Exon loss accounts for differential sorting of Na-K-Cl cotransporters in polarized epithelial cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Carmosino M
PROVIDER: S-EPMC2555935 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Precise regulation of the intracellular concentration of chloride [Cl-]i is necessary for proper cell volume regulation, transepithelial transport, and GABA neurotransmission. The Na-K-2Cl (NKCCs) and K-Cl (KCCs) cotransporters, related SLC12A transporters mediating cellular chloride influx and efflux, respectively, are key determinants of [Cl-]i in numerous cell types, including red blood cells, epithelial cells, and neurons. A common "chloride/volume-sensitive kinase", or related system of kinases, has long been hypothesized to mediate the reciprocal but coordinated phosphoregulation of the NKCCs and the KCCs, but the identity of these kinase(s) has remained unknown. Recent evidence suggests that the WNK (with no lysine = K) serine-threonine kinases directly or indirectly via the downstream Ste20-type kinases SPAK/OSR1, are critical components of this signaling pathway. Hypertonic stress (cell shrinkage), and possibly decreased [Cl-]i, triggers the phosphorylation and activation of specific WNKs, promoting NKCC activation and KCC inhibition via net transporter phosphorylation. Silencing WNK kinase activity can promote NKCC inhibition and KCC activation via net transporter dephosphorylation, revealing a dynamic ability of the WNKs to modulate [Cl-]. This pathway is essential for the defense of cell volume during osmotic perturbation, coordination of epithelial transport, and gating of sensory information in the peripheral system. Commiserate with their importance in serving these critical roles in humans, mutations in WNKs underlie two different Mendelian diseases, pseudohypoaldosteronism type II (an inherited form of salt-sensitive hypertension), and hereditary sensory and autonomic neuropathy type 2. WNKs also regulate ion transport in lower multicellular organisms, including Caenorhabditis elegans, suggesting that their functions are evolutionarily-conserved. An increased understanding of how the WNKs regulate the Na-K-2Cl and K-Cl cotransporters may provide novel opportunities for the selective modulation of these transporters, with ramifications for common human diseases like hypertension, sickle cell disease, neuropathic pain, and epilepsy.
| S-EPMC3529164 | biostudies-literature
Project description:1. Electrophysiological methods were used to investigate the interaction of inhibitors with the human Na(+)/glucose (hSGLT1) and Na(+)/Cl(-)/GABA (hGAT1) cotransporters. Inhibitor constants were estimated from both inhibition of substrate-dependent current and inhibitor-induced changes in cotransporter conformation. 2. The competitive, non-transported inhibitors are substrate derivatives with inhibition constants from 200 nM (phlorizin) to 17 mM (esculin) for hSGLT1, and 300 nM (SKF89976A) to 10 mM (baclofen) for hGAT1. At least for hSGLT1, values determined using either method were proportional over 5-orders of magnitude. 3. Correlation of inhibition to structure of the inhibitors resulted in a pharmacophore for glycoside binding to hSGLT1: the aglycone is coplanar with the pyranose ring, and binds to a hydrophobic/aromatic surface of at least 7x12A. Important hydrogen bond interactions occur at five positions bordering this surface. 4. In both hSGLT1 and hGAT1 the data suggests that there is a large, hydrophobic inhibitor binding site approximately 8A from the substrate binding site. This suggests an architectural similarity between hSGLT1 and hGAT1. There is also structural similarity between non-competitive and competitive inhibitors, e.g., phloretin is the aglycone of phlorizin (hSGLT1) and nortriptyline resembles SKF89976A without nipecotic acid (hGAT1). 5. Our studies establish that measurement of the effect of inhibitors on presteady state currents is a valid non-radioactive method for the determination of inhibitor binding constants. Furthermore, analysis of the presteady state currents provide novel insights into partial reactions of the transport cycle and mode of action of the inhibitors.
| S-EPMC1572974 | biostudies-other
Project description:The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain, which are separated by tight junctions. The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules. This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers. Here we review the recent advances in the field of polarized sorting in epithelial cells. We especially highlight the role of lipid rafts in apical sorting.
| S-EPMC3343658 | biostudies-other
Project description:The trans-Golgi network must coordinate sorting and secretion of proteins and lipids to intracellular organelles and the plasma membrane. During polarization of epithelial cells, changes in the lipidome and the expression and distribution of proteins contribute to the formation of apical and basolateral plasma membrane domains. Previous studies using HeLa cells show that the syndecan-1 transmembrane domain confers sorting within sphingomyelin-rich vesicles in a sphingomyelin secretion pathway. In polarized Madin-Darby canine kidney cells, we reveal differences in the sorting of syndecan-1, whereupon the correct trafficking of the protein is not dependent on its transmembrane domain and changes in sphingomyelin content of cells during polarization. Instead, we reveal that correct basolateral targeting of syndecan-1 requires a full-length PDZ motif in syndecan-1 and the PDZ domain golgin protein GOPC. Moreover, we reveal changes in Golgi morphology elicited by GOPC overexpression. These results suggest that the role of GOPC in sorting syndecan-1 is indirect and likely due to GOPC effects on Golgi organization.
