Accelerated Current Decay Kinetics of a Rare Human Acid-Sensing ion Channel 1a Variant That Is Used in Many Studies as Wild Type

Human acid-sensing ion channel 1b (hASIC1b) is a H+-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1b's COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment.

Download Full-text

Fluorescence Decay Kinetics of Wild Type and D2-H117N Mutant Photosystem II Reaction Centers Isolated fromChlamydomonas reinhardtii

The Journal of Physical Chemistry B ◽

10.1021/jp993556l ◽

2000 ◽

Vol 104 (19) ◽

pp. 4777-4781 ◽

Cited By ~ 12

Author(s):

Heather G. Johnston ◽

Jun Wang ◽

Stuart V. Ruffle ◽

Richard T. Sayre ◽

Terry L. Gustafson

Keyword(s):

Photosystem Ii ◽

Fluorescence Decay ◽

Reaction Centers ◽

Wild Type ◽

Decay Kinetics ◽

Fluorescence Decay Kinetics ◽

Kinetics Of

Download Full-text

High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

PLoS Biology ◽

10.1371/journal.pbio.3001321 ◽

2021 ◽

Vol 19 (9) ◽

pp. e3001321 ◽

Cited By ~ 1

Author(s):

Nina Braun ◽

Søren Friis ◽

Christian Ihling ◽

Andrea Sinz ◽

Jacob Andersen ◽

...

Keyword(s):

Ion Channels ◽

Ion Channel ◽

High Throughput ◽

Mammalian Cells ◽

Noncanonical Amino Acids ◽

Current Decay ◽

Human Acid ◽

Automated Patch Clamp ◽

Insight Into

Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a–psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.

Download Full-text

Kinetics of Neutralizing Antibodies of COVID-19 Patients Tested Using Clinical D614G, B.1.1.7 and B 1.351 Isolates in Microneutralization Assays

Viruses ◽

10.3390/v13060996 ◽

2021 ◽

Vol 13 (6) ◽

pp. 996

Author(s):

Jenni Virtanen ◽

Ruut Uusitalo ◽

Essi M. Korhonen ◽

Kirsi Aaltonen ◽

Teemu Smura ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Acute Infection ◽

Clinical Isolates ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Kinetics Of

Increasing evidence suggests that some newly emerged SARS-CoV-2 variants of concern (VoCs) resist neutralization by antibodies elicited by the early-pandemic wild-type virus. We applied neutralization tests to paired recoveree sera (n = 38) using clinical isolates representing the first wave (D614G), VoC1, and VoC2 lineages (B.1.1.7 and B 1.351). Neutralizing antibodies inhibited contemporary and VoC1 lineages, whereas inhibition of VoC2 was reduced 8-fold, with 50% of sera failing to show neutralization. These results provide evidence for the increased potential of VoC2 to reinfect previously SARS-CoV-infected individuals. The kinetics of NAbs in different patients showed similar decline against all variants, with generally low initial anti-B.1.351 responses becoming undetectable, but with anti-B.1.1.7 NAbs remaining detectable (>20) for months after acute infection.

Download Full-text

A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium

The Journal of Cell Biology ◽

10.1083/jcb.200611056 ◽

2007 ◽

Vol 176 (3) ◽

pp. 263-268 ◽

Cited By ~ 107

Author(s):

Adam C. Smith ◽

Won Do Heo ◽

Virginie Braun ◽

Xiuju Jiang ◽

Chloe Macrae ◽

...

Keyword(s):

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Dominant Negative ◽

Rab Gtpases ◽

Wild Type ◽

Intracellular Growth ◽

Non Invasive ◽

Serovar Typhimurium ◽

Guanosine Triphosphatase ◽

Kinetics Of

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome–lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.

