Is the chemical gate of connexins voltage sensitive? Behavior of Cx32 wild-type and mutant channels

1999 ◽  
Vol 276 (6) ◽  
pp. C1361-C1373 ◽  
Author(s):  
Camillo Peracchia ◽  
Xiao G. Wang ◽  
Lillian L. Peracchia
Keyword(s):  
Xenopus Oocytes ◽  
Wild Type ◽  
Cytoplasmic Loop ◽  
Terminal Domain ◽  
Cytosolic Protein ◽  
Gating Mechanism ◽  
Chemical Gating ◽  
Sensitive Behavior ◽  
Pore Plugging

Connexin channels are gated by transjunctional voltage ( V j) or CO2 via distinct mechanisms. The cytoplasmic loop (CL) and arginines of a COOH-terminal domain (CT1) of connexin32 (Cx32) were shown to determine CO2sensitivity, and a gating mechanism involving CL-CT1 association-dissociation was proposed. This study reports that Cx32 mutants, tandem, 5R/E, and 5R/N, designed to weaken CL-CT1interactions, display atypical V jand CO2 sensitivities when tested heterotypically with Cx32 wild-type channels in Xenopus oocytes. In tandems, two Cx32 monomers are linked NH2-to-COOH terminus. In 5R/E and 5R/N mutants, glutamates or asparagines replace CT1 arginines. On the basis of the intriguing sensitivity of the mutant-32 channel to V jpolarity, the existence of a “slow gate” distinct from the conventional V jgate is proposed. To a lesser extent the slow gate manifests itself also in homotypic Cx32 channels. Mutant-32 channels are more CO2 sensitive than homotypic Cx32 channels, and CO2-induced chemical gating is reversed with relative depolarization of the mutant oocyte, suggesting V jsensitivity of chemical gating. A hypothetical pore-plugging model involving an acidic cytosolic protein (possibly calmodulin) is discussed.

Molecular dissection of a basic COOH-terminal domain of Cx32 that inhibits gap junction gating sensitivity

1998 ◽  
Vol 275 (5) ◽  
pp. C1384-C1390 ◽  
Author(s):  
Xiao Guang Wang ◽  
Camillo Peracchia
Keyword(s):  
Gap Junction ◽  
Electrostatic Interactions ◽  
Xenopus Oocytes ◽  
Voltage Sensitivity ◽  
Wild Type ◽  
Cytoplasmic Loop ◽  
Terminal Domain ◽  
Gap Junction Channel ◽  
Chemical Gating

Connexin32 (Cx32) mutants were studied by double voltage clamp in Xenopus oocytes to determine the role of basic COOH-terminal residues in gap junction channel gating by CO2 and transjunctional voltage. Replacement of five arginines with N (5R/N) or T residues in the initial COOH-terminal domain (CT1) of Cx32 enhanced CO2 sensitivity. The positive charge, rather than the R residue per se, is responsible for the inhibitory role of CT1, because mutants replacing the five R residues with K (5R/K) or H (5R/H) displayed CO2 sensitivity comparable to that of wild-type Cx32. Mutants replacing R with N residues four at a time (4R/N) showed that CO2 sensitivity is strongly inhibited by R215 and mildly by R219, whereas R220, R223, and R224 may slightly increase sensitivity. Neither the 5R/N nor the 4R/N mutants differed in voltage sensitivity from wild-type Cx32. The possibility that inhibition of gating sensitivity results from electrostatic interactions between CT1 and the cytoplasmic loop is discussed as part of a model that envisions the cytoplasmic loop of Cx32 as a key element of chemical gating.

scholarly journals Tyrosine Latching of a Regulatory Gate Affords Allosteric Control of Aromatic Amino Acid Biosynthesis

2011 ◽  
Vol 286 (12) ◽  
pp. 10216-10224 ◽  
Author(s):  
Penelope J. Cross ◽  
Renwick C. J. Dobson ◽  
Mark L. Patchett ◽  
Emily J. Parker
Keyword(s):  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Shikimate Pathway ◽  
Amino Acid Biosynthesis ◽  
Wild Type ◽  
Acid Biosynthesis ◽  
Allosteric Control ◽  
Terminal Domain ◽  
Aromatic Amino Acid Biosynthesis ◽  
Gating Mechanism

The first step of the shikimate pathway for aromatic amino acid biosynthesis is catalyzed by 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Thermotoga maritima DAH7PS (TmaDAH7PS) is tetrameric, with monomer units comprised of a core catalytic (β/α)8 barrel and an N-terminal domain. This enzyme is inhibited strongly by tyrosine and to a lesser extent by the presence of phenylalanine. A truncated mutant of TmaDAH7PS lacking the N-terminal domain was catalytically more active and completely insensitive to tyrosine and phenylalanine, consistent with a role for this domain in allosteric inhibition. The structure of this protein was determined to 2.0 Å. In contrast to the wild-type enzyme, this enzyme is dimeric. Wild-type TmaDAH7PS was co-crystallized with tyrosine, and the structure of this complex was determined to a resolution of 2.35 Å. Tyrosine was found to bind at the interface between two regulatory N-terminal domains, formed from diagonally located monomers of the tetramer, revealing a major reorganization of the regulatory domain with respect to the barrel relative to unliganded enzyme. This significant conformational rearrangement observed in the crystal structures was also clearly evident from small angle X-ray scattering measurements recorded in the presence and absence of tyrosine. The closed conformation adopted by the protein on tyrosine binding impedes substrate entry into the neighboring barrel, revealing an unusual tyrosine-controlled gating mechanism for allosteric control of this enzyme.

scholarly journals Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation

2003 ◽  
Vol 185 (23) ◽  
pp. 6801-6808 ◽  
Author(s):  
Shannon A. Carroll ◽  
Torsten Hain ◽  
Ulrike Technow ◽  
Ayub Darji ◽  
Philippos Pashalidis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Separation ◽  
Bacterial Species ◽  
Surface Protein ◽  
Wild Type ◽  
Terminal Domain ◽  
Cell Wall Hydrolase ◽  
Characteristic Features ◽  
Type Gene

ABSTRACT A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes.

scholarly journals HOPS Proofreads the trans-SNARE Complex for Yeast Vacuole Fusion

2008 ◽  
Vol 19 (6) ◽  
pp. 2500-2508 ◽  
Author(s):  
Vincent J. Starai ◽  
Christopher M. Hickey ◽  
William Wickner
Keyword(s):  
Snare Complex ◽  
Wild Type ◽  
Fusion Event ◽  
Sm Proteins ◽  
Terminal Domain ◽  
Domain Structures ◽  
Vacuole Fusion ◽  
Protein Receptors ◽  
Optimal Fusion ◽  
Guanosine Triphosphatase

The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the Km value for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but it has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at two levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, because it suppresses the fusion of trans-SNARE complexes formed without the N-terminal Phox homology domain of Vam7p (Qc). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Qa) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.

scholarly journals State‐dependent barium block of wild‐type and inactivation‐deficient HERG channels in Xenopus oocytes

2000 ◽  
Vol 526 (2) ◽  
pp. 265-278 ◽  
Author(s):  
Manjula Weerapura ◽  
Stanley Nattel ◽  
Marc Courtemanche ◽  
David Doern ◽  
Nathalie Ethier ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Wild Type ◽  
State Dependent ◽  
Barium Block ◽  
Herg Channels

Effect of PCMBS on CO2permeability of Xenopus oocytes expressing aquaporin 1 or its C189S mutant

1998 ◽  
Vol 275 (6) ◽  
pp. C1481-C1486 ◽  
Author(s):  
Gordon J. Cooper ◽  
Walter F. Boron
Keyword(s):  
Carbonic Anhydrase ◽  
Intracellular Ph ◽  
Xenopus Oocytes ◽  
Membrane Lipid ◽  
Wild Type ◽  
Aquaporin 1 ◽  
Gas Channel ◽  
A Cell ◽  
Pass Through ◽  
Rule Out

A recent study on Xenopus oocytes [N. L. Nakhoul, M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 ( Cell Physiol. 43): C543–548, 1998] injected with carbonic anhydrase showed that expressing aquaporin 1 (AQP1) increases by ∼40% the rate at which exposing the cell to CO2 causes intracellular pH to fall. This observation is consistent with several interpretations. Overexpressing AQP1 might increase apparent CO2 permeability by 1) allowing CO2 to pass through AQP1, 2) stimulating injected carbonic anhydrase, 3) enhancing the CO2 solubility of the membrane’s lipid, or 4) increasing the expression of a native “gas channel.” The purpose of the present study was to distinguish among these possibilities. We found that expressing the H2O channel AQP1 in Xenopus oocytes increases the CO2 permeability of oocytes in an expression-dependent fashion, whereas expressing the K+ channel ROMK1 has no effect. The mercury derivative p-chloromercuriphenylsulfonic acid (PCMBS), which inhibits the H2O movement through AQP1, also blocks the AQP1-dependent increase in CO2 permeability. The mercury-insensitive C189S mutant of AQP1 increases the CO2 permeability of the oocyte to the same extent as does the wild-type channel. However, the C189S-dependent increase in CO2permeability is unaffected by treatment with PCMBS. These data rule out options 2–4 listed above. Thus our results suggest that CO2passes through the pore of AQP1 and are the first data to demonstrate that a gas can enter a cell by a means other than diffusing through the membrane lipid.

scholarly journals Increased metal tolerance and bioaccumulation of zinc and cadmium in Chlamydomonas reinhardtii expressing a AtHMA4 C-terminal domain protein

2020 ◽  
Author(s):  
Aniefon Ibuot ◽  
Rachel E. Webster ◽  
Lorraine E. Williams ◽  
Jon K. Pittman
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Metal Binding ◽  
Metal Tolerance ◽  
Alginate Beads ◽  
Full Length ◽  
Wild Type ◽  
Microalgal Biomass ◽  
Terminal Domain ◽  
Metal Biosorption ◽  
Transgenic Cells

