The cross-correlation-based analysis to digest the conformational dynamics of the mitoBK channels in terms of their modulation by flavonoids.

The activity of mitochondrial large-conductance voltage- and [Formula: see text]-activated [Formula: see text] channels (mitoBK) is regulated by a number of biochemical factors, including flavonoids. In particular, naringenin (Nar) and quercetin (Que) reached reasonable scientific attention due to their well-pronounced channel-activating effects. The open-reinforcing outcomes of Nar and Que on the mitoBK channel gating have been already reported. Nevertheless, the molecular picture of the corresponding channel-ligand interactions remains still to be revealed. In this work, we investigate the effects of the Nar and Que on the conformational dynamics of the mitoBK channel. In this aim, the cross-correlation-based analysis of the single-channel signals recorded by the patch-clamp method is performed. The obtained results in the form of phase space diagrams enable us to visually monitor the effects exerted by the considered flavonoids at the level of temporal characteristics of repetitive sequences of channel conformations. It turns out that the mitoBK channel activation by naringenin and quercetin does not lead to the change in the number of clusters within the phase space diagrams, which can be related to the constant number of available channel macroconformations regardless of the flavonoid administration. The localization and occupancy of the clusters of cross-correlated sequences suggest that mitoBK channel stimulation by flavonoids affects the relative stability of channel conformations and the kinetics of switching between them. For most clusters, greater net effects are observed in terms of quercetin administration in comparison with naringenin. It indicates stronger channel interaction with Que than Nar.

New Diagnostic Tool for Ion Channel Activity Hidden Behind the Dwell-Time Correlations.

The patch-clamp technique is a powerful tool that allows for a long observation of transport protein activity in real time. Experimental traces of single-channel currents can be considered as a record of the channel's conformational switching related to its activation and gating. In this work, we present a mathematically simple method of patch-clamp data analysis that assesses the connectivity and occupancy of distinct conformational substates of the channel. The proposed approach appears to be a big step forward due to its possible applications in the determination of channel substates related to disease and in the analysis of drug-channel interactions on the level of repetitive sequences of channel conformations. This is especially important in cases when molecular dynamics docking is impossible and Markovian modeling requires ambiguous optimization tasks.

Activity of TREK-2-like Channels in the Pyramidal Neurons of Rat Medial Prefrontal Cortex Depends on Cytoplasmic Calcium.

TREK-2-like channels in the pyramidal neurons of rat prefrontal cortex are characterized by a wide range of spontaneous activity-from very low to very high-independent of the membrane potential and the stimuli that are known to activate TREK-2 channels, such as temperature or membrane stretching. The aim of this study was to discover what factors are involved in high levels of TREK-2-like channel activity in these cells. Our research focused on the PI(4,5)P2-dependent mechanism of channel activity. Single-channel patch clamp recordings were performed on freshly dissociated pyramidal neurons of rat prefrontal cortexes in both the cell-attached and inside-out configurations. To evaluate the role of endogenous stimulants, the activity of the channels was recorded in the presence of a PI(4,5)P2 analogue (PI(4,5)P2DiC8) and Ca2+. Our research revealed that calcium ions are an important factor affecting TREK-2-like channel activity and kinetics. The observation that calcium participates in the activation of TREK-2-like channels is a new finding. We showed that PI(4,5)P2-dependent TREK-2 activity occurs when the conditions for PI(4,5)P2/Ca2+ nanocluster formation are met. We present a possible model explaining the mechanism of calcium action.

Differences in Gating Dynamics of BK Channels in Cellular and Mitochondrial Membranes from Human Glioblastoma Cells Unraveled by Short- and Long-Range Correlations Analysis.

The large-conductance voltage- and Ca2+-activated K+ channels (BK) are encoded in humans by the Kcnma1 gene. Nevertheless, BK channel isoforms in different locations can exhibit functional heterogeneity mainly due to the alternative splicing during the Kcnma1 gene transcription. Here, we would like to examine the existence of dynamic diversity of BK channels from the inner mitochondrial and cellular membrane from human glioblastoma (U-87 MG). Not only the standard characteristics of the spontaneous switching between the functional states of the channel is discussed, but we put a special emphasis on the presence and strength of correlations within the signal describing the single-channel activity. The considered short- and long-range memory effects are here analyzed as they can be interpreted in terms of the complexity of the switching mechanism between stable conformational states of the channel. We calculate the dependencies of mean dwell-times of (conducting/non-conducting) states on the duration of the previous state, Hurst exponents by the rescaled range R/S method and detrended fluctuation analysis (DFA), and use the multifractal extension of the DFA (MFDFA) for the series describing single-channel activity. The obtained results unraveled statistically significant diversity in gating machinery between the mitochondrial and cellular BK channels.

Multifractal Properties of BK Channel Currents in Human Glioblastoma Cells.

Potassium channels play an important physiological role in glioma cells. In particular, voltage- and Ca2+-activated large-conductance BK channels (gBK in gliomas) are involved in the intensive growth and extensive migrating behavior of the mentioned tumor cells; thus, they may be considered as a drug target for the therapeutic treatment of glioblastoma. To enable appropriate drug design, molecular mechanisms of gBK channel activation by diverse stimuli should be unraveled as well as the way that the specific conformational states of the channel relate to its functional properties (conducting/nonconducting). There is an open debate about the actual mechanism of BK channel gating, including the question of how the channel proteins undergo a range of conformational transitions when they flicker between nonconducting (functionally closed) and conducting (open) states. The details of channel conformational diffusion ought to have its representation in the properties of the experimental signal that describes the ion-channel activity. Nonlinear methods of analysis of experimental nonstationary series can be useful for observing the changes in the number of channel substates available from geometrical and energetic points of view at given external conditions. In this work, we analyze whether the multifractal properties of the activity of glioblastoma BK channels depend on membrane potential, and which states, conducting or nonconducting, affect the total signal to a larger extent. With this aim, we carried out patch-clamp experiments at different levels of membrane hyper- and depolarization. The obtained time series of single channel currents were analyzed using the multifractal detrended fluctuation analysis (MFDFA) method in a standard form and incorporating focus-based multifractal (FMF) formalism. Thus, we show the applicability of a modified MFDFA technique in the analysis of an experimental patch-clamp time series. The obtained results suggest that membrane potential strongly affects the conformational space of the gBK channel proteins and the considered process has nonlinear multifractal characteristics. These properties are the inherent features of the analyzed signals due to the fact that the main tendencies vanish after shuffling the data.

Monitoring of dynamic ATP level changes by oligomycin-modulated ATP synthase inhibition in SW480 cancer cells using fluorescent "On-Off" switching DNA aptamer.

Adenosine triphosphate (ATP) is the main energy source in cells and an important biomolecule participating in cellular reactions in living organisms. Since the ATP level changes dynamically reflecting the development of a debilitating disease or carcinogenesis, we have focused in this work on monitoring of the oligomycin (OMC)-modulated ATP synthase inhibition using a fluorescent-switching DNA aptamer designed for the detection of ATP (Apt(ATP)), as the model for studies of dynamic ATP level variation. The behavior of the ATP aptamer has been characterized using fluorescence spectroscopy. The Intramolecular fluorescence resonance energy transfer (iFRET) operates in the proposed aptamer from the FAM dye moiety to guanines of the aptamer G-quadruplex when the target ATP is present and binds to the aptamer changing its conformation. The iFRET process enables the detection of ATP down to the limit of detection, LOD = 17 µM, without resorting to any extra chemi-amplification schemes. The selectivity coefficients for relevant interferent triphosphates (UTP, GTP, and CTP) are low for the same concentration as that of ATP. We have demonstrated an efficient transfection of intact cells and OMC-treated SW480 colon cancer cells with Apt(ATP), using microscopic imaging, iFRET measurements, and cell viability testing with MTT method. The applicability of the switching DNA aptamer for the analysis of real samples, obtained by lysis of SW480 cells, was also tested. The proposed Apt(ATP) may be considered as a viable candidate for utilization in measurements of dynamic ATP level modulation in cells in different stages of cancer development and testing of new drugs in pharmacological studies. Graphical abstract.

Mechanosensitivity of the BK Channels in Human Glioblastoma Cells: Kinetics and Dynamical Complexity.

BK channels are potassium selective and exhibit large single-channel conductance. They play an important physiological role in glioma cells: they are involved in cell growth and extensive migrating behavior. Due to the fact that these processes are accompanied by changes in membrane stress, here, we examine mechanosensitive properties of BK channels from human glioblastoma cells (gBK channels). Experiments were performed by the use of patch-clamp method on excised patches under membrane suction (0-40 mmHg) at membrane hyper- and depolarization. We have also checked whether channel's activity is affected by possible changes of membrane morphology after a series of long impulses of suction. Unconventionally, we also analyzed internal structure of the experimental signal to make inferences about conformational dynamics of the channel in stressed membranes. We examined the fractal long-range memory effect (by R/S Hurst analysis), the rate of changes in information by sample entropy, or correlation dimension, and characterize its complexity over a range of scales by the use of Multiscale Entropy method. The obtained results indicate that gBK channels are mechanosensitive at membrane depolarization and hyperpolarization. Prolonged suction of membrane also influences open-closed fluctuations-it decreases channel's activity at membrane hyperpolarization and, in contrary, increases channel's activity at high voltages. Both membrane strain and its "fatigue" reduce dynamical complexity of channel gating, which suggest decrease in the number of available open conformations of channel protein in stressed membranes.

Optical Biosensing System for the Detection of Survivin mRNA in Colorectal Cancer Cells Using a Graphene Oxide Carrier-Bound Oligonucleotide Molecular Beacon.

The anti-apoptotic protein survivin is one of the most promising cancer biomarkers owing to its high expression in human cancers and rare occurrence in normal adult tissues. In this work, we have investigated the role of supramolecular interactions between a graphene oxide (GO) nanosheet nanocarrier and a survivin molecular beacon (SurMB), functionalized by attaching fluorophore Joe and quencher Dabcyl (SurMB-Joe). Molecular dynamics simulations revealed hydrogen bonding of Joe moiety and Dabcyl to GO carriers that considerably increase the SurMB-GO bonding strength. This was confirmed in experimental work by the reduced fluorescence background in the OFF state, thereby increasing the useful analytical signal range for mRNA detection. A new mechanism of hairpin⁻hairpin interaction of GO@SurMB with target oligonucleotides has been proposed. A low limit of detection, LOD = 16 nM (S/N = 3), has been achieved for complementary tDNA using GO@SurMB-Joe nanocarriers. We have demonstrated an efficient internalization of SurMB-Joe-loaded GO nanocarriers in malignant SW480 cells. The proposed tunability of the bonding strength in the attached motifs for MBs immobilized on nanocarriers, via structural modifications, should be useful in gene delivery systems to enhance the efficacy of gene retention, cell transfection and genomic material survivability in the cellular environment.

Hydrocortisone inhibition of wild-type and αD200Q nicotinic acetylcholine receptors.

Short-term treatment with large doses of corticosteroids can result in acute weakness of muscles in processes that have not yet been fully characterized. Corticosteroids have been shown to exert direct inhibitory action on the muscle-type nicotinic acetylcholine receptor (AChR), and therefore can promote pharmacological muscle denervation. The mechanism of hydrocortisone (HC) blockage of AChR has not been fully established yet. It is uncommon for an electrically neutral molecule, for example, HC, to induce voltage-dependent changes in AChR kinetics. Our experiments aimed to determine the source of voltage-dependency in HC action. Wild-type (WT) and αD200Q receptors were transiently expressed in HEK293 cells. Recordings were performed in either the presence or absence of HC. We showed that the D-to-Q substitution is capable of suppressing the voltage dependency in the HC-induced block. We conclude that the distance between αD200 and the agonist-binding site depends on the membrane potential. The voltage-dependent changes of the αD200 position have not been considered yet. To our knowledge, the ability to induce voltage-dependency in blocker action has not been shown previously for an amino acid located outside the transmembrane portion of the receptor. Possible mechanisms of HC block (allosteric and knocking) in WT and αD200Q receptors are discussed.

Hairpin-Hairpin Molecular Beacon Interactions for Detection of Survivin mRNA in Malignant SW480 Cells.

Cancer biomarkers offer unique prospects for the development of cancer diagnostics and therapy. One of such biomarkers, protein survivin (Sur), exhibits strong antiapoptotic and proliferation-enhancing properties and is heavily expressed in multiple cancers. Thus, it can be utilized to provide new modalities for modulating the cell-growth rate, essential for effective cancer treatment. Herein, we have focused on the development of a new survivin-based cancer detection platform for colorectal cancer cells SW480 using a turn-on fluorescence oligonucleotide molecular beacon (MB) probe, encoded to recognize Sur messenger RNA (mRNA). Contrary to the expectations, we have found that both the complementary target oligonucleotide strands as well as the single- and double-mismatch targets, instead of exhibiting the anticipated simple random conformations, preferentially formed secondary structure motifs by folding into small-loop hairpin structures. Such a conformation may interfere with, or even undermine, the biorecognition process. To gain better understanding of the interactions involved, we have replaced the classical Tyagi-Kramer model of interactions between a straight target oligonucleotide strand and a hairpin MB with a new model to account for the hairpin-hairpin interactions as the biorecognition principle. A detailed mechanism of these interactions has been proposed. Furthermore, in experimental work, we have demonstrated an efficient transfection of malignant SW480 cells with SurMB probes containing a fluorophore Joe (SurMB-Joe) using liposomal nanocarriers. The green emission from SurMB-Joe in transfected cancer cells, due to the hybridization of the SurMB-Joe loop with Sur mRNA hairpin target, corroborates Sur overexpression. On the other hand, healthy human-colon epithelial cells CCD 841 CoN show only negligible expression of survivin mRNA. These experiments provide the proof-of-concept for distinguishing between the cancer and normal cells by the proposed hairpin-hairpin interaction method. The single nucleotide polymorphism sensitivity and a low detection limit of 26 nM (S/N = 3σ) for complementary targets have been achieved.

The temperature dependence of the BK channel activity - kinetics, thermodynamics, and long-range correlations.

Large-conductance, voltage dependent, Ca2+-activated potassium channels (BK) are transmembrane proteins that regulate many biological processes by controlling potassium flow across cell membranes. Here, we investigate to what extent temperature (in the range of 17-37°C with ΔT=5°C step) is a regulating parameter of kinetic properties of the channel gating and memory effect in the series of dwell-time series of subsequent channel's states, at membrane depolarization and hyperpolarization. The obtained results indicate that temperature affects strongly the BK channels' gating, but, counterintuitively, it exerts no effect on the long-range correlations, as measured by the Hurst coefficient. Quantitative differences between dependencies of appropriate channel's characteristics on temperature are evident for different regimes of voltage. Examining the characteristics of BK channel activity as a function of temperature allows to estimate the net activation energy (Eact) and changes of thermodynamic parameters (ΔH, ΔS, ΔG) by channel opening. Larger Eact corresponds to the channel activity at membrane hyperpolarization. The analysis of entropy and enthalpy changes of closed to open channel's transition suggest the entropy-driven nature of the increase of open state probability during voltage activation and supports the hypothesis about the voltage-dependent geometry of the channel vestibule.

Restoration of the response of the middle cerebral artery of the rat to acidosis in hyposmotic hyponatremia by the opener of large-conductance calcium sensitive potassium channels (BKCa).

Hyposmotic hyponatremia (the decrease of extracellular concentration of sodium ions from 145 to 121 mM and the decrease of hyposmolality from 300 to 250 mOsm/kg H2O) impairs response of the middle cerebral artery (MCA) to acetylcholine and NO donor (S-nitroso-N-acetyl-DL-penicillamine). Since acidosis activates a similar intracellular signaling pathway, the present study was designed to verify the hypothesis that the response of the MCA to acidosis is impaired during acute hyposmotic hyponatremia due to abnormal NO-related signal transduction in vascular smooth muscle cells. Studies performed on isolated, cannulated, and pressurized rat MCA revealed that hyposmotic hyponatremia impaired the response of the MCA to acidosis and this was associated with hyposmolality rather than with decreased sodium ion concentration. Response to acidosis was restored by the BKCa but not by the KATP channel activator. Patch-clamp electrophysiology performed on myocytes freshly isolated from MCAs, demonstrated that hyposmotic hyponatremia does not affect BKCa currents but decreases the voltage-dependency of the activation of the BKCa channels in the presence of a specific opener of these channels. Our study suggests that reduced sensitivity of BKCa channels in the MCA to agonists results in the lack of response of this artery to acidosis during acute hyposmotic hyponatremia.

Mitochondria-based biosensors with piezometric and RELS transduction for potassium uptake and release investigations.

Dysfunctional mitochondria appear to be involved in many diseases through their role in respiration, reactive oxygen species generation, and energy production. To aid in the design of new biosensors based on mitochondria (MT), we have investigated the feasibility of detecting ion fluxes through the MT-membrane K+-ion channels using piezosensors with MTs immobilized either by hydrogen bonding or thin polypyrrole (PPy) binding film. We have demonstrated for the first time that the mitochondria-based piezosensors are able to detect ion fluxes and thus be utilized for drug development aimed at ion channel opener- or inhibitor-function. The quartz crystal resonator responding only to mass changes in the lower part of the MT film, penetrated by the acoustic wave, is able to detect a pronounced cationic dynamics in PPy-bonded MT piezosensors despite of the undoped-PPy preference for pure anion dynamics. The control experiments performed by resonance elastic light scattering (RELS) confirmed MT swelling/shrinking, ion dynamics, and osmotic water transfer in MTs, as well as the effects of exposure to a drug valinomycin at sub-nanomolar concentrations.

Piezometric biosensors for anti-apoptotic protein survivin based on buried positive-potential barrier and immobilized monoclonal antibodies.

The anti-apoptotic protein survivin (Sur) plays an important role in the regulation of cell division and inducing the chemotherapeutic drug resistance. The Sur protein and its mRNA have recently been studied as cancer biomarkers and potential targets for cancer therapy. In this work, we have focused on the design of immunosensors for the detection of Sur based on buried positive-potential barrier layer structure and anti-survivin antibody. The modification of solid AuQC piezoelectrodes was monitored by recording the resonance frequency shift and electrochemical measurements during each step of the sensor preparation. Our results indicate that the immunosensor with covalently bound monoclonal anti-survivin antibody can detect Sur with the limit of detection, LOD=1.7nM (S/N=3σ). The immunosensor applicability for the analysis of real samples was assessed by testing samples of cell lysate solutions obtained from human astrocytoma (glioblastoma) U-87MG cell line, with the experiments performed using the standard addition method. The good linearity of the calibration curves for PBS and lysate solutions at low Sur concentrations confirm the high specificity of the proposed biosensor and good discrimination against nonspecific interactions with lysate components. The calculations indicate that there is still room to increase the Sur capture capacity for Sur while miniaturizing the sensor. The important advantage of the sensor is that it can be reused by a simple regeneration procedure.

On the simple random-walk models of ion-channel gate dynamics reflecting long-term memory.

Several approaches to ion-channel gating modelling have been proposed. Although many models describe the dwell-time distributions correctly, they are incapable of predicting and explaining the long-term correlations between the lengths of adjacent openings and closings of a channel. In this paper we propose two simple random-walk models of the gating dynamics of voltage and Ca(2+)-activated potassium channels which qualitatively reproduce the dwell-time distributions, and describe the experimentally observed long-term memory quite well. Biological interpretation of both models is presented. In particular, the origin of the correlations is associated with fluctuations of channel mass density. The long-term memory effect, as measured by Hurst R/S analysis of experimental single-channel patch-clamp recordings, is close to the behaviour predicted by our models. The flexibility of the models enables their use as templates for other types of ion channel.

The H-loop in the second nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator is required for efficient chloride channel closing.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR-->AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR-->AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.

Potassium currents in human myogenic cells from healthy and congenital myotonic dystrophy foetuses.

The whole-cell patch clamp technique was used to record potassium currents in in vitro differentiating myoblasts isolated from healthy and myotonic dystrophy type 1 (DM1) foetuses carrying 2000 CTG repeats. The fusion of the DM1 myoblasts was reduced in comparison to that of the control cells. The dystrophic muscle cells expressed less voltage-activated K(+) (delayed rectifier and non-inactivating delayed rectifier) and inward rectifier channels than the age-matched control cells. However, the resting membrane potential was not significantly different between the control and the DM1 cells. After four days in a differentiation medium, the dystrophic cells expressed the fast-inactivating transient outward K(+) channels, which were not observed in healthy cells. We suggest that the low level of potassium currents measured in differentiated DM1 cells could be related to their impaired fusion.

Potassium currents in human myogenic cells from donors of different ages.

Ageing in humans is accompanied by a reduction in the capacity of satellite cells to proliferate and the forming myoblasts to fuse. The processes of myoblast differentiation and fusion are associated with specific changes in the cells electrical properties. We wanted to elucidate the possible effects of ageing on these parameters and performed whole-cell patch-clamp recordings on human myoblasts obtained from biopsies of skeletal muscles from 2-, 48- and 76-year-old donors. First, we found that resting membrane potential on the 4th day of differentiation in vitro is less negative in the older than in the younger cells. Moreover, the oldest cells showed a smaller density of outward and inward potassium currents. More cells from the old and middle-age donors have a low (less than -40 mV) potential of activation for the outward potassium current. We conclude that in human myoblasts biophysical properties of potassium currents change with donor age.

Large-conductance K+ channel openers NS1619 and NS004 as inhibitors of mitochondrial function in glioma cells.

Recently, it has been reported that large-conductance Ca(2+)-activated potassium channels, also known as BK(Ca)-type potassium channels, are present in the inner mitochondrial membrane of the human glioma LN229 cell line. Hence, in the present study, we have investigated whether BK(Ca)-channel openers (BK(Ca)COs), such as the benzimidazolone derivatives NS004 (5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazole-2-one) and NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one), affect the functioning of LN229 glioma cell mitochondria in situ. We examined the effect of BK(Ca)COs on mitochondrial membrane potential, mitochondrial respiration and plasma membrane potassium current in human glioma cell line LN229. We found that BK(Ca)COs decrease the mitochondrial membrane potential with an EC(50) value of 3.6+/-0.4 microM for NS1619 and 5.4+/-0.8 microM for NS004. This mitochondrial depolarization was accompanied by an inhibition of the mitochondrial respiratory chain. Both BK(Ca)COs induced whole-cell potassium current blocked by charybdotoxin, as measured by the patch-clamp technique. The BK(Ca)COs had no effect on membrane bilayer conductance. Moreover, the inhibition of mitochondrial function by NS004 and NS1619 was without effect on cell survival, as measured by lactate dehydrogenase release from the cells.

[Role and function of voltage-gated chloride channels of the CIC family and their defects leading to genetic diseases]. / Rola i funkcja kanalów chlorkowych sterowanych potencjalem z rodziny ClC i ich defekty prowadzace do chorób genetycznych.

There are 9 channels of the ClC family in mammals and few others in fishes, plants, yeast and bacteria. The ClC channels are present in different tissues and play a role in transmembrane potential stabilization, transepithelial transport, cell volume regulation, acidification of intracellular organelles. The genetic defects of ClC-1 chloride channel lead to myotonias, the defect in ClC-5 channel to the formation of stones in kidney, while the defect in ClC-Kb channel leads to the Bartter's syndrome.