Hill Coefficient Research Articles

Copper Increases the Cooperative Gating of Rat P2X2a Receptor Channels

Background/Objectives: P2X receptor channels are widely expressed in the CNS, where they have multiple functions in health and disease. The rat P2X2a (rP2X2a) receptor channel is modulated by copper, an essential trace element that plays important roles in synaptic modulation and neurodegenerative disorders. Although essential extracellular amino acids that coordinate copper have been identified, the exact mechanism of copper-induced modulation has not been yet elucidated. Methods: We used HEK293T cells expressing rP2X2a channel(s) and performed outside-out single-channel and whole-cell recordings to explore copper’s effects on rP2X2 currents and determine whether this metal can increase the cooperative gating of rP2X2a channel. Results: In whole-cell recordings and in patches containing 2 or 3 rP2X2a channels, copper enhanced the ATP-induced currents, significantly reducing the ATP EC50 and increasing the Hill coefficient. Moreover, copper increased the apparent Po in patches containing two or three channels. By contrast, in patches containing only one rP2X2a channel, we did not observe any significant changes in ATP EC50, the Hill coefficient, or Po. Conclusions: Copper modulates the gating of rP2X2a channels, enhancing interchannel cooperativity without altering single-channel conductance or Po. This novel regulatory mechanism could be relevant for understanding the role of P2X2 channels in physiological and pathological processes.

Role of ryanodine receptor cooperativity in Ca2+-wave-mediated triggered activity in cardiomyocytes.

Ca2+ waves are known to trigger delayed after-depolarizations that can cause malignant cardiac arrhythmias. However, modelling Ca2+ waves using physiologically realistic models has remained a major challenge. Existing models with low Ca2+ sensitivity of ryanodine receptors (RyRs) necessitate large release currents, leading to an unrealistically large Ca2+ transient amplitude incompatible with the experimental observations. Consequently, current physiologically detailed models of delayed after-depolarizations resort to unrealistic cell architectures to produce Ca2+ waves with a normal Ca2+ transient amplitude. Here, we address these challenges by incorporating RyR cooperativity into a physiologically detailed model with a realistic cell architecture. We represent RyR cooperativity phenomenologically through a Hill coefficient within the sigmoid function of RyR open probability. Simulations in permeabilized myocytes with high Ca2+ sensitivity reveal that a sufficiently large Hill coefficient is required for Ca2+ wave propagation via the fire-diffuse-fire mechanism. In intact myocytes, propagating Ca2+ waves can occur only within an intermediate Hill coefficient range. Within this range, the spark rate is neither too low, enabling Ca2+ wave propagation, nor too high, allowing for the maintenance of a high sarcoplasmic reticulum load during diastole of the action potential. Moreover, this model successfully replicates other experimentally observed manifestations of Ca2+-wave-mediated triggered activity, including phase 2 and phase 3 early after-depolarizations and high-frequency voltage-Ca2+ oscillations. These oscillations feature an elevated take-off potential with depolarization mediated by the L-type Ca2+ current. The model also sheds light on the roles of luminal gating of RyRs and the mobile buffer ATP in the genesis of these arrhythmogenic phenomena. KEY POINTS: Existing mathematical models of Ca2+ waves use an excessively large Ca2+-release current or unrealistic diffusive coupling between release units. Our physiologically realistic model, using a Hill coefficient in the ryanodine receptor (RyR) gating function to represent RyR cooperativity, addresses these limitations and generates organized Ca2+ waves at Hill coefficients ranging from ∼5 to 10, as opposed to the traditional value of 2. This range of Hill coefficients gives a spark rate neither too low, thereby enabling Ca2+ wave propagation, nor too high, allowing for the maintenance of a high sarcoplasmic reticulum load during the plateau phase of the action potential. Additionally, the model generates Ca2+-wave-mediated phase 2 and phase 3 early after-depolarizations, and coupled membrane voltage with Ca2+ oscillations mediated by the L-type Ca2+ current. This study suggests that pharmacologically targeting RyR cooperativity could be a promising strategy for treating cardiac arrhythmias linked to Ca2+-wave-mediated triggered activity.

Anisotropy of plastic flow in Zr-2.5Nb pressure tube material analysed using a viscoplastic self-consistent approach

Plastic anisotropy is observed during plastic flow in samples from the axial and transverse directions of Zr-2.5Nb pressure tubes used in CANDU11CANada Deuterium Uranium - Pressurized Heavy Water power reactors. nuclear reactors tested in uniaxial tension. Plastic anisotropy was also measured in shear by testing in torsion ‘mini’ pressure tubes from the same material. Room temperature results from these experiments were analysed using a visco-plastic self-consistent model that takes into account the crystallographic texture of the material and allows for the single crystal work hardening behaviour to be described by means of a deformation law specific to each slip system.The visco-plastic self-consistent model was used to derive: (i) the evolution with strain of the critical resolved shear stress values consistent with prismatic, basal and pyramidal dislocation glide, (ii) the evolution of slip system activity as a function of strain, and (iii) the values of Hill's plastic anisotropy coefficients that are consistent with the observed anisotropy of yielding and their dependence on accumulated strain. The model also allows for the prediction of the components of the flow stress tensor that cannot be measured experimentally and their dependence on the work hardening behaviour of pressure tube material. Moreover, the yield surface after different amounts of plastic strain was calculated using the visco-plastic self-consistent model, compared to the one that results from using the Hill's anisotropy coefficients. Our work exposes the limitations of the Hill ellipsoid for describing plastic yield when microstructural evolution is present.

Use of a PK/PD Model to Select Cetagliptin Dosages for Patients with Type 2 Diabetes in Phase 3 Trials.

Cetagliptin is a novel dipeptidyl peptidase-4 (DPP-4) inhibitor developed for the treatment of patients with type 2 diabetes (T2D). Several phase 1 studies have been conducted in China. Modelling and simulation were used to obtain cetagliptin dose for phase 3 trials in T2D patients. A pharmacokinetic (PK)/pharmacodynamic (PD) model and model-based analysis of the relationship between hemoglobin A1c (HbA1c) and dosage was explored to guide dose selection of cetagliptin for phase 3 trials. The PK/PD data were derived from four phase 1 clinical studies, and sitagliptin 100 mg was employed as a positive control in studies 1, 3, and 4. The PK profiles of cetagliptin were well described by a two-compartment model with first-order absorption, saturated efflux, and first-order elimination. The final PD model was a sigmoid maximum inhibitory efficacy (Emax) model with the Hill coefficient. The final model accurately captured cetagliptin PK/PD, demonstrated by goodness-of-fit plots. Based on weighted average inhibition (WAI), the relationship between HbA1c and dose was well displayed. Cetagliptin 50 mg once daily or above as monotherapy or as add-on therapy appeared more effective in HbA1c reduction than sitagliptin 100 mg. Cetagliptin 50 mg or 100 mg once daily was selected as the dose for phase 3 trials of cetagliptin in T2D patients. The PK/PD model supports dose selection of cetagliptin for phase 3 trials. A model‑informed approach can be used to replace a dose-finding trial and accelerate cetagliptin's development.

Carbohydrate-Binding Mechanism of the Coagulant Lectin from Moringa oleifera Seeds (cMoL) Is Related to the Dimeric Protein Structure

This study characterized the binding mechanisms of the lectin cMoL (from Moringa oleifera seeds) to carbohydrates using spectroscopy and molecular dynamics (MD). The interaction with carbohydrates was studied by evaluating lectin fluorescence emission after titration with glucose or galactose (2.0–11 mM). The Stern–Volmer constant (Ksv), binding constant (Ka), Gibbs free energy (∆G), and Hill coefficient were calculated. After the urea-induced denaturation of cMoL, evaluations were performed using fluorescence spectroscopy, circular dichroism (CD), and hemagglutinating activity (HA) evaluations. The MD simulations were performed using the Amber 20 package. The decrease in Ksv revealed that cMoL interacts with carbohydrates via a static mechanism. The cMoL bound carbohydrates spontaneously (ΔG < 0) and presented a Ka on the order of 102, with high selectivity for glucose. Protein–ligand complexes were stabilized by hydrogen bonds and hydrophobic interactions. The Hill parameter (h~2) indicated that the binding occurs through the cMoL dimer. The loss of HA at urea concentrations at which the fluorescence and CD spectra indicated protein monomerization confirmed these results. The MD simulations revealed that glucose bound to the large cavity formed between the monomers. In conclusion, the biotechnological application of cMoL lectin requires specific methods or media to improve its dimeric protein structure.