| S-EPMC9582621 | biostudies-literature
Project description:K(+)-Cl(-) cotransporters (KCCs) were originally characterized as regulators of red blood cell (RBC) volume. Since then, four distinct KCCs have been cloned, and their importance for volume regulation has been demonstrated in other cell types. Genetic models of certain KCCs, such as KCC3, and their inhibitory WNK-STE20/SPS1-related proline/alanine-rich kinase (SPAK) serine-threonine kinases, have demonstrated the evolutionary necessity of these molecules for nervous system cell volume regulation, structure, and function, and their involvement in neurological disease. The recent characterization of a swelling-activated dephosphorylation mechanism that potently stimulates the KCCs has pinpointed a potentially druggable switch of KCC activity. An improved understanding of WNK/SPAK-mediated KCC cell volume regulation in the nervous system might reveal novel avenues for the treatment of multiple neurological diseases.
| S-EPMC4834970 | biostudies-literature
Project description:The neuronal K(+)-Cl(-) cotransporter (KCC2) is a membrane transport protein that extrudes Cl(-) from neurons and helps maintain low intracellular [Cl(-)] and hyperpolarizing GABAergic synaptic potentials. Depolarizing gamma-aminobutyric acid (GABA) responses in neonatal neurons and following various forms of neuronal injury are associated with reduced levels of KCC2 expression. Despite the importance for plasticity of inhibitory transmission, less is known about cellular mechanisms involved in more dynamic changes in KCC2 function. In this study, we investigated the role of tyrosine phosphorylation in KCC2 localization and function in hippocampal neurons and in cultured GT1-7 cells. Mutation to the putative tyrosine phosphorylation site within the long intracellular carboxyl terminus of KCC2(Y1087D) or application of the tyrosine kinase inhibitor genistein shifted the GABA reversal potential (E(GABA)) to more depolarized values, indicating reduced KCC2 function. This was associated with a change in the expression pattern of KCC2 from a punctate distribution to a more uniform distribution, suggesting that functional tyrosine-phosphorylated KCC2 forms clusters in restricted membrane domains. Sodium vanadate, a tyrosine phosphatase inhibitor, increased the proportion of KCC2 associated with lipid rafts membrane domains. Loss of tyrosine phosphorylation also reduced oligomerization of KCC2. A loss of the punctuate distribution and oligomerization of KCC2 and a more depolarized E(GABA) were seen when the 28-amino-acid carboxyl terminus of KCC2 was deleted. These results indicate that direct tyrosine phosphorylation of KCC2 results in membrane clusters and functional transport activity, suggesting a mechanism by which intracellular Cl(-) concentrations and GABA responses can be rapidly modulated.
| S-EPMC2788850 | biostudies-literature
Project description:Sodium bicarbonate has long been used to treat chronic kidney disease. It has been demonstrated to slow the decline in glomerular filtration rate in chronic kidney disease patient; however, the mechanisms are not completely understood. We hypothesized that NaHCO3 dilates afferent arterioles (Af-Art) by stimulating nitric oxide (NO) release mediated by the Na+/HCO3- cotransporter (NBC) contributing to the elevation in glomerular filtration rate. Isolated microperfused mouse renal Af-Art, preconstricted with norepinephrine (1 µmol/L), dilated 45±2% (n=6, P<0.05) in response to NaHCO3 (44 mmol/L). Whereas, NaCl solution containing the same Na+ concentration was not effective. The mRNA for NBCn1 and NBCe1 were detected in microdissected Af-Art using reverse transcription-polymerase chain reaction and quantitative polymerase chain reaction. The Af-Art intracellular pH measured with 2',7'-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein, acetoxymethyl ester increased significantly by 0.29±0.02 (n=6; P<0.05) in the presence of NaHCO3, which was blunted by N-cyanosulphonamide compound (S0859) that is an inhibitor of the NBC family. After clamping the intracellular pH with 10 ?M nigericin, changing the bath solution pH from 7.4 to 7.8 still dilates the Af-Art by 53±4% (n=7; P<0.005) and increases NO generation by 22±3% (n=7; P<0.005). Both pH-induced NO generation and vasodilation were blocked by L-NG-Nitroarginine Methyl Ester. NaHCO3 increased NO generation in Af-Art by 19±4% (n=5; P<0.005) and elevated glomerular filtration rate in conscious mice by 36% (233 versus 318 ul/min; n=9-10; P<0.0001). S0859 and L-NG-nitroarginine methyl ester blocked NaHCO3-induced increases in NO generation and vasodilation. We conclude that NBCn1 and NBCe1 are expressed in Af-Art and that NaHCO3 dilates Af-Art via NBCs mediated by NO that increases the glomerular filtration rate.