AbstractThe use of microalgal biomass for metal pollutant bioremediation might be improved by genetic engineering to modify the selectivity or capacity of metal biosorption. A plant cadmium (Cd) and zinc (Zn) transporter (AtHMA4) was used as a transgene to increase the ability of Chlamydomonas reinhardtii to tolerate 0.2 mM Cd and 0.3 mM Zn exposure. The transgenic cells showed increased accumulation and internalisation of both metals compared to wild type. AtHMA4 was expressed either as the full-length protein or just the C-terminal tail, which is known to have metal binding sites. Similar Cd and Zn tolerance and accumulation was observed with expression of either the full-length protein or C-terminal domain, suggesting that enhanced metal tolerance was mainly due to increased metal binding rather than metal transport. The effectiveness of the transgenic cells was further examined by immobilisation in calcium alginate to generate microalgal beads that could be added to a metal contaminated solution. Immobilisation maintained metal tolerance, while AtHMA4-expressing cells in alginate showed a concentration-dependent increase in metal biosorption that was significantly greater than alginate beads composed of wild type cells. This demonstrates that expressing AtHMA4 full-length or C-terminus has great potential as a strategy for bioremediation using microalgal biomass.

A Dictystelium mutant with defective aggregate size determination

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2569-2578 ◽  
Author(s):  
D.A. Brock ◽  
G. Buczynski ◽  
T.P. Spann ◽  
S.A. Wood ◽  
J. Cardelli ◽  
...  
Keyword(s):  
Mrna Level ◽  
Phosphodiesterase Inhibitor ◽  
Aggregate Size ◽  
Conditioned Media ◽  
Wild Type ◽  
Significant Similarity ◽  
Western Blots ◽  
Cytosolic Protein ◽  
Large Numbers ◽  
Cell Fractions

Starved Dictyostelium cells aggregate into groups of roughly 10(5) cells. We have identified a gene which, when repressed by antisense transformation or homologous recombination, causes starved cells to form large numbers of small aggregates. We call the gene smlA for small aggregates. A roughly 1.0 kb smlA mRNA is expressed in vegetative and early developing cells, and the mRNA level then decreases at about 10 hours of development. The sequence of the cDNA and the derived amino acid sequence of the SmlA protein show no significant similarity to any known sequence. There are no obvious motifs in the protein or large regions of hydrophobicity or charge. Immunofluorescence and staining of Western blots of cell fractions indicates that SmlA is a 35x10(3) Mr cytosolic protein present in all vegetative and developing cells and is absent from smlA cells. The absence of SmlA does not affect the growth rate, cell cycle, motility, differentiation, or developmental speed of cells. Synergy experiments indicate that mixing 5% smlA cells with wild-type cells will cause the wild-type cells to form smaller fruiting bodies and aggregates. Although there is no detectable SmlA protein secreted from cells, starvation medium conditioned by smlA cells will cause wild-type cells to form large numbers of small aggregates. The component in the smlA-conditioned media that affects aggregate size is a molecule with a molecular mass greater than 100x10(3) Mr that is not conditioned media factor, phosphodiesterase or the phosphodiesterase inhibitor. The data thus suggest that the cytosolic protein SmlA regulates the secretion or processing of a secreted factor that regulates aggregate size.

scholarly journals Protective role for the N-terminal domain of α-dystroglycan in Influenza A virus proliferation

2019 ◽  
Vol 116 (23) ◽  
pp. 11396-11401 ◽  
Author(s):  
Jessica C. de Greef ◽  
Bram Slütter ◽  
Mary E. Anderson ◽  
Rebecca Hamlyn ◽  
Raul O’Campo Landa ◽  
...  
Keyword(s):  
Puerto Rico ◽  
Basement Membrane ◽  
Influenza A Virus ◽  
Influenza A ◽  
Protective Role ◽  
Wild Type ◽  
Terminal Domain ◽  
Bodily Fluids ◽  
Control Virus

α-Dystroglycan (α-DG) is a highly glycosylated basement membrane receptor that is cleaved by the proprotein convertase furin, which releases its N-terminal domain (α-DGN). Before cleavage, α-DGN interacts with the glycosyltransferase LARGE1 and initiates functional O-glycosylation of the mucin-like domain of α-DG. Notably, α-DGN has been detected in a wide variety of human bodily fluids, but the physiological significance of secreted α-DGN remains unknown. Here, we show that mice lacking α-DGN exhibit significantly higher viral titers in the lungs after Influenza A virus (IAV) infection (strain A/Puerto Rico/8/1934 H1N1), suggesting an inability to control virus load. Consistent with this, overexpression of α-DGN before infection or intranasal treatment with recombinant α-DGN prior and during infection, significantly reduced IAV titers in the lungs of wild-type mice. Hemagglutination inhibition assays using recombinant α-DGN showed in vitro neutralization of IAV. Collectively, our results support a protective role for α-DGN in IAV proliferation.

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