Inhibition of hERG K channels by verapamil at physiological temperature: Implications for the CiPA initiative

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative reassesses using the inhibition of hERG potassium channels by drugs as the major determinant for the potential to cause drug-induced Torsades de Pointes (TdP) cardiac arrhythmias. Here we report our findings on the next phase of CiPA: Determination of hERG inhibitory properties using the standard CiPA-defined data acquisition protocol, here called the standard protocol, at physiological temperature (37 degrees Celsius). To do this, we measured inhibition of hERG1a potassium channels stably expressed in HEK293 cells by the small molecule verapamil, using manual whole-cell patch-clamp electrophysiology recordings with the standard protocol, which is characterized, in part, by a series of 10 s duration voltage steps to 0 mV, ultimately leading to a cumulative recording time of approximately 30 min. Using the standard protocol, we measured an IC50 for verapamil of 225 nM, a Hill coefficient of 1, and time constant of inhibition at 0 mV of 0.64 s. But, using the standard protocol resulted in a very low (5 %) experimental success rate per cell, which had low practicality for future experiments. To address the 5 % success rate, we generated a revised protocol characterized, in part, by a series of 3 s duration voltage steps to 0 mV, leading to a cumulative recording time of approximately 10 min. Using the revised protocol, we found an IC50 for verapamil of 252 nM, a Hill coefficient of 0.8, and time constant of inhibition at 0 mV of 0.67 s. The values measured with the revised protocol were similar to those measured using the standard protocol and, furthermore, our success rate using the revised protocol rose to 25 %, an increase of 5-fold over the standard protocol, and more in line with the success rate for biophysical studies. In summary, we captured key pharmacological data for subsequent analysis in CiPA using a revised protocol with an increased success rate and an overall enhanced feasibility and practicality. We propose that the revised protocol may be more pragmatic for generation of some hERG channel drug inhibition data for CiPA and other regulatory sciences.

Electron transfer in the respiratory chain at low salinity

Recent studies have established that cellular electrostatic interactions are more influential than assumed previously. Here, we use cryo-EM and perform steady-state kinetic studies to investigate electrostatic interactions between cytochrome (cyt.) c and the complex (C) III2-IV supercomplex from Saccharomyces cerevisiae at low salinity. The kinetic studies show a sharp transition with a Hill coefficient ≥2, which together with the cryo-EM data at 2.4 Å resolution indicate multiple cyt. c molecules bound along the supercomplex surface. Negatively charged loops of CIII2 subunits Qcr6 and Qcr9 become structured to interact with cyt. c. In addition, the higher resolution allows us to identify water molecules in proton pathways of CIV and, to the best of our knowledge, previously unresolved cardiolipin molecules. In conclusion, the lowered electrostatic screening renders engagement of multiple cyt. c molecules that are directed by electrostatically structured CIII2 loops to conduct electron transfer between CIII2 and CIV.