| S-EPMC6785401 | biostudies-literature
Project description:Fluxes of monovalent ions through the multiple pathways of the plasma membrane are highly interdependent, and their assessment by direct measurement is difficult or even impossible. Computation of the entire flux balance helps to identify partial flows and study the functional expression of individual transporters. Our previous computation of unidirectional fluxes in real cells ignored the ubiquitous cotransporters NKCC and KCC. Here, we present an analysis of the entire balance of unidirectional Na+, K+, and Cl- fluxes through the plasma membrane in human lymphoid U937 cells, taking into account not only the Na/K pump and electroconductive channels but all major types of cotransporters NC, NKCC, and KCC. Our calculations use flux equations based on the fundamental principles of macroscopic electroneutrality of the system, water balance, and the generally accepted thermodynamic dependence of ion fluxes on the driving force, and they do not depend on hypotheses about the molecular structure of the channel and transporters. A complete list of the major inward and outward Na+, K+, and Cl- fluxes is obtained for human lymphoid U937 cells at rest and during changes in the ion and water balance for the first 4 h of staurosporine-induced apoptosis. It is shown how the problem of the inevitable multiplicity of solutions to the flux equations, which arises with an increase in the number of ion pathways, can be solved in real cases by analyzing the ratio of ouabain-sensitive and ouabain-resistant parts of K+ (Rb+) influx (OSOR) and using additional experimental data on the effects of specific inhibitors. It is found that dynamics of changes in the membrane channels and transporters underlying apoptotic changes in the content of ions and water in cells, calculated without taking into account the KCC and NKCC cotransporters, differs only in details from that calculated for cells with KCC and NKCC. The developed approach to the assessment of unidirectional fluxes may be useful for understanding functional expression of ion channels and transporters in other cells under various conditions. Attached software allows reproduction of all calculated data under presented conditions and to study the effects of the condition variation.
| S-EPMC7677585 | biostudies-literature
Project description:Cotransport proteins are responsible for the active accumulation of organic substrates in cells. Na+ gradients provide the driving force for uptake of most substrates into eukaryotes and for a few substrates in some prokaryotes. We report here the cloning and sequencing of the human intestinal Na+/glucose cotransporter (SGLT1) and compare its structure with other cloned transporters. At the DNA level and the predicted amino acid and secondary structure levels, close homology is evident between the human and rabbit intestinal Na+/glucose cotransporters, and a significant homology is found between these and the Escherichia coli Na+/proline cotransporter (putP). No homology is detectible with other known proteins. We infer from these results that the mammalian Na+/glucose and prokaryote Na+/proline cotransporters share a common ancestral gene.
| S-EPMC297707 | biostudies-other
Project description:The routes water takes through membrane barriers is still a matter of debate. Although aquaporins only allow transmembrane water movement along an osmotic gradient, cotransporters are believed to be capable of water transport against the osmotic gradient. Here we show that the renal potassium-chloride-cotransporter (KCC1) does not pump a fixed amount of water molecules per movement of one K(+) and one Cl(-), as was reported for the analogous transporter in the choroid plexus. We monitored water and potassium fluxes through monolayers of primary cultured renal epithelial cells by detecting tiny solute concentration changes in the immediate vicinity of the monolayer. KCC1 extruded K(+) ions in the presence of a transepithelial K(+) gradient, but did not transport water. KCC1 inhibition reduced epithelial osmotic water permeability P(f) by roughly one-third, i.e., the effect of inhibitors was small in resting cells and substantial in hormonal stimulated cells that contained high concentrations of aquaporin-2 in their apical membranes. The furosemide or DIOA (dihydroindenyl-oxy-alkanoic acid)-sensitive water flux was much larger than expected when water passively followed the KCC1-mediated ion flow. The inhibitory effect of these drugs on water flux was reversed by the K(+)-H(+) exchanger nigericin, indicating that KCC1 affects water transport solely by K(+) extrusion. Intracellular K(+) retention conceivably leads to cell swelling, followed by an increased rate of endocytic AQP2 retrieval from the apical membrane.
| S-EPMC2998630 | biostudies-literature