scholarly journals A Serine Residue in ClC-3 Links Phosphorylation–Dephosphorylation to Chloride Channel Regulation by Cell Volume

1999 ◽  
Vol 113 (1) ◽  
pp. 57-70 ◽  
Author(s):  
Dayue Duan ◽  
Suzanne Cowley ◽  
Burton Horowitz ◽  
Joseph R. Hume
Keyword(s):  
Cell Volume ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Phosphorylation Site ◽  
Cardiac Cells ◽  
Site Directed Mutagenesis ◽  
Cell Swelling ◽  
Transport Systems ◽  
Serine Residue ◽  
Channel Regulation

In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, ICl.vol.

scholarly journals Hydrogen, Bicarbonate, and Their Associated Exchangers in Cell Volume Regulation

2021 ◽  
Vol 9 ◽  
Author(s):  
Yizeng Li ◽  
Xiaohan Zhou ◽  
Sean X. Sun
Keyword(s):  
Ion Channels ◽  
Cell Volume ◽  
Volume Regulation ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Cell Function ◽  
Water Flux ◽  
Cell Volume Regulation ◽  
Sodium Potassium ◽  
Before And After

Cells lacking a stiff cell wall, e.g., mammalian cells, must actively regulate their volume to maintain proper cell function. On the time scale that protein production is negligible, water flow in and out of the cell determines the cell volume variation. Water flux follows hydraulic and osmotic gradients; the latter is generated by various ion channels, transporters, and pumps in the cell membrane. Compared to the widely studied roles of sodium, potassium, and chloride in cell volume regulation, the effects of proton and bicarbonate are less understood. In this work, we use mathematical models to analyze how proton and bicarbonate, combined with sodium, potassium, chloride, and buffer species, regulate cell volume upon inhibition of ion channels, transporters, and pumps. The model includes several common, widely expressed ion transporters and focuses on obtaining generic outcomes. Results show that the intracellular osmolarity remains almost constant before and after cell volume change. The steady-state cell volume does not depend on water permeability. In addition, to ensure the stability of cell volume and ion concentrations, cells need to develop redundant mechanisms to maintain homeostasis, i.e., multiple ion channels or transporters are involved in the flux of the same ion species. These results provide insights for molecular mechanisms of cell volume regulation with additional implications for water-driven cell migration.

Post-translational modifications of the β-1 subunit of AMP-activated protein kinase affect enzyme activity and cellular localization

2001 ◽  
Vol 354 (2) ◽  
pp. 275-283 ◽  
Author(s):  
Scott M. WARDEN ◽  
Christine RICHARDSON ◽  
John O'DONNELL ◽  
David STAPLETON ◽  
Bruce E. KEMP ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Cellular Localization ◽  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Β Subunit ◽  
Α Subunit ◽  
Amp Activated Protein Kinase ◽  
The Impact

The AMP-activated protein kinase (AMPK) is a ubiquitous mammalian protein kinase important in the adaptation of cells to metabolic stress. The enzyme is a heterotrimer, consisting of a catalytic α subunit and regulatory β and γ subunits, each of which is a member of a larger isoform family. The enzyme is allosterically regulated by AMP and by phosphorylation of the α subunit. The β subunit is post-translationally modified by myristoylation and multi-site phosphorylation. In the present study, we have examined the impact of post-translational modification of the β-1 subunit on enzyme activity, heterotrimer assembly and subcellular localization, using site-directed mutagenesis and expression of subunits in mammalian cells. Removal of the myristoylation site (G2A mutant) results in a 4-fold activation of the enzyme and relocalization of the β subunit from a particulate extranuclear distribution to a more homogenous cell distribution. Mutation of the serine-108 phosphorylation site to alanine is associated with enzyme inhibition, but no change in cell localization. In contrast, the phosphorylation site mutations, SS24,25AA and S182A, while having no effects on enzyme activity, are associated with nuclear redistribution of the subunit. Taken together, these results indicate that both myristoylation and phosphorylation of the β subunit of AMPK modulate enzyme activity and subunit cellular localization, increasing the complexity of AMPK regulation.

Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket

2012 ◽  
Vol 302 (1) ◽  
pp. C257-C266 ◽  
Author(s):  
Pedro Cano-Soldado ◽  
Edurne Gorraitz ◽  
Ekaitz Errasti-Murugarren ◽  
F. Javier Casado ◽  
M. Pilar Lostao ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Hill Coefficient ◽  
Thymidine Uptake ◽  
Nucleoside Transporter ◽  
Nucleotide Polymorphisms ◽  
Loss Of Function ◽  
Serine Residue ◽  
Concentrative Nucleoside Transporter

SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na+/1 nucleoside stoichiometry, showed altered Na+ binding properties associated with a shift in the Hill coefficient, consistent with one Na+ binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na+ leak, which was dependent on the concentration of extracellular Na+ indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.

Cell swelling increases membrane conductance of canine cardiac cells: evidence for a volume-sensitive Cl channel

1992 ◽  
Vol 262 (4) ◽  
pp. C1056-C1068 ◽  
Author(s):  
G. N. Tseng
Keyword(s):  
Kinase Inhibitor ◽  
Membrane Conductance ◽  
Cardiac Cells ◽  
Cell Swelling ◽  
Transport Systems ◽  
Ca Channel ◽  
Induced Current ◽  
Pipette Solution ◽  
Cl Channel ◽  
Current Voltage

Cardiac cell swelling occurs under abnormal conditions. Currents through volume-sensitive channels, if present in heart, will affect the cardiac electrical activity. Single canine ventricular myocytes were voltage clamped under conditions that largely suppressed Na, K, and Ca channel currents and currents generated by electrogenic transport systems. Cell width and membrane conductance were monitored continuously. Swelling was induced by increasing the osmolarity of the pipette solution or by decreasing the osmolarity of the external solution. During cell swelling, the cell widened and membrane conductance increased. This increase in membrane conductance was sensitive to Cl channel blockers and to external Cl removal, suggesting that a major component was provided by a Cl channel. The current-voltage relationship of the swelling-induced current displayed an outward rectification, with an average zero-current voltage of -60 mV. The activation of the swelling-induced current did not seem to depend on external or internal Ca and was not sensitive to a protein kinase inhibitor (H-8). Shape-altering agents chlorpromazine decreased while dipyridamole and trinitrophenol increased the membrane conductance without osmotic perturbations, suggesting that changes in tension in the cell membrane may play a role in opening and closing of the swelling-induced channels.

scholarly journals Cryo-EM structure of the volume-regulated anion channel LRRC8

2018 ◽  
Author(s):  
Go Kasuya ◽  
Takanori Nakane ◽  
Takeshi Yokoyama ◽  
Yanyan Jia ◽  
Masato Inoue ◽  
...  
Keyword(s):  
Cell Volume ◽  
Molecular Mechanisms ◽  
Anion Channel ◽  
Membrane Topology ◽  
Cell Swelling ◽  
Cell Functions ◽  
Gap Junction Channel ◽  
Electron Microscopy Analysis ◽  
Extracellular Side ◽  
And Migration

AbstractMaintenance of cell volume against osmotic change is crucial for proper cell functions, such as cell proliferation and migration. The leucine-rich repeat-containing 8 (LRRC8) proteins are anion selective channels, and were recently identified as pore components of the volume-regulated anion channels (VRACs), which extrude anions to decrease the cell volume upon cell-swelling. Here, we present the human LRRC8A structure, determined by a single-particle cryo-electron microscopy analysis. The sea anemone-like structure represents a trimer of dimers assembly, rather than a symmetrical hexameric assembly. The four-spanning transmembrane region has a gap junction channel-like membrane topology, while the LRR region containing 15 leucine-rich repeats forms a long twisted arc. The channel pore is along the central axis and constricted on the extracellular side, where the highly conserved polar and charged residues at the tip of the extracellular helix contribute to the anion and other osmolyte permeability. Comparing the two structural populations facilitated the identification of both compact and relaxed conformations, suggesting that the LRR region is flexible and mobile with rigid-body motions, which might be implicated in structural transitions upon pore opening. Overall, our structure provides a framework for understanding the molecular mechanisms of this unique class of ion channels.

scholarly journals The O-GlcNAc transferase OGT is a conserved and essential regulator of the cellular and organismal response to hypertonic stress

2020 ◽  
Author(s):  
Sarel J. Urso ◽  
Marcella Comly ◽  
John A. Hanover ◽  
Todd Lamitina
Keyword(s):  
Stress Response ◽  
Cell Volume ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Cell Physiology ◽  
Hypertonic Stress ◽  
C Elegans ◽  
Intracellular Proteins ◽  
Molecular Functions ◽  
Glcnac Transferase