Transglutaminase–mucin binding dynamics in gastrointestinal mucus: Interfacial behaviour, thermodynamics and gelation mechanism

Transglutaminase, an enzyme present in epithelial cells and as a residual component in processed foods, is hypothesised to interact with gastrointestinal mucus, impacting its structure and function. To test the physical validity of this phenomenon, this study investigates the binding kinetics and thermodynamics between porcine gastric mucin (PGM) and microbial transglumatinse (TGM) in a model gastrointestinal mucus, followed by the rheological analysis of the resulting systems. At pH 7, TGM exhibited pronounced binding with the PGM interface, characterised by a high surface density (55.34 ± 1.86 µg/m−2) and a low dissociation constant (KD ∼ 4.03 ± 0.10 μM) as determined by surface plasma resonance (SPR). Conversely, at pH 3, TGM showed weak adhesion onto PGM, resulting in a less stable binding, reflected by a lower surface density (10.94 ± 0.67 µg/m−2) and a higher dissociation constant (KD ∼ 6.24 ± 0.22 μM). Regardless of the nature of interaction, the Hill coefficients (nH ≥ 1) indicated that the binding sites were markedly denser at pH 7 (1383.38 ± 46.47 pmol/m−2), than at pH 3 (273.68 ± 16.84 pmol/m−2). Fluorimetry analysis suggested temperature-dependent dynamic binding between PGM and TGM. The resulting Benesi – Hildebrand plots illustrated a linear correlation between PGM and increasing TGM concentration, suggesting a single-step interaction mechanism. The calculated thermodynamic parameters indicated spontaneous interaction between PGM and TGM (ΔG < 0) via endothermic (entropic) interactions (ΔH > 0). Notably, hydrophobic forces played a significant role in the network stabilisation of PGM−TGM complex (ΔS > 0). Rheometry analysis elucidates that the interaction maxima within TGM−PGM systems substantially elevate both shear viscosity (η), and the melting point (Tm), shifting from 6.75 ± 0.28 to 70.94 ± 2.21 (×10−3) Pa s (at 1 s−1), and 36.80 ± 0.80 to 48.20 ± 0.11 °C, respectively. Furthermore, the coexistence of these two macromolecular species results in a 3- to 4-fold dramatic increase in the viscoelastic moduli of the binary complex. These findings build a strong physicochemical basis for the interaction between TGM and PGM, with profound effects on the rhological behaviour of the latter; it also highlights the need to examine the biochemical/enzymatic aspects of said interactions, and their potential effects on mucosa and human physiology.

Preliminary Study on Polymerization between Hemoglobin and Enzymes during the Preparation of PolyHb-SOD-CAT-CA.

The objective of this study was to explore the influence of different factors on the aggregation effect on hemoglobin (Hb) and enzymes during the preparation of Polyhemoglobin-Superoxide dismutase-Catalase-Carbonic anhydrase (PolyHb-SOD-CAT-CA). Several factors including temperatures, pH values, Glutaraldehyde (GDA) amounts and enzymes amounts were investigated systematically to study their effects on the enzymes recoveries and polymerization rates including the Superoxide dismutase (SOD), Catalase (CAT) and Carbonic anhydrase (CA), as well as their effects on the molecular weight distribution of PolyHb-SOD-CAT-CA. Then the oxygen affinity and methemoglobin (MetHb) contents of obtained PolyHb-SOD-CAT-CA were measured to evaluate the effects of enzyme crosslinking on the properties of Polyhemoglobin (PolyHb) moieties in the molecular structure of obtained PolyHb-SOD-CAT-CA conjugate. The results showed that the enzyme recoveries and polymerization rates could be decreased with the temperatures increasing and could be generally kept stable in the physiological pH conditions, but presented only slight changes among the investigated enzyme amounts ranges. Although the GDA concentration increasing could promote the enzyme polymerization rates, the enzyme recoveries decreased in whole. The polymerization rate and molecular size of PolyHb-SOD-CAT-CA conjugate increased with the elevation of temperature and the concentration of GDA. Lastly, the P50 values, Hill coefficients, and MetHb contents of PolyHb-SOD-CAT-CA conjugate with different enzyme crosslinking degrees exhibited no obvious differences with each other. In conclusion, the polymerization reactions between enzymes and Hb molecules could be remarkably affected by temperatures, pH values, and GDA amounts, and the enzyme crosslinking presented no obvious effects on the Hb properties, especially about the oxygen affinity and oxidation degrees.

Stimuli‐Responsive Molecular Duplexes Displaying Duplex‐to‐Duplex Switching

AbstractSynthetic duplexes with high stabilities have promising potential for mimicking biomolecular functions and developing supramolecular smart materials. Herein, we describe the synthesis and stimuli‐responsive properties of molecular duplexes derived from indolocarbazole–pyridine (I−P) oligomers. These duplexes adopt nonclassical helical structures, stabilized by I−P hydrogen‐bonding pairs in anhydrous chlorinated solvents. Notably, the longest duplex 62 (11‐mer)2 displays remarkable stability, forming twenty hydrogen bonds; its exchange energy barrier was determined to be ΔG≠=22.0 kcal ⋅ mol−1 at 75 °C in anhydrous (CDCl2)2. Upon the addition of water, a hydrated duplex 62 (11‐mer)2⊃10H2O was formed, with one water molecule inserted between each I−P hydrogen‐bonding pair. The Hill coefficient (n) for this process is 6.1, demonstrating extremely positive cooperativity. Conversely, the hydrated duplex 62 (11‐mer)2⊃10H2O was completely converted into the original anhydrous duplex 62 (11‐mer)2 when the temperature was increased. Interconversion between these two distinct duplexes can be repeatedly carried out by varying the temperature. Furthermore, reversible switching between hetero‐duplexes and homo‐duplexes was also demonstrated by controlling the temperature, with concomitant changes in the characteristic emission signals.

Stimuli-Responsive Molecular Duplexes Displaying Duplex-to-Duplex Switching.

Synthetic duplexes with high stabilities have promising potential for mimicking biomolecular functions and developing supramolecular smart materials. Herein, we describe the synthesis and stimuli-responsive properties of molecular duplexes derived from indolocarbazole-pyridine (I-P) oligomers. These duplexes adopt nonclassical helical structures, stabilized by I-P hydrogen-bonding pairs in anhydrous chlorinated solvents. Notably, the longest duplex 62 (11-mer)2 displays remarkable stability, forming twenty hydrogen bonds; its exchange energy barrier was determined to be ΔG≠=22.0 kcal ⋅ mol-1 at 75 °C in anhydrous (CDCl2)2. Upon the addition of water, a hydrated duplex 62 (11-mer)2⊃10H2O was formed, with one water molecule inserted between each I-P hydrogen-bonding pair. The Hill coefficient (n) for this process is 6.1, demonstrating extremely positive cooperativity. Conversely, the hydrated duplex 62 (11-mer)2⊃10H2O was completely converted into the original anhydrous duplex 62 (11-mer)2 when the temperature was increased. Interconversion between these two distinct duplexes can be repeatedly carried out by varying the temperature. Furthermore, reversible switching between hetero-duplexes and homo-duplexes was also demonstrated by controlling the temperature, with concomitant changes in the characteristic emission signals.

Comparison of clot waveform analysis with or without adjustment between prothrombin time and activated partial thromboplastin time assays to assess in vitro effects of direct oral anticoagulants

BackgroundClot waveform analysis (CWA) reportedly enhances the interpretation of clotting time measurement. This study aimed to compare CWA between prothrombin time (PT) and activated partial thromboplastin time (APTT) assays for better understanding how to apply CWA for assessing effects of direct oral anticoagulants (DOACs). MethodsSamples were prepared by spiking plasma with rivaroxaban, apixaban, edoxaban, or dabigatran. To compensate the influence of fibrinogen, CWA parameters were adjusted by unifying maximum changes in transmittance in clotting reaction curves detected by the optical system. ResultsNon-adjusted PT-CWA parameters unexpectedly rose at low drug concentrations but declined at high drug concentrations while adjusted PT-CWA parameters exhibited dose-dependent decrease. Both non-adjusted and adjusted APTT-CWA parameters showed dose-dependent decrease. Adjusted CWA parameters were applicable to Hill plot analysis. All DOACs exhibited Hill coefficients indicating positively cooperative effects regarding most adjusted PT-CWA parameters. Regarding adjusted APTT-CWA parameters, rivaroxaban, apixaban, and edoxaban exhibited Hill coefficients indicating no or negatively cooperative effects. The observed differences between PT-CWA and APTT-CWA suggested the implication of thrombin positive feedback in DOAC effects. ConclusionThe results revealed distinct features of DOAC effects in extrinsic and intrinsic pathways. To ascertain the clinical implication, further studies using clinical samples are needed.

Cytostatic Bacterial Metabolites Interfere with 5-Fluorouracil, Doxorubicin and Paclitaxel Efficiency in 4T1 Breast Cancer Cells.

The microbiome is capable of modulating the bioavailability of chemotherapy drugs, mainly due to metabolizing these agents. Multiple cytostatic bacterial metabolites were recently identified that have cytostatic effects on cancer cells. In this study, we addressed the question of whether a set of cytostatic bacterial metabolites (cadaverine, indolepropionic acid and indoxylsulfate) can interfere with the cytostatic effects of the chemotherapy agents used in the management of breast cancer (doxorubicin, gemcitabine, irinotecan, methotrexate, rucaparib, 5-fluorouracil and paclitaxel). The chemotherapy drugs were applied in a wide concentration range to which a bacterial metabolite was added in a concentration within its serum reference range, and the effects on cell proliferation were assessed. There was no interference between gemcitabine, irinotecan, methotrexate or rucaparib and the bacterial metabolites. Nevertheless, cadaverine and indolepropionic acid modulated the Hill coefficient of the inhibitory curve of doxorubicin and 5-fluorouracil. Changes to the Hill coefficient implicate alterations to the kinetics of the binding of the chemotherapy agents to their targets. These effects have an unpredictable significance from the clinical or pharmacological perspective. Importantly, indolepropionic acid decreased the IC50 value of paclitaxel, which is a potentially advantageous combination.

Eugenol and lidocaine inhibit voltage-gated Na+ channels from dorsal root ganglion neurons with different mechanisms.

Eugenol (EUG) is a bioactive monoterpenoid used as an analgesic, preservative, and flavoring agent. Our new data show EUG as a voltage-gated Na+ channel (VGSC) inhibitor, comparable but not identical to lidocaine (LID). EUG inhibits both total and only TTX-R voltage-activated Na+ currents (INa) recorded from VGSCs naturally expressed on dorsal root ganglion sensory neurons in rats. Inhibition is quick, fully reversible, and dose-dependent. Our biophysical and pharmacological analyses showed that EUG and LID inhibit VGSCs with different mechanisms. EUG inhibits VGSCs with a dose-response relationship characterized by a Hill coefficient of 2, while this parameter for the inhibition by LID is 1. Furthermore, in a different way from LID, EUG modified the voltage dependence of both the VGSC activation and inactivation processes and the recovery from fast inactivated states and the entry to slow inactivated states. In addition, we suggest that EUG, but not LID, interacts with VGSC pre-open-closed states, according to our data.

The Self-Assembly of Cationic Metal Complexes on Gold Nanoparticle Surface.

This work aims to study the interaction between cationic metal complexes (M z +) and gold nanoparticles (AuNPs z- ). The M z + complexes were chosen from previous works described in the literature and were synthesized as defined. For example, they are as follows: 1 = [RuCl(dppb)(bipy)(py)](PF6); 2 = [RuCl(dppb)(bipy)(vpy)](PF6); 3 = [RuCl(dppb)(bipy)(mepy)](PF6); 4 = [RuCl(dppb)(bipy)(tbpy)](PF6); 5 = [RuCl2(dppb)(bipy)](PF6); 6 = [Fe(bipy)3]Cl2; 7 = [Ru(bipy)3](PF6)2; 8 = [TPyP{RuCl(dppb)(bipy)}4](PF6)4; and 9 = [RuCl(p-cymene)(Diipmp)](PF6). The interactions between M z + and AuNPs z- were carried out using conductometry and UV-vis spectroscopy. These experiments allowed determination of kinetic parameters, revealing three different steps in the interaction process: induction time, flocculation, and agglomeration. The self-assembly between M z + and AuNPs z- was investigated using three different models of binding site, namely, Langmuir or direct plot, Benesi-Hildebrand, and Scatchard. These models provide the fraction of total binding sites occupied (θ), the formation constant (K f), which is dependent on the temperature and geometric structure of each group of M z +, and the Gibbs free energy of reaction (ΔG r ), which was negative for each pair of M z + and AuNPs z- , revealing a spontaneous agglomeration process. The Hill coefficient (n) was 1 for almost all complexes, indicating that agglomeration is an independent process, except for 5, where n = 2, suggesting a positive propensity to bind onto the AuNPs z- surface. The models have confirmed a noncovalent interaction between these species. The relative error in site binding does not show any variation with changes in the temperature, but a fine-tuning of the n value to 1.00 was observed with the increase of the temperature. Finally, the reduction reaction of the 4-nitrophenolate anion (4-NP-) by NaBH4 catalyzed by AuNPs z- was used in the presence of M z + as an evaluation test to show how the M z + species will disturb the 4-NP- binding site on the surface of gold nanoparticles.

Using Fluorescent GAP Indicators to Monitor ER Ca2+

AbstractThe endoplasmic reticulum (ER) is the main reservoir of Ca2+ of the cell. Accurate and quantitative measuring of Ca2+ dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca2+ indicators have been developed, including a family of fluorescent Ca2+ indicators, dubbed GFP‐Aequorin Proteins (GAPs). They are based on the fusion of two jellyfish proteins, the green fluorescent protein (GFP) and the Ca2+‐binding protein aequorin. GAP Ca2+ indicators exhibit a combination of several features: they are excitation ratiometric indicators, with reciprocal changes in the fluorescence excited at 405 and 470 nm, which is advantageous for imaging experiments; they exhibit a Hill coefficient of 1, which facilitates the calibration of the fluorescent signal into Ca2+ concentrations; they are insensible to variations in the Mg2+ concentrations or pH variations (in the 6.5‐8.5 range); and, due to the lack of mammalian homologues, these proteins have a favorable expression in transgenic animals. A low Ca2+ affinity version of GAP, GAP3 (KD ≅ 489 µM), has been engineered to conform with the estimated [Ca2+] in the ER. GAP3 targeted to the lumen of the ER (erGAP3) can be utilized for imaging intraluminal Ca2+. The ratiometric measurements provide a quantitative method to assess accurate [Ca2+]ER, both dynamically and at rest. In addition, erGAP3 can be combined with synthetic cytosolic Ca2+ indicators to simultaneously monitor ER and cytosolic Ca2+. Here, we provide detailed methods to assess erGAP3 expression and to perform Ca2+ imaging, either restricted to the ER lumen, or simultaneously in the ER and the cytosol. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Detection of erGAP3 in the ER by immunofluorescenceBasic Protocol 2: Monitoring ER Ca2+Basic Protocol 3: Monitoring ER‐ and cytosolic‐Ca2+Support Protocol: Generation of a stable cell line expressing erGAP3

A novel copper ion enhanced electrochemical DNA biosensor for the determination of epinephrine

A novel electrochemical biosensor was developed for the detection of epinephrine (EP) by immobilizing double-strand DNA (dsDNA) bound with copper ions on a gold electrode (Cu2+/dsDNA/MCH/AuE). The electrochemical behavior of EP at Cu2+/dsDNA/MCH/AuE was examined, and the results demonstrated a significant enhancement in the electrocatalytic oxidation peak current of EP due to the formation of a stable G-Cu(II)-G sandwich structure between Cu2+ and guanine at the modified electrode. The modification process of the electrode was characterized by scanning electron microscopy, infrared spectroscopy, electrochemical impedance spectroscopy, and differential pulse voltammetry. A study on the effect of pH in phosphate buffer solution on the electrochemical oxidation of EP indicated that the catalytic oxidation process was pH-dependent. A plot of catalytic current versus EP concentration exhibited a dual-linear relationship within two ranges: 1.0–12.5 μM and 12.5–1000.0 μM, with correlation coefficients of 0.995 and 0.997, respectively. The limit of detection was determined to be 47 nM (S/N = 3). According to the calculated Hill coefficient (0.99), it can be concluded that the electrocatalytic process followed the Michaelis-Menten kinetic mechanism. The maximum catalytic current Im was 25 μA, while the apparent Michaelis-Menten constant Km was 1.425 mM. These findings indicated excellent electrocatalytic activity of the modified electrode towards oxidation of EP. The developed biosensor successfully detected EP in spiked mouse serum as well as epinephrine hydrochloride injection with high selectivity, sensitivity, stability, and accuracy.