Download Full-text

Fundamental Mechanisms of Copper CMP – Passivation Kinetics of Copper in CMP Slurry Constituents

MRS Proceedings ◽

10.1557/proc-1157-e06-02 ◽

2009 ◽

Vol 1157 ◽

Cited By ~ 6

Author(s):

Shantanu Tripathi ◽

Fiona M. Doyle ◽

David A. Dornfeld

Keyword(s):

Electrochemical Impedance ◽

Protective Film ◽

Current Decay ◽

Glycine Solution ◽

Current Densities ◽

Circuit Elements ◽

Copper Cmp ◽

Cu Cmp ◽

Kinetics Of ◽

Capacitive Charging

AbstractDuring copper CMP, abrasives and asperities interact with the copper at the nano-scale, partially removing protective films. The local Cu oxidation rate increases, then decays with time as the protective film reforms. In order to estimate the copper removal rate and other Cu-CMP output parameters with a mechanistic model, the passivation kinetics of Cu, i.e. the decay of the oxidation current with time after an abrasive/copper interaction, are needed. For the first time in studying Cu-CMP, microelectrodes were used to reduce interference from capacitive charging, IR drops and low diffusion limited currents, problems typical with traditional macroelectrodes. Electrochemical impedance spectroscopy (EIS) was used to obtain the equivalent circuit elements associated with different electrochemical phenomena (capacitive, kinetics, diffusion etc.) at different polarization potentials. These circuit elements were used to interpret potential-step chronoamperometry results in inhibiting and passivating solutions, notably to distinguish between capacitive charging and Faradaic currents.Chronoamperometry of Cu in acidic aqueous glycine solution containing the corrosion inhibitor benzotriazole (BTA) displayed a very consistent current decay behavior at all potentials, indicating that the rate of current decay was controlled by diffusion of BTA to the surface. In basic aqueous glycine solution, Cu (which undergoes passivation by a mechanism similar to that operating in weakly acidic hydrogen peroxide slurries) displayed similar chronoamperometric behavior for the first second or so at all anodic potentials. Thereafter, the current densities at active potentials settled to values around those expected from polarization curves, whereas the current densities at passive potentials continued to decline. Oxidized Cu species typically formed at ‘active’ potentials were found to cause significant current decay at active potentials and at passive potentials before more protective passive films form. This was established from galvanostatic experiments.

Download Full-text

Bactericidal Activity, Absence of Serum Effect, and Time-Kill Kinetics of Ceftazidime-Avibactam against β-Lactamase-Producing Enterobacteriaceae and Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02894-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5297-5305 ◽

Cited By ~ 55

Author(s):

Tiffany R. Keepers ◽

Marcela Gomez ◽

Chris Celeri ◽

Wright W. Nichols ◽

Kevin M. Krause

Keyword(s):

Pseudomonas Aeruginosa ◽

Human Serum ◽

Bactericidal Activity ◽

Minimum Bactericidal Concentration ◽

Wild Type ◽

Content Type ◽

Positive Isolate ◽

New Treatment ◽

Time Kill ◽

Kinetics Of

ABSTRACTAvibactam, a non-β-lactam β-lactamase inhibitor with activity against extended-spectrum β-lactamases (ESBLs), KPC, AmpC, and some OXA enzymes, extends the antibacterial activity of ceftazidime against most ceftazidime-resistant organisms producing these enzymes. In this study, the bactericidal activity of ceftazidime-avibactam against 18Pseudomonas aeruginosaisolates and 15Enterobacteriaceaeisolates, including wild-type isolates and ESBL, KPC, and/or AmpC producers, was evaluated. Ceftazidime-avibactam MICs (0.016 to 32 μg/ml) were lower than those for ceftazidime alone (0.06 to ≥256 μg/ml) against all isolates except for 2P. aeruginosaisolates (1blaVIM-positive isolate and 1blaOXA-23-positive isolate). The minimum bactericidal concentration/MIC ratios of ceftazidime-avibactam were ≤4 for all isolates, indicating bactericidal activity. Human serum and human serum albumin had a minimal effect on ceftazidime-avibactam MICs. Ceftazidime-avibactam time-kill kinetics were evaluated at low MIC multiples and showed time-dependent reductions in the number of CFU/ml from 0 to 6 h for all strains tested. A ≥3-log10decrease in the number of CFU/ml was observed at 6 h for allEnterobacteriaceae, and a 2-log10reduction in the number of CFU/ml was observed at 6 h for 3 of the 6P. aeruginosaisolates. Regrowth was noted at 24 h for some of the isolates tested in time-kill assays. These data demonstrate the potent bactericidal activity of ceftazidime-avibactam and support the continued clinical development of ceftazidime-avibactam as a new treatment option for infections caused byEnterobacteriaceaeandP. aeruginosa, including isolates resistant to ceftazidime by mechanisms dependent on avibactam-sensitive β-lactamases.

Download Full-text