 microM) also significantly reduced cough, whereas the GABAA receptor agonist muscimol potentiated acid-evoked responses. These data suggest that vagal sensory neurons may accumulate Cl- due to the expression of the furosemide-sensitive Cl- transporter, NKCC1. Efflux of intracellular Cl-, in part through calcium-activated Cl- channels, may play an important role in regulating airway afferent neuron activity.     

Spatially distributed alternative splice variants of the renal Na-K-Cl cotransporter exhibit dramatically different affinities for the transported ions

Three splice variants of the renal Na-K-Cl cotransporter (NKCC2 F, A, and B) are spatially distributed along the thick ascending limb of the mammalian kidney. To test whether NKCC2 splice variants differ in ion transport characteristics we expressed cDNAs encoding rabbit NKCC2 F, A, and B in Xenopus oocytes and determined the ion dependence of bumetanide-sensitive (86)Rb influx. The three splice variants of NKCC2 showed dramatic differences in their kinetic behavior. The medullary variant F exhibited 3-4-fold lower affinity than variants A and B for Na(+) and K(+). Chloride affinities also markedly distinguish the three variants (K(m)F = 111.3, K(m)A = 44.7, and K(m)B = 8.9 mm Cl(-)). Thus, the kinetic properties of the NKCC2 splice variants are consistent with the spatial distribution of the variants along the thick ascending limb as they are involved in reabsorbing Na(+), K(+), and Cl(-) from a progressively diluted fluid in the tubule lumen. Variant B also showed an anomalous inhibition of rubidium influx at high extracellular Na(+) concentrations, possibly important in its highly specialized role in the macula densa. The adaptation of the kinetic characteristics of the NKCC2 variants to the luminal concentrations of substrate represents an excellent example of functional specialization and diversity that can be achieved through alternative mRNA splicing.     

Molecular mechanisms of Cl- transport by the renal Na(+)-K(+)-Cl- cotransporter. Identification of an intracellular locus that may form part of a high affinity Cl(-)-binding site

The 2nd transmembrane domain (tm) of the secretory Na(+)-K(+)-Cl(-) cotransporter (NKCC1) and of the kidney-specific isoform (NKCC2) has been shown to play an important role in cation transport. For NKCC2, by way of illustration, alternative splicing of exon 4, a 96-bp sequence from which tm2 is derived, leads to the formation of the NKCC2A and F variants that both exhibit unique affinities for cations. Of interest, the NKCC2 variants also exhibit substantial differences in Cl- affinity as well as in the residue composition of the first intracellular connecting segment (cs1a), which immediately follows tm2 and which too is derived from exon 4. In this study, we have prepared chimeras of the shark NKCC2A and F (saA and saF) to determine whether cs1a could play a role in Cl- transport; here, tm2 or cs1a in saF was replaced by the corresponding domain from saA (generating saA/F or saF/A, respectively). Functional analyses of these chimeras have shown that cs1a-specific residues account for most of the A-F difference in Cl- affinity. For example, Km(Cl-)s were approximately 8 mm for saF/A and saA, and approximately 70 mm for saA/F and saF. Intriguingly, variant residues in cs1a also affected cation transport; here, Km(Na+)s for the chimeras and for saA were all approximately 20 mM, and Km(Rb+) all approximately 2 mM. Regarding tm2, our studies have confirmed its importance in cation transport and have also identified novel properties for this domain. Taken together, our results demonstrate for the first time that an intracellular loop in NKCC contributes to the transport process perhaps by forming a flexible structure that positions itself between membrane spanning domains.     

