Modulation of channel activity and gadolinium block of MscL by static magnetic fields

The magnetic field of the Earth has for long been known to influence the behaviour and orientation of a variety of living organisms. Experimental studies of the magnetic sense have, however, been impaired by the lack of a plausible cellular and/or molecular mechanism providing meaningful explanation for detection of magnetic fields by these organisms. Recently, mechanosensitive (MS) ion channels have been implied to play a role in magnetoreception. In this study we have investigated the effect of static magnetic fields (SMFs) of moderate intensity on the activity and gadolinium block of MscL, the bacterial MS channel of large conductance, which has served as a model channel to study the basic physical principles of mechanosensory transduction in living cells. In addition to showing that direct application of the magnetic field decreased the activity of the MscL channel, our study demonstrates for the first time that SMFs can reverse the effect of gadolinium, a well-known blocker of MS channels. The results of our study are consistent with a notion that (1) the effects of SMFs on the MscL channels may result from changes in physical properties of the lipid bilayer due to diamagnetic anisotropy of phospