scholarly journals Residues in Transmembrane Segments of the P2X4 Receptor Contribute to Channel Function and Ethanol Sensitivity

2020 ◽  
Vol 21 (7) ◽  
pp. 2471 ◽  
Author(s):  
Maya Popova ◽  
Larry Rodriguez ◽  
James R. Trudell ◽  
Sylvia Nguyen ◽  
Michael Bloomfield ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Molecular Models ◽  
Ethanol Sensitivity ◽  
Wild Type ◽  
Transmembrane Segments ◽  
Channel Opening ◽  
Promising Target ◽  
Electrode Voltage ◽  
P2x4 Receptors ◽  
Open States

Mouse models of alcohol use disorder (AUD) revealed purinergic P2X4 receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)–ectodomain interface and within the TM1 segment contribute to the formation of an ethanol action pocket in P2X4Rs. In the present study, we tested the hypothesis that there are more residues in TM1 and TM2 segments that are important for the ethanol sensitivity of P2X4Rs. Using site-directed mutagenesis and two electrode voltage-clamp electrophysiology in Xenopus oocytes, we found that arginine at position 33 (R33) in the TM1 segment plays a role in the ethanol sensitivity of P2X4Rs. Molecular models in both closed and open states provided evidence for interactions between R33 and aspartic acid at position 354 (D354) of the neighboring TM2 segment. The loss of ethanol sensitivity in mixtures of wild-type (WT) and reciprocal single mutants, R33D:WT and D354R:WT, versus the WT-like response in R33D-D354R:WT double mutant provided further support for this interaction. Additional findings indicated that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility/stability during channel opening. Collectively, these findings identified new activity sites and suggest the importance of TM1-TM2 interaction for the function and ethanol sensitivity of P2X4Rs.

scholarly journals Residues in Transmembrane Segments of the P2X4 Receptor Contribute to Channel Function and Ethanol Sensitivity

2020 ◽  
Author(s):  
Maya Popova ◽  
Larry Rodriguez ◽  
James R. Trudell ◽  
Sylvia Nguyen ◽  
Michael Bloomfield ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Molecular Models ◽  
Ethanol Sensitivity ◽  
Wild Type ◽  
Transmembrane Segments ◽  
Channel Function ◽  
Channel Opening ◽  
Promising Target ◽  
Electrode Voltage ◽  
Open States

Mouse models of alcohol use disorder (AUD) revealed a subtype of purinergic receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)-ectodomain interface and within TM1 segment contribute to the formation of an ethanol action pocket in P2X4Rs. In the present study, we tested the hypothesis that there are more residues in TM segments, which are important for ethanol sensitivity of P2X4Rs. Using site-directed mutagenesis and two-electrode voltage-clamp electrophysiology in Xenopus oocytes, we found that arginine at position 33 (R33) in the TM1 segment plays a role in ethanol sensitivity of P2X4Rs. Molecular models in both closed and open states provided evidence for interactions between R33 and aspartic acid at position 354 (D354) of the neighboring TM2 segment. Further work with mixtures of wild-type (WT) and reciprocal single (R33D:WT, D354R:WT) and double (R33D-D354R:WT) mutants confirmed the importance of this interaction for ethanol sensitivity, ivermectin action and channel function. Additionally, our findings suggest that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility during channel opening. Collectively, these findings identified new activity sites, and suggest the importance of TM1-TM2 interaction for channel function and ethanol sensitivity of P2X4Rs.

scholarly journals Major transmembrane movement associated with colicin Ia channel gating.

1996 ◽  
Vol 107 (3) ◽  
pp. 313-328 ◽  
Author(s):  
X Q Qiu ◽  
K S Jakes ◽  
P K Kienker ◽  
A Finkelstein ◽  
S L Slatin
Keyword(s):  
Water Soluble ◽  
Site Directed Mutagenesis ◽  
Lipid Bilayer Membranes ◽  
Transmembrane Segment ◽  
Transmembrane Segments ◽  
Protein Toxin ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Opening And Closing ◽  
Colicin Ia

Colicin Ia, a bacterial protein toxin of 626 amino acid residues, forms voltage-dependent channels in planar lipid bilayer membranes. We have exploited the high affinity binding of streptavidin to biotin to map the topology of the channel-forming domain (roughly 175 residues of the COOH-terminal end) with respect to the membrane. That is, we have determined, for the channel's open and closed states, which parts of this domain are exposed to the aqueous solutions on either side of the membrane and which are inserted into the bilayer. This was done by biotinylating cysteine residues introduced by site-directed mutagenesis, and monitoring by electrophysiological methods the effect of streptavidin addition on channel behavior. We have identified a region of at least 68 residues that flips back and forth across the membrane in association with channel opening and closing. This identification was based on our observations that for mutants biotinylated in this region, streptavidin added to the cis (colicin-containing) compartment interfered with channel opening, and trans streptavidin interfered with channel closing. (If biotin was linked to the colicin by a disulfide bond, the effects of streptavidin on channel closing could be reversed by detaching the streptavidin-biotin complex from the colicin, using a water-soluble reducing agent. This showed that the cysteine sulfur, not just the biotin, is exposed to the trans solution). The upstream and downstream segments flanking the translocated region move into and out of the bilayer during channel opening and closing, forming two transmembrane segments. Surprisingly, if any of several residues near the upstream end of the translocated region is held on the cis side by streptavidin, the colicin still forms voltage-dependent channels, indicating that a part of the protein that normally is fully translocated across the membrane can become the upstream transmembrane segment. Evidently, the identity of the upstream transmembrane segment is not crucial to channel formation, and several open channel structures can exist.

scholarly journals Topology of the Erwinia chrysanthemi oligogalacturonate porin KdgM

2003 ◽  
Vol 372 (2) ◽  
pp. 329-334 ◽  
Author(s):  
Teijo PELLINEN ◽  
Helena AHLFORS ◽  
Nicolas BLOT ◽  
Guy CONDEMINE
Keyword(s):  
Sequence Similarity ◽  
Site Directed Mutagenesis ◽  
Erwinia Chrysanthemi ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Transmembrane Segments

The Erwinia chrysanthemi oligogalacturonate-specific monomeric porin, KdgM, does not present homology with any porins of known structure. A model of this protein, based on sequence similarity and the amphipathy profile, was constructed. The model depicts a β-barrel composed of 14 antiparallel β-strands. The accuracy of this model was tested by the chemical labelling of cysteine residues introduced by site-directed mutagenesis. The protein has seven surface-exposed loops. They are rather small with the exception of one, loop L6. Deletion of this loop allowed the entry of maltopentaose into the bacteria, a molecule too large to enter through the wild-type KdgM. Loop L6 could fold back into the lumen of the pore and play the role of the constriction loop L3 of general porins. With 14 transmembrane segments, the KdgM porin family could represent the smallest porin characterized to date.

Expression and Characterization of Clustered Charge-to-Alanine Mutants of Low Mr Single-Chain Urokinase-Type Plasminogen Activator

1994 ◽  
Vol 71 (01) ◽  
pp. 134-140 ◽  
Author(s):  
S Ueshima ◽  
P Holvoet ◽  
H R Lijnen ◽  
L Nelles ◽  
V Seghers ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Solvent Accessibility ◽  
Site Directed Mutagenesis ◽  
Clot Lysis ◽  
Wild Type ◽  
Single Chain ◽  
Charged Amino Acids ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Pharmacokinetic Properties

SummaryIn an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M r single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala).The rscu-PA 32k moictic3 were expressed in High Five Ttichoplasiani cells, and purified to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/1. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion In lwo chain moieties by plasmin were comparable for mutant and wild-type rscu PA 32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 pi 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant lysis with 16 pg/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min). In hamsters with pulmonary embolism, continuous intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type rscu-PA-32k.These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed to eliminate charged regions with the highest solvent accessibility, do not have significantly improved functional, fibrinolytic or pharmacokinetic properties.

scholarly journals Unique Cellular and Biochemical Features of Human Mitochondrial Peroxiredoxin 3 Establish the Molecular Basis for Its Specific Reaction with Thiostrepton

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 150
Author(s):  
Kimberly J. Nelson ◽  
Terri Messier ◽  
Stephanie Milczarek ◽  
Alexis Saaman ◽  
Stacie Beuschel ◽  
...  
Keyword(s):  
Oxygen Species ◽  
Mitochondrial Reactive Oxygen Species ◽  
Wild Type ◽  
Cellular Models ◽  
Chemotherapeutic Approach ◽  
Promising Target ◽  
Redox Responsive ◽  
Catalytic Cysteine ◽  
Increased Sensitivity ◽  
Peroxiredoxin 3

A central hallmark of tumorigenesis is metabolic alterations that increase mitochondrial reactive oxygen species (mROS). In response, cancer cells upregulate their antioxidant capacity and redox-responsive signaling pathways. A promising chemotherapeutic approach is to increase ROS to levels incompatible with tumor cell survival. Mitochondrial peroxiredoxin 3 (PRX3) plays a significant role in detoxifying hydrogen peroxide (H2O2). PRX3 is a molecular target of thiostrepton (TS), a natural product and FDA-approved antibiotic. TS inactivates PRX3 by covalently adducting its two catalytic cysteine residues and crosslinking the homodimer. Using cellular models of malignant mesothelioma, we show here that PRX3 expression and mROS levels in cells correlate with sensitivity to TS and that TS reacts selectively with PRX3 relative to other PRX isoforms. Using recombinant PRXs 1–5, we demonstrate that TS preferentially reacts with a reduced thiolate in the PRX3 dimer at mitochondrial pH. We also show that partially oxidized PRX3 fully dissociates to dimers, while partially oxidized PRX1 and PRX2 remain largely decameric. The ability of TS to react with engineered dimers of PRX1 and PRX2 at mitochondrial pH, but inefficiently with wild-type decameric protein at cytoplasmic pH, supports a novel mechanism of action and explains the specificity of TS for PRX3. Thus, the unique structure and propensity of PRX3 to form dimers contribute to its increased sensitivity to TS-mediated inactivation, making PRX3 a promising target for prooxidant cancer therapy.

scholarly journals Suberoylanilide hydroxamic acid enhances the radiosensitivity of lung cancer cells through acetylated wild-type and mutant p53-dependent modulation of mitochondrial apoptosis

2021 ◽  
Vol 49 (2) ◽  
pp. 030006052098154
Author(s):  
Kan Wu ◽  
Xueqin Chen ◽  
Xufeng Chen ◽  
Shirong Zhang ◽  
Yasi Xu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Cells ◽  
Hydroxamic Acid ◽  
Site Directed Mutagenesis ◽  
Lung Cancer Cells ◽  
Mutant P53 ◽  
Suberoylanilide Hydroxamic Acid ◽  
Mitochondrial Apoptosis ◽  
Wild Type ◽  
Clonogenic Cell

Objective Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has shown potential as a candidate radiosensitizer for many types of cancers. This study aimed to explore the radiosensitization mechanism of SAHA in lung cancer cells. Methods Mutations in p53 were generated by site-directed mutagenesis using polymerase chain reaction. Transfection was performed to generate H1299 cells carrying wild-type or mutant p53. The radiosensitizing enhancement ratio was determined by clonogenic assays. Mitochondrial apoptosis was detected using JC-1 staining and flow cytometry analysis. Results Our results showed that SAHA induced radiosensitization in H1299 cells expressing wild-type p53, p53R175H or p53P223L, but this enhanced clonogenic cell death was not observed in parental H1299 (p53-null) cells or H1299 cells expressing p53 with K120R, A161T and V274R mutations. In SAHA-sensitized cells, mitochondrial apoptosis was induced following exposure to irradiation. Additionally, we observed that a secondary mutation at K120 (K120R) could eliminate p53-mediated radiosensitization and mitochondrial apoptosis. Conclusions The results of this study suggest that wild-type and specific mutant forms of p53 mediate SAHA-induced radiosensitization by regulating mitochondrial apoptosis, and the stabilization of K120 acetylation by SAHA is the molecular basis contributing to radiosensitization in lung cancer cells.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

scholarly journals Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels

2016 ◽  
Vol 147 (6) ◽  
pp. 437-449 ◽  
Author(s):  
Petronel Tuluc ◽  
Bruno Benedetti ◽  
Pierre Coste de Bagneaux ◽  
Manfred Grabner ◽  
Bernhard E. Flucher
Keyword(s):  
Calcium Channels ◽  
Molecular Mechanisms ◽  
Voltage Dependence ◽  
Specific Interaction ◽  
Site Directed Mutagenesis ◽  
Control Voltage ◽  
Voltage Sensitivity ◽  
Voltage Sensing ◽  
Activation Kinetics ◽  
Transmembrane Segments

Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.

scholarly journals N-terminal fatty acylation of the α-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation

1994 ◽  
Vol 303 (3) ◽  
pp. 697-700 ◽  
Author(s):  
F Galbiati ◽  
F Guzzi ◽  
A I Magee ◽  
G Milligan ◽  
M Parenti
Keyword(s):  
Palmitic Acid ◽  
G Protein ◽  
Fatty Acyl ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Α Subunit ◽  
Metabolic Labelling ◽  
Fatty Acylation ◽  
Absolute Requirement ◽  
Cdna Construct

The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.

scholarly journals Mutations in the Escherichia coli UvrB ATPase motif compromise excision repair capacity

1989 ◽  
Vol 86 (17) ◽  
pp. 6577-6581 ◽  
Author(s):  
T W Seeley ◽  
L Grossman
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Excision Repair ◽  
Site Directed Mutagenesis ◽  
Sequence Motif ◽  
Amino Acid Side Chain ◽  
Uv Resistance ◽  
Wild Type ◽  
General Applicability ◽  
Repair Capacity

The Escherichia coli UvrB protein possesses an amino acid sequence motif common to many ATPases. The role of this motif in UvrB has been investigated by site-directed mutagenesis. Three UvrB mutants, with amino acid replacements at lysine-45, failed to confer UV resistance when tested in the UV-sensitive strain N364 (delta uvrB), while five other mutants constructed near this region of UvrB confer wild-type levels of UV resistance. Because even the conservative substitution of arginine for lysine-45 in UvrB results in failure to confer UV resistance, we believe we have identified an amino acid side chain in UvrB essential to nucleotide excision repair in E. coli. The properties of two purified mutant UvrB proteins, lysine-45 to alanine (K45A) and asparagine-51 to alanine (N51A), were analyzed in vitro. While the K45A mutant is fully defective in incision of UV-irradiated DNA, K45A is capable of interaction with UvrA in forming an ATP-dependent nucleoprotein complex. The K45A mutant, however, fails to activate the characteristic increase in ATPase activity observed with the wild-type UvrB in the presence of UvrA and DNA. From these results we conclude that there is a second nucleotide-dependent step in incision following initial complex formation, which is defective in the K45A mutant. This experimental approach may prove of general applicability in the study of function and mechanism of other ATPase motif proteins.

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