Role of Cl- in cerebral vascular tone and expression of Na+-K+-2Cl- co-transporter after neonatal hypoxia-ischemia

Chloride (Cl-) efflux induces depolarization and contraction of vascular smooth muscle cells. In the basilar arteries from the New Zealand white rabbits, the role of Cl- flux in serotonin-induced contraction was demonstrated by (i) inhibition of Na+-K+-2Cl- co-transporter (NKCC1) to decreased Cl- influx with bumetanide; (ii) a disabled Cl-/HCO3- exchanger with bicarbonate free HEPES solution; (iii) blockade of Cl- channels using 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and indanyloxyacetic acid 94, R-(+)-methylindazone (R-(+)-IAA-94); and (iv) substitution of extracellular Cl- with methanesulfonate acid (113 mmol/L; Cl-, 10 mmol/L). In addition, the expression of NKCC1 in brain tissues after neonatal hypoxia-ischemia was examined at mRNA and protein levels using RT-PCR and Western blotting techniques. NKCC1 mRNA and protein expressions were increased at 24 and 48 h and returned to normal levels at 72 h after hypoxia insult when compared with the control littermates. In conclusion, Cl- efflux regulates cerebral circulation and the up-regulation of NKCC1 after neonatal hypoxia-ischemia may contribute to brain injury.     

Prolonged interferon-gamma exposure decreases ion transport,NKCC1, and Na+-K+-ATPase expression in human intestinal xenografts in vivo

IFN-gamma is elevated in intestinal inflammation and alters barrier and transport functions in human colonic epithelial cell lines, but its effects on normal human small intestinal epithelium in vivo are poorly defined. We investigated effects of prolonged IFN-gamma exposure on ion transport and expression of transporters by using human fetal small intestinal xenografts. Xenograft-bearing mice were injected with IFN-gamma, and 24 h later xenografts were harvested and mounted in Ussing chambers. Baseline potential difference (PD) was not affected by IFN-gamma treatment. However, conductance was enhanced and agonist-stimulated ion transport was decreased. IFN-gamma also decreased expression of the Na+-K+-2Cl- cotransporter and the alpha-subunit of Na+-K+-ATPase compared with controls, whereas levels of the calcium-activated Cl- channel and CFTR were unaltered. Thus prolonged exposure to IFN-gamma leads to decreased ion secretion due, in part, to decreased ion transporter levels. These findings demonstrate the implications of elevated IFN-gamma levels in human small intestine and validate the human intestinal xenograft as a model to study chronic effects of physiologically relevant stimuli.     

The role of volume-sensitive ion transport systems in regulation of epithelial transport

This review focuses on using the knowledge on volume-sensitive transport systems in Ehrlich ascites tumour cells and NIH-3T3 cells to elucidate osmotic regulation of salt transport in epithelia. Using the intestine of the European eel (Anguilla anguilla) (an absorptive epithelium of the type described in the renal cortex thick ascending limb (cTAL)) we have focused on the role of swelling-activated K+- and anion-conductive pathways in response to hypotonicity, and on the role of the apical (luminal) Na+-K+-2Cl- cotransporter (NKCC2) in the response to hypertonicity. The shrinkage-induced activation of NKCC2 involves an interaction between the cytoskeleton and protein phosphorylation events via PKC and myosin light chain kinase (MLCK). Killifish (Fundulus heteroclitus) opercular epithelium is a Cl(-)-secreting epithelium of the type described in exocrine glands, having a CFTR channel on the apical side and the Na+/K+ ATPase, NKCC1 and a K+ channel on the basolateral side. Osmotic control of Cl- secretion across the operculum epithelium includes: (i) hyperosmotic shrinkage activation of NKCC1 via PKC, MLCK, p38, OSR1 and SPAK; (ii) deactivation of NKCC by hypotonic cell swelling and a protein phosphatase, and (iii) a protein tyrosine kinase acting on the focal adhesion kinase (FAK) to set levels of NKCC activity.     

