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A protocol for refolding of thioredoxin-fused cysteine-rich peptides via addition of oxidized/reduced glutathione reagent directly to unfolded soluble fused protein has been ...developed. This procedure allows one to skip the steps of inclusion bodies purification, denaturation/disulfide reduction as well as lyophilization before oxidative folding, and thus to improve the yield of cysteine-rich peptides in their production using E. coli expression system.
Potassium voltage-gated channels (Kv) are considered as molecular targets in a number of serious neuronal, immune, and cardiac disorders. Search for efficient low-molecular weight modulators of Kv ...channel function provides a basis for the development of an appropriate therapy for various Kv-mediated diseases. We report here on a new bacterial cell-based system, which is suitable for study of interactions between ligands and ligand-binding sites of eukaryotic Kv1.3 and Kv1.1 channels. To create this system, high-level expression of KcsA-Kv1.3 and KcsA-Kv1.1 hybrid proteins (ligand-binding sites of Kv1.3 or Kv1.1 fused with prokaryotic KcsA potassium channel) was achieved in the plasma membrane of
Escherichia coli
. An efficient procedure of
E. coli
conversion to intact spheroplasts was developed. We demonstrate that fluorescently labeled agitoxin 2 binds specifically to high-affinity and lower-affinity sites of KcsA-Kv1.3 and KcsA-Kv1.1, respectively, at the membrane of spheroplasts. Number of binding sites per cell is estimated to be (1.0 ± 0.6) ×10
5
and (0.3 ± 0.2) ×10
5
for KcsA-Kv1.3- and KcsA-Kv1.1-presenting cells, respectively, that allows reliable detection of ligand–receptor interactions by confocal laser scanning microscopy. This bacterial cell-based system is intended for screening of ligands to membrane-embedded pharmaceutical targets.
CO
inhalation is currently the most common method of euthanasia for laboratory rats and mice, and it is often used for further terminal blood sampling for clinical biochemical assays. Lately, this ...method has been criticized due to animal welfare issues associated with some processes that develop after CO
inhalation. The stress reaction and the value of the clinical laboratory parameters significantly depend on the used anesthetics, method, and the site of blood sampling. Especially in small rodents, an acute terminal state followed by a cascade of metabolic reactions that can affect the studied biochemical profile may develop and cause unnecessary suffering of animals. The aim of this study was to compare the stability of biochemical parameters of outbred Sprague Dawley rats and CD-1 mice serum collected after CO
inhalation or the intramuscular injection of tiletamine-zolazepam-xylazine (TZX). The serum content of total protein and albumin, cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotr ansferase (ALT), alkaline phosphatase (ALP), total bilirubin, and creatinine was decreased by the injection of TZX in comparison with CO
inhalation. In addition, the levels of calcium, phosphates, chlorides and potassium were lowered by TZX vs. CO
administration, while the level of sodium increased. Finally, the level of the majority of serum clinical biochemical parameters in rats and mice tend to be overestimated after CO
inhalation, which may lead to masking the possible effect of anti-inflammatory drugs in animal tests. Injection anesthesia for small rodents with TZX is a more feasible method for terminal blood sampling, which also reduces the suffering of animals.
The wild-type scorpion toxin BeKm-1, which selectively blocks human ether-a-go-go related (hERG) channels, was radiolabeled with iodine at tyrosine 11. Both the mono- and di-iodinated derivatives ...were found to be biologically active. In electrophysiological patch-clamp recordings mono-127I-BeKm-1 had a concentration of half-maximal inhibition (IC50 value) of 27 nM, while wild-type BeKm-1 inhibited hERG channels with an IC50 value of 7 nM. Mono-125I-BeKm-1 was found to bind in a concentration-dependent manner and with picomolar affinity to hERG channel protein in purified membrane vesicles from transfected human embryonic kidney cells (HEK-293). Under optimized conditions the equilibrium dissociation constant ( Kd) values from saturation and kinetic binding analysis were 13 and 14 pM, respectively. Both the association and dissociation of (125)I-BeKm-1 were fast (association rate constant, k(on)=3.6 x 10(7) M(-1)s(-1); dissociation rate constant, k(off)=0.005 s(-1)). Wild-type BeKm-1 displaced binding of 125I-BeKm-1 with half-maximal inhibitory concentrations of 44 pM. In contrast, competition experiments with a BeKm-1 mutant BeKm-1-K18A, in which the toxin interaction site is disrupted, resulted in a drop in affinity by more than 300-fold as compared to the wild-type toxin. Iberiotoxin and apamin, peptide inhibitors of Ca2+-activated K+-channels, had no effect on 125I-BeKm-1 binding. Adding the classical rapid delayed rectifier current (IKr) blocker E-4031 reduced binding of 125I-BeKm-1 to the hERG channel to an IC50 of 7 nM. In autoradiographic studies on rat hearts, binding of 125I-BeKm-1 was dose-dependent and could partially be displaced by the addition of excess amounts of non-radioactive BeKm-1. The density of the radioactive signal was equally distributed in the myocardium of both the ventricle and atria indicating a homogenous expression of hERG channels throughout the heart.