AbstractThe conserved O-GlcNAc transferase OGT O-GlcNAcylates serine and threonine residues of intracellular proteins to regulate their function. OGT is required for viability in mammalian cells, but its specific roles in cellular physiology are poorly understood. Here we describe a conserved requirement for OGT in an essential aspect of cell physiology: the hypertonic stress response. Through a forward genetic screen in Caenorhabditis elegans, we discovered OGT is acutely required for osmoprotective protein expression and adaptation to hypertonic stress. Gene expression analysis shows that ogt-1 functions through a post-transcriptional mechanism. Human OGT partially rescues the C. elegans phenotypes, suggesting that the osmoregulatory functions of OGT are ancient. Intriguingly, mutations that ablate O-GlcNAcylation activity in either human or C. elegans OGT rescue the hypertonic stress response phenotype. Our findings are among the first to demonstrate a specific physiological role for OGT at the organismal level and demonstrate that OGT engages in important molecular functions outside of its well described roles in post-translational O-GlcNAcylation of intracellular proteins.Author SummaryThe ability to sense and adapt to changes in the environment is an essential feature of cellular life. Changes in environmental salt and water concentrations can rapidly cause cell volume swelling or shrinkage and, if left unchecked, will lead to cell and organismal death. All organisms have developed similar physiological strategies for maintaining cell volume. However, the molecular mechanisms that control these physiological outputs are not well understood in animals. Using unbiased genetic screening in C. elegans, we discovered that a highly conserved enzyme called O-GlcNAc transferase (OGT) is essential for regulating physiological responses to increased environmental solute levels. A human form of OGT can functionally substitute for worm OGT, showing that this role is conserved across evolution. Surprisingly, the only known enzymatic activity of OGT was not required for this role, suggesting this enzyme has important undescribed molecular functions. Our studies reveal a new animal-specific role for OGT in the response to osmotic stress and show that C. elegans is an important model for defining the conserved molecular mechanisms that respond to alterations in cell volume.

scholarly journals PKC-mediated phosphorylation of nuclear lamins at a single serine residue regulates interphase nuclear size in Xenopus and mammalian cells

2017 ◽  
Vol 28 (10) ◽  
pp. 1389-1399 ◽  
Author(s):  
Lisa J. Edens ◽  
Matthew R. Dilsaver ◽  
Daniel L. Levy
Keyword(s):  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Mechanistic Explanation ◽  
Nuclear Size ◽  
The Novel ◽  
Serine Residue ◽  
Size Regulation ◽  
Nuclear Lamins ◽  
Kinase C ◽  
Xenopus Laevis Embryos

How nuclear size is regulated is a fundamental cell-biological question with relevance to cancers, which often exhibit enlarged nuclei. We previously reported that conventional protein kinase C (cPKC) contributes to nuclear size reductions that occur during early Xenopus development. Here we report that PKC-mediated phosphorylation of lamin B3 (LB3) contributes to this mechanism of nuclear size regulation. By mapping PKC phosphorylation sites on LB3 and testing the effects of phosphomutants in Xenopus laevis embryos, we identify the novel site S267 as being an important determinant of nuclear size. Furthermore, FRAP studies demonstrate that phosphorylation at this site increases lamina dynamics, providing a mechanistic explanation for how PKC activity influences nuclear size. We subsequently map this X. laevis LB3 phosphorylation site to a conserved site in mammalian lamin A (LA), S268. Manipulating PKC activity in cultured mammalian cells alters nuclear size, as does expression of LA-S268 phosphomutants. Taken together, these data demonstrate that PKC-mediated lamin phosphorylation is a conserved mechanism of nuclear size regulation.

scholarly journals A plate reader-based method for cell water permeability measurement

2010 ◽  
Vol 298 (1) ◽  
pp. F224-F230 ◽  
Author(s):  
R. A. Fenton ◽  
H. B. Moeller ◽  
S. Nielsen ◽  
B. L. de Groot ◽  
M. Rützler
Keyword(s):  
Cell Volume ◽  
Water Permeability ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Type I ◽  
Volume Changes ◽  
Average Cell ◽  
Compound Libraries ◽  
Plate Reader

Cell volume and water permeability measurements in cultured mammalian cells are typically conducted under a light microscope. Many of the employed approaches are time consuming and not applicable to a study of confluent epithelial cell monolayers. We present here an adaptation of a calcein-quenching-based approach for a plate reader. A standard curve of fluorescence intensities at equilibrium has been recorded, following a shift from 285 mosmol/kgH2O to a series of altered extracellular osmolyte concentrations, ranging from final concentrations of 185 to 585 mosmol/kgH2O, by changing buffer d-mannitol concentrations. Similarly, according average cell volumes have been measured in suspension in a Coulter counter (particle-sizing device). Based on these measurements, we have derived an equation that facilitates the modeling of cell volume changes based on fluorescence intensity changes. We have utilized the method to study the role of a carboxyl-terminus aquaporin (AQP)-2 phosphorylation site, which is known to affect AQP2 membrane trafficking, in heterologous type I Madin-Darby canine kidney cells. We find that water permeability in cells expressing phosphorylation site mutants was in the following order: AQP2-S256D > AQP2 wild-type > AQP2-S256A. We propose that the method can be applied to study AQP function and more generally to study cell volume changes in adherent cell lines. Furthermore, it should be adaptable for AQP inhibitor screening in chemical compound libraries.

5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB)

2001 ◽  
Vol 353 (2) ◽  
pp. 307-316 ◽  
Author(s):  
Luciana E. GIONO ◽  
Cecilia L. VARONE ◽  
Eduardo T. CÁNEPA
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Phosphorylation Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Putative Transcription Factor

The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

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