Novel insights regarding the operational characteristics and teleological purpose of the renal Na+-K+-Cl2 cotransporter (NKCC2s) splice variants

The absorptive Na(+)-K(+)-Cl(-) cotransporter (NKCC2) is a polytopic protein that forms homooligomeric complexes in the apical membrane of the thick ascending loop of Henle (TAL). It occurs in at least four splice variants (called B, A, F, and AF) that are identical to one another except for a short region in the membrane-associated domain. Although each of these variants exhibits unique functional properties and distributions along the TAL, their teleological purpose and structural organization remain poorly defined. In the current work, we provide additional insight in these regards by showing in mouse that the administration of either furosemide or an H(2)O-rich diet, which are predicted to alter NKCC2 expression in the TAL, exerts differential effects on mRNA levels for the variants, increasing those of A (furosemide) but decreasing those of F and AF (furosemide or H(2)O). Based on a yeast two-hybrid mapping analysis, we also show that the formation of homooligomeric complexes is mediated by two self-interacting domains in the COOH terminus (residues 671 to 816 and 910 to 1098), and that these complexes could probably include more than one type of variant. Taken together, the data reported here suggest that A, F, and AF each play unique roles that are adapted to specific physiological needs, and that the accomplishment of such roles is coordinated through the splicing machinery as well as complex NKCC2-NKCC2 interactions.     

Activation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells by aluminum fluoride and phosphatase inhibitors.

The bumetanide-sensitive component of pHi recovery from an NH4Cl-induced acute alkaline load was used as a measure of Na(+)-K(+)-2Cl- cotransport activity in rat parotid acini. Acinar treatment with NaF/AlCl3 (15 mM NaF plus 10 microM AlCl3) induced a 5-fold stimulation in the initial rate of bumetanide-sensitive pHi recovery. This effect was dependent on NaF concentration (K1/2 approximately 7 mM) and was blunted in the presence of the Al3+ chelator desferal mesylate suggesting that it might be due to the aluminofluoride ion, AlF-4. NaF/AlCl3 treatment did not increase acinar intracellular cAMP levels but did result in an increase in intracellular calcium concentration (from 87 +/- 5 to 181 +/- 2 nM) and in acinar cell shrinkage (12 +/- 1%). But the stimulation of the Na(+)-K(+)-2Cl- cotransporter by NaF/AlCl3 persisted in acini which had been depleted of their intracellular Ca2+ stores. In these acini no effect of NaF/AlCl3 on intracellular calcium or cell volume was observed, indicating that stimulation of the cotransporter was not secondary to either of these phenomena. The effect of NaF/AlCl3 on the cotransporter was blocked by the protein kinase inhibitor K252a indicating the involvement of a protein phosphorylation event. This result is consistent with either NaF/AlCl3-dependent protein kinase activation or phosphatase inhibition. The stimulation of the cotransporter by NaF/AlCl3 was mimicked by the protein phosphatase inhibitor calyculin A; however, this effect was not blocked by K252a suggesting that a different protein kinase from that associated with NaF/AlCl3 may be involved. The data indicate that the Na(+)-K(+)-2Cl- cotransporter in this tissue is under tight regulatory control, in all likelihood via multiple protein kinase/phosphatase systems. The physiological roles of these regulatory events in modulating acinar fluid secretion driven by the Na(+)-K(+)-2Cl- cotransporter remain to be elucidated.     

Molecular mechanisms of cation transport by the renal Na+-K+-Cl- cotransporter: structural insight into the operating characteristics of the ion transport sites

Two variants of the renal Na(+)-K(+)-Cl(-) cotransporter (NKCC2), called NKCC2A and NKCC2F, display marked differences in Na(+), Rb(+), and Cl(-) affinities, yet are identical to one another except for a 23-residue membrane-associated domain that is derived from alternatively spliced exons. The proximal portion of these exons is predicted to encode the second transmembrane domain (tm2) in the form of an alpha-helix, and the distal portion, part of the following connecting segment (cs1a). In recent studies, we have taken advantage of the A-F differences in kinetic behavior to determine which regions in tm2-cs1a are involved in ion transport. Functional characterizations of chimeras in which tm2 or cs1a were interchanged between the variants showed that both regions are important in specifying ion affinities, but did not allow delineating the contribution of individual residues. Here, we have extended these structure-function analyses by studying additional mutants in which variant residues between A and F were interchanged individually in the tm2-cs1a region (amino acid number 216, 220, 223, 229, or 233 in NKCC2). None of the substitutions were found to affect K(m (C1-)), suggesting that the affinity difference for anion transport is conveyed by a combination of variant residues in this domain. However, 2 substitutions in the tm2 of F were found to affect cation constants specifically; interestingly, one of these mutations (residue 216) only affected K(m (Rb+)) while the other (residue 220) only affected K(m (Na+)). We have thus identified two novel residues in NKCC2 that play a key role in cation transport. Because such residues should be adjacent to one another on the vertical axis of the tm2 alpha-helix, our results imply, furthermore, that the ion transport sites in NKCC2 could be physically linked.     

Electroneutral Cation-Chloride Cotransporters in the Central Nervous System

Several members of the cation-chloride cotransporter (solute carrier family 12, SLC12) gene family are expressed within the central nervous system, with one family member, the K+-Cl- cotransporter KCC2, exclusive to neurons. These transporters are best known for their roles in cell volume regulation and epithelial salt transport, but are increasingly receiving attention in neuroscience. In particular, intracellular chloride activity and hence the neuronal response to GABA and glycine appears to be determined by a balance between chloride efflux and influx through KCC2 and the Na+-K+-2Cl- cotransporter NKCC1, respectively. This relationship has important implications for neuronal development, sensory perception, neuronal excitability, and the response to neuronal injury. Finally, the association between loss of function in the K+-Cl- cotransporter KCC3, with a severe peripheral neuropathy associated with agenesis of the corpus callosum, has revealed an unexpected role for K+-Cl- cotransport in the development and/or maintenance of both the central and peripheral nervous systems.     

The ins and outs of intracellular chloride in olfactory receptor neurons

Stimulation of olfactory receptor neurons with odors culminates in opening of a ciliary Ca2+-activated Cl- channel. Because intracellular Cl- ([Cl-]i) is above electrochemical equilibrium in these cells, the result is cell depolarization that triggers action potentials that carry information to the olfactory bulb. In this issue of Neuron, Reisert and coworkers use combined pharmacological and mouse genetic approaches to show that the transporter responsible for maintaining Cl- above electrochemical equilibrium is NKCC1, a (Na+)(2Cl-)(K+) cotransporter found in other tissues, including neurons.     

Homooligomeric and heterooligomeric associations between K+-Cl- cotransporter isoforms and between K+-Cl- and Na+-K+-Cl- cotransporters

Little is known regarding the quaternary structure of cation-Cl- cotransporters (CCCs) except that the Na+-dependent CCCs can exist as homooligomeric units. Given that each of the CCCs exhibits unique functional properties and that several of these carriers coexist in various cell types, it would be of interest to determine whether the four K+-Cl- cotransporter (KCC) isoforms and their splice variants can also assemble into such units and, more importantly, whether they can form heterooligomers by interacting with each other or with the secretory Na+-K+-Cl- cotransporter (NKCC1). In the present work, we have addressed these questions by conducting two groups of analyses: 1) yeast two-hybrid and pull-down assays in which CCC-derived protein segments were used as both bait and prey and 2) coimmunoprecipitation and functional studies of intact CCCs coexpressed in Xenopus laevis oocytes. Through a combination of such analyses, we have found that KCC2 and KCC4 could adopt various oligomeric states (in the form of KCC2-KCC2, KCC4-KCC4, KCC2-KCC4, and even KCC4-NKCC1 complexes), that their carboxyl termini were probably involved in carrier assembly, and that the KCC4-NKCC1 oligomers, more specifically, could deploy unique functional features. Through additional coimmunoprecipitation studies, we have also found that KCC1 and KCC3 had the potential of assembling into various types of CCC-CCC oligomers as well, although the interactions uncovered were not characterized as extensively, and the protein segments involved were not identified in yeast two-hybrid assays. Taken together, these findings could change our views on how CCCs operate or are regulated in animal cells by suggesting, in particular, that cation-Cl- cotransport achieves higher levels of functional diversity than foreseen.