In heterologous expression systems, KCNE1 and KCNE2 each can associate with KCNQ1 and exert apparently opposite effects on its channel function. KCNQ1 and KCNE1 associate to form the slow delayed ...rectifier I(Ks) channels in the heart. Whether KCNE2 plays any role in I(Ks) function is not clear.
The purpose of this study was to study whether KCNE2 can associate with KCNQ1 in the presence of KCNE1 and modulate its function.
Voltage clamp methods were used to study channel function in cardiomyocytes and in oocytes or COS-7 cells and immunocytochemistry/coimmunoprecipitation was used to study protein colocalization/association.
Adult rat ventricular myocytes express functional I(Ks), and KCNE2 is colocalized with KCNQ1 and KCNE1 at surface membrane and t-tubules. A detailed study of KCNQ1 modulation by KCNE2 at different KCNE2 expression levels reveals that, surprisingly, KCNE2 and KCNE1 share the major features in modulating KCNQ1 gating kinetics: slowing of activation, positive shift in the voltage range of activation, and suppression of inactivation. However, KCNE2 reduces KCNQ1 current amplitude whereas KCNE1 increases it, and KCNE2 induces a constitutively active KCNQ1 component whereas KCNE1 does not. Coimmunoprecipitation suggests that KCNQ1, KCNE1, and KCNE2 can form a tripartite complex, indicating that KCNE2 can bind to KCNQ1 in the presence of KCNE1. Coexpressing KCNE2 with KCNQ1 and KCNE1 leads to a decrease in the I(Ks) current amplitude without altering the gating kinetics.
Our data suggest that KCNE2 is in close proximity to KCNQ1 and KCNE1 in cardiomyocytes and may participate in dynamic regulation of I(Ks) current amplitude in the heart.
A novel inhibitor of voltage-gated K+ channels has been purified to homogeneity from the venom of the black scorpion Orthochirus scrobiculosus. This toxin, named OsK2, has been characterized as a ...28-residue peptide, containing six conserved cysteine residues and was shown to be a potent and selective blocker of Kv1.2 channels (Kd = 97 nM). OsK2 is the second member of the 13th subfamily of short-chain K+ channel-blocking peptides known thus far and is therefore called α-KTx 13.2.
The ether-a-go-go-related gene (erg) K super(+) channels are known to be crucial for life in Caenorhabditis elegans (mating), Drosophila melanogaster (seizure), and humans (LQT syndrome). The erg ...genes known to date (erg1, erg2, and erg3) are highly expressed in various areas of the rat and mouse central nervous system (CNS), and ERG channel blockers alter firing accommodation. To assign physiological roles to each isoform, it is necessary to design pharmacological strategies to distinguish individual currents. To this purpose, we have investigated the blocking properties of specific peptide inhibitors of hERG1 channels on the human and rat isoforms. In particular, we have tested ErgTx1 (from the scorpion Centruroides noxious), BeKm-1 (from the scorpion Buthus eupeus), and APETx1 (from the sea anemone Anthopleura elegantissima). Because these peptides had different species-specific effects on the six different channels, we have also carried out a biophysical characterization of hERG2 and hERG3 channels that turned out to be different from the rat homologs. It emerged that APETx1 is exquisitely selective for ERG1 and does not compete with the other two toxins. BeKm-1 discriminates well among the three rat members. ErgTx1 is unable to block hERG2, but blocks rERG2 and has the lowest K sub(D) for hERG3. BeKm-1 and ErgTx1 compete for hERG3 but not for rERG2 blockade. Our findings should be helpful for structure-function studies and for novel CNS ERG-specific drug design.