Inhibition of voltage-gated potassium channel by aripiprazole in rabbit coronary arterial smooth muscle cells

Aripiprazole, a third-generation antipsychotic, has been widely used to treat schizophrenia. In this study, we evaluated the effect of aripiprazole on voltage-gated potassium (Kv) channels in rabbit coronary arterial smooth muscle cells using the patch clamp technique. Aripiprazole reduced the Kv current in a concentration-dependent manner with a half-maximal inhibitory concentration of 0.89 ± 0.20 μM and a Hill coefficient of 1.30 ± 0.25. The inhibitory effect of aripiprazole on Kv channels was voltage-dependent, and an additional aripiprazole-induced decrease in the Kv current was observed in the voltage range of full channel activation. The decay rate of Kv channel inactivation was accelerated by aripiprazole. Aripiprazole shifted the steady-state activation curve to the right and the inactivation curve to the left. Application of a repetitive train of pulses (1 and 2 Hz) promoted inhibition of the Kv current by aripiprazole. Furthermore, the recovery time constant from inactivation increased in the presence of aripiprazole. Pretreatment of Kv1.5 subtype inhibitor reduced the inhibitory effect of aripiprazole. However, pretreatment with Kv 7 and Kv2.1 subtype inhibitors did not change the degree of aripiprazole-induced inhibition of the Kv current. We conclude that aripiprazole inhibits Kv channels in a concentration-, voltage-, time-, and use (state)-dependent manner by affecting the gating properties of the channels.

Bounds on the Ultrasensitivity of Biochemical Reaction Cascades.

The ultrasensitivity of a dose response function can be quantifiably defined using the generalized Hill coefficient of the function. We examined an upper bound for the Hill coefficient of the composition of two functions, namely the product of their individual Hill coefficients. We proved that this upper bound holds for compositions of Hill functions, and that there are instances of counterexamples that exist for more general sigmoidal functions. Additionally, we tested computationally other types of sigmoidal functions, such as the logistic and inverse trigonometric functions, and we provided computational evidence that in these cases the inequality also holds. We show that in large generality there is a limit to how ultrasensitive the composition of two functions can be, which has applications to understanding signaling cascades in biochemical reactions.

Insights into the binding of buspirone to human serum albumin using multi-spectroscopic and molecular docking techniques

Buspirone is an anxiolytic drug that plays a significant role in managing anxiety disorders and alleviating their symptoms as well. Several techniques were utilized to study the interaction between buspirone and human serum albumin under physiological conditions, including UV–vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism, Fourier transform infrared spectroscopy (FT-IR), equilibrium dialysis, and molecular docking. The results of this study demonstrated that buspirone quenched the intrinsic fluorescence of human serum albumin through a mixed mechanism. Moreover, the binding constants (Kb), the quenching constants (Ksv), and thermodynamic parameters were calculated at various temperatures. The binding process of buspirone to human serum albumin showed a cooperative binding pattern, confirmed by the Scatchard diagram and Hill coefficient. Molecular docking results showed that buspirone interacted with the IIA, IIIA, and IIB subdomains of human serum albumin and slightly changed its conformation. It was also found that hydrophobic forces played a major role in this interaction. This study consequently proves that BSH as a drug can be transported by blood albumin. Additionally, due to its ratiometric response in absorbance upon binding to a biological target, HSA can be used as a molecular probe to follow biomolecular interactions.