Cotransporters KCC1 Research Articles

Glial regulators of ions and solutes required for specific chemosensory functions in Caenorhabditis elegans.

Glia and accessory cells regulate the microenvironment around neurons and primary sensory cells. However, the impact of specific glial regulators of ions and solutes on functionally diverse primary cells is poorly understood. Here, we systemically investigate the requirement of ion channels and transporters enriched in Caenorhabditis elegans Amsh glia for the function of chemosensory neurons. Although Amsh glia ablated worms show reduced function of ASH, AWC, AWA, and ASE neurons, we show that the loss of glial enriched ion channels and transporters impacts these neurons differently, with nociceptor ASH being the most affected. Furthermore, our analysis underscores the importance of K+, Cl-, and nucleoside homeostasis in the Amphid sensory organ and uncovers the contribution of glial genes implicated in neurological disorders. Our findings build a unique fingerprint of each glial enriched ion channel and transporter and may provide insights into the function of supporting cells of mammalian sensory organs.

Expression and Purification of the Human Cation-chloride Cotransporter KCC1 from HEK293F Cells for Structural Studies

Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na+ and/or K+ with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl- by the transmembrane K+ gradient. In humans, these KCCs play vital roles in the physiology of the nervous system and kidney. However, mechanisms underlying the KCCs specific properties remain poorly understood, partly because purification of membrane proteins is challenging. Here, we present the protocol for purifying the full-length KCC1 from HEK293F cells used in our recent publication ( Liu et al., 2019 ). The procedure may be adapted for functional and structural studies.

Structures and an activation mechanism of human potassium-chloride cotransporters.

Potassium-chloride cotransporters KCC1 to KCC4 mediate the coupled export of potassium and chloride across the plasma membrane and play important roles in cell volume regulation, auditory system function, and γ-aminobutyric acid (GABA) and glycine-mediated inhibitory neurotransmission. Here, we present 2.9- to 3.6-Å resolution structures of full-length human KCC2, KCC3, and KCC4. All three KCCs adopt a similar overall architecture, a domain-swap dimeric assembly, and an inward-facing conformation. The structural and functional studies reveal that one unexpected N-terminal peptide binds at the cytosolic facing cavity and locks KCC2 and KCC4 at an autoinhibition state. The C-terminal domain (CTD) directly interacts with the N-terminal inhibitory peptide, and the relative motions between the CTD and the transmembrane domain (TMD) suggest that CTD regulates KCCs' activities by adjusting the autoinhibitory effect. These structures provide the first glimpse of full-length structures of KCCs and an autoinhibition mechanism among the amino acid-polyamine-organocation transporter superfamily.

Genetic disruption of KCC cotransporters in a mouse model of thalassemia intermedia

β-thalassemia (β-Thal) is caused by defective β-globin production leading to globin chain imbalance, aggregation of free alpha chain in developing erythroblasts, reticulocytes, and mature circulating red blood cells. The hypochromic thalassemic red cells exhibit increased cell dehydration in association with elevated K+ leak and increased K-Cl cotransport activity, each of which has been linked to globin chain imbalance and related oxidative stress.We therefore tested the effect of genetic inactivation of K-Cl cotransporters KCC1 and KCC3 in a mouse model of β-thalassemia intermedia. In the absence of these transporters, the anemia of β-Thal mice was ameliorated, in association with increased MCV and reductions in CHCM and hyperdense cells, as well as in spleen size. The resting K+ content of β-Thal red cells was greatly increased, and Thal-associated splenomegaly slightly decreased. Lack of KCC1 and KCC3 activity in Thal red cells reduced red cell density and improved β-Thal-associated osmotic fragility. We conclude that genetic inactivation of K-Cl cotransport can reverse red cell dehydration and partially attenuate the hematologic phenotype in a mouse model of β-thalassemia.

Cryo-EM structures of the human cation-chloride cotransporter KCC1.

Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations with chloride across the plasma membrane and are vital for cell volume regulation, salt reabsorption in the kidney, and γ-aminobutyric acid (GABA)-mediated modulation in neurons. Here we present cryo-electron microscopy (cryo-EM) structures of human potassium-chloride cotransporter KCC1 in potassium chloride or sodium chloride at 2.9- to 3.5-angstrom resolution. KCC1 exists as a dimer, with both extracellular and transmembrane domains involved in dimerization. The structural and functional analyses, along with computational studies, reveal one potassium site and two chloride sites in KCC1, which are all required for the ion transport activity. KCC1 adopts an inward-facing conformation, with the extracellular gate occluded. The KCC1 structures allow us to model a potential ion transport mechanism in KCCs and provide a blueprint for drug design.

Physiological role of SLC12 family members in the kidney.

The solute carrier family 12, as numbered according to Human Genome Organisation (HUGO) nomenclature, encodes the electroneutral cation-coupled chloride cotransporters that are expressed in many cells and tissues; they play key roles in important physiological events, such as cell volume regulation, modulation of the intracellular chloride concentration, and transepithelial ion transport. Most of these family members are expressed in specific regions of the nephron. The Na-K-2Cl cotransporter NKCC2, which is located in the thick ascending limb, and the Na-Cl cotransporter, which is located in the distal convoluted tubule, play important roles in salt reabsorption and serve as the receptors for loop and thiazide diuretics, respectively (Thiazide diuretics are among the most commonly prescribed drugs in the world.). The activity of these transporters correlates with blood pressure levels; thus, their regulation has been a subject of intense research for more than a decade. The K-Cl cotransporters KCC1, KCC3, and KCC4 are expressed in several nephron segments, and their role in renal physiology is less understood but nevertheless important. Evidence suggests that they are involved in modulating proximal tubule glucose reabsorption, thick ascending limb salt reabsorption and collecting duct proton secretion. In this work, we present an overview of the physiological roles of these transporters in the kidney, with particular emphasis on the knowledge gained in the past few years.

Activation of the erythroid K-Cl cotransporter Kcc1 enhances sickle cell disease pathology in a humanized mouse model

AbstractWe used an N-ethyl-N-nitrosurea-based forward genetic screen in mice to identify new genes and alleles that regulate erythropoiesis. Here, we describe a mouse line expressing an activated form of the K-Cl cotransporter Slc12a4 (Kcc1), which results in a semi-dominant microcytosis of red cells. A missense mutation from methionine to lysine in the cytoplasmic tail of Kcc1 impairs phosphorylation of adjacent threonines required for inhibiting cotransporter activity. We bred Kcc1M935K mutant mice with a humanized mouse model of sickle cell disease to directly explore the relevance of the reported increase in KCC activity in disease pathogenesis. We show that a single mutant allele of Kcc1 induces widespread sickling and tissue damage, leading to premature death. This mouse model reveals important new insights into the regulation of K-Cl cotransporters and provides in vivo evidence that increased KCC activity worsened end-organ damage and diminished survival in sickle cell disease.

WNK3 is a Putative Chloride-sensing Kinase

The with-no-lysine kinase 3 (WNK3) is a serine/threonine kinase that modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC). Using the Xenopus laevis oocyte heterologous expression system, it has been shown that WNK3 activates the Na⁺-coupled chloride cotransporters NKCC1, NKCC2, and NCC and inhibits the K⁺-coupled chloride cotransporters KCC1 through KCC4. Interestingly, the effect of catalytically inactive WNK3 is opposite to that of wild type WNK3: inactive WNK3 inhibits NKCCs and activates KCCs. In doing so, wild type and catalytically inactive WNK3 bypass the tonicity requirement for activation/inhibition of the cotransporter. Thus, WNK3 modulation of the electroneutral cotransporters promotes Cl^- influx and prevents Cl^- efflux, thus fitting the profile for a putative “Cl^--sensing kinase”. Other kinases that potentially have these properties are the Ste20-type kinases, SPAK/OSR1, which become phosphorylated in response to reductions in intracellular chloride concentration and regulate the activity of NKCC1. It has been demonstrated that WNKs lie upstream of SPAK/OSR1 and that the activity of these kinases is activated by phosphorylation of threonines in the T-loop by WNKs. It is possible that a protein phosphatase is also involved in the WNK3 effects on its associated cotransporters because activation of KCCs and inhibition of NKCCs by inactive WNK3 can be prevented by known inhibitors of protein phosphatases, such as calyculin A and cyclosporine, suggesting that a protein phosphatase is also involved in the protein complex.

Chemical crosslinking studies with the mouse Kcc1 K–Cl cotransporter

Oligomerization, function, and regulation of unmodified mouse Kcc1 K–Cl cotransporter were studied by chemical crosslinking. Treatment of Xenopus oocytes and 293T cells expressing K–Cl cotransporter Kcc1 with several types of chemical cross-linkers shifted Kcc1 polypeptide to higher molecular weight forms. More extensive studies were performed with the amine-reactive disuccinyl suberate (DSS) and with the sulfhydryl-reactive bis-maleimidohexane (BMH). Kcc1 cross-linking was time-dependent in intact oocytes, and was independent of protein concentration in detergent lysates from oocytes or 293T cells. Kcc1 cross-linking by the cleavable cross-linker DTME was reversible. The N-terminal and C-terminal cytoplasmic tails of Kcc1 were not essential for Kcc1 crosslinking. PFO-PAGE and gel filtration revealed oligomeric states of uncrosslinked KCC1 corresponding in mobility to that of cross-linked protein. DSS and BMH each inhibited KCC1-mediated 86Rb + uptake stimulated by hypotonicity or by N-ethylmaleimide (NEM) without reduction in nominal surface abundance of KCC1. These data add to evidence supporting the oligomeric state of KCC polypeptides.

Amino acid transporter (VIAAT, VGLUT2) and chloride cotransporter (KCC1, KCC2 and NKCC1) expression in the vestibular nuclei of intact and labyrinthectomized rat

We report the first investigation of whether unilateral labyrinthectomy in adult rats affects the expression of two amino acid transporters, vesicular glutamate transporter 2 (VGLUT2) and vesicular inhibitory amino acid transporter (VIAAT) and of chloride cotransporters (KCC1, KCC2 and NKCC1) in the intact and deafferented medial vestibular nuclei (MVN). In situ hybridization with specific radioactive oligonucleotide probes and immunofluorescent methods were used in normal and unilaterally labyrinthectomized rats at various times following the lesion: 5 h, and 1, 3 and 8 days. In normal animals, several brainstem regions including the lateral, medial, superior and inferior vestibular nuclei contained VGLUT2, VIAAT and KCC2 mRNA. In contrast, no or a very faint labeling was observed with KCC1 and NKCC1 probes. In unilaterally lesioned rats, there was no asymmetry between the two MVN with any of the oligonucleotide probes at any time after the lesion. Similarly, there were no differences in the intensity of MVN labeling between controls and lesioned animals. Finally, no over-expression of the cotransporter KCC1 and NKCC1 was found in ipsilateral or controlateral MVN in lesioned rats at any time. Immunohistochemical experiments gave similar conclusions. Our findings suggest that the recovery of the resting discharge of the deafferented MVN neurons, and consequently the functional compensation of the deficits, are not dependent on changes in the expression of amino acid transporters (VIAAT, VGLUT2), and chloride cotransporters (KCC1, KCC2 and NKCC1) or on their mRNAs.

WNK4 Kinase is a Negative Regulator of K + ‐Cl − Cotransporters

WNK kinases (with no lysine kinase(K)) are regulators of several membrane transport proteins in which these kinases act as a molecular switches that coordinate the activity of several players. Members of the Solute Carrier Family number 12 are one of the main targets. WNK3 activates the Na+-driven NCC, NKCC1, and NKCC2 and inhibits the K+-driven KCC1 to KCC4. WNK4 inhibits the activity of NCC and NKCC1, while in the presence of the STE20-related proline-alanine rich kinase SPAK activates NKCC1. Nothing is known, however, regarding the effect of WNK4 upon the K+-Cl− cotransporters. Using the heterologous expression system of Xenopus laevis oocytes, here we show that WNK4 inhibits the activity of the K+-Cl− cotransporters KCC1, KCC3, and KCC4 by 50 to 60% under cell swelling, a condition in which these cotransporters are maximally active. The effect of WNK4 requires its catalytic activity because it was lost by the substitution of aspartate 318 for alanine (WNK4-D318A) that renders WNK4 catalytically inactive. WNK4 effect upon K+-Cl− cotransporters is not affected by co-injection with wild type SPAK cRNA or its catalytically inactive form (SPAK-K104R). In contrast, WNK4 missense mutations that cause pseudohypoaldosteronism type II do not affect the WNK4-induced inhibition of KCC4. Finally, we observed that catalytically inactive WNK4-D318A is able to bypass the tonicity requirements for KCC3 activation in isotonic conditions. This effect is enhanced by the presence of catalytically inactive SPAK and was not present in KCC1 and KCC4. Our results reveal that WNK4 regulates the activity of the K+-Cl− cotransporters.

WNK4 kinase is a negative regulator of K+-Cl−cotransporters

WNK kinases [with no lysine (K) kinase] are emerging as regulators of several membrane transport proteins in which WNKs act as molecular switches that coordinate the activity of several players. Members of the cation-coupled chloride cotransporters family (solute carrier family number 12) are one of the main targets. WNK3 activates the Na(+)-driven cotransporters NCC, NKCC1, and NKCC2 and inhibits the K(+)-driven cotransporters KCC1 to KCC4. WNK4 inhibits the activity of NCC and NKCC1, while in the presence of the STE20-related proline-alanine-rich kinase SPAK activates NKCC1. Nothing is known, however, regarding the effect of WNK4 on the K(+)-Cl(-) cotransporters. Using the heterologous expression system of Xenopus laevis oocytes, here we show that WNK4 inhibits the activity of the K(+)-Cl(-) cotransporters KCC1, KCC3, and KCC4 under cell swelling, a condition in which these cotransporters are maximally active. The effect of WNK4 requires its catalytic activity because it was lost by the substitution of aspartate 318 for alanine (WNK4-D318A) that renders WNK4 catalytically inactive. In contrast, three different WNK4 missense mutations that cause pseudohypoaldosteronism type II do not affect the WNK4-induced inhibition of KCC4. Finally, we observed that catalytically inactive WNK4-D318A is able to bypass the tonicity requirements for KCC2 and KCC3 activation in isotonic conditions. This effect is enhanced by the presence of catalytically inactive SPAK, was prevented by the presence of protein phosphatase inhibitors, and was not present in KCC1 and KCC4. Our results reveal that WNK4 regulates the activity of the K(+)-Cl(-) cotransporters expressed in the kidney.

Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation.

Although K-Cl cotransporter (KCC1) mRNA is expressed in many tissues, K-Cl cotransport activity has been measured in few cell types, and detection of endogenous KCC1 polypeptide has not yet been reported. We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flanking genomic regions and mapped the mKCC1 gene to chromosome 8. Three anti-peptide antibodies raised against recombinant mKCC1 function as immunoblot and immunoprecipitation reagents. The tissue distributions of mKCC1 mRNA and protein are widespread, and mKCC1 RNA is constitutively expressed during erythroid differentiation of ES cells. KCC1 polypeptide or related antigen is present in erythrocytes of multiple species in which K-Cl cotransport activity has been documented. Erythroid KCC1 polypeptide abundance is elevated in proportion to reticulocyte counts in density-fractionated cells, in bleeding-induced reticulocytosis, in mouse models of sickle cell disease and thalassemia, and in the corresponding human disorders. mKCC1-mediated uptake of (86)Rb into Xenopus oocytes requires extracellular Cl(-), is blocked by the diuretic R(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2, 3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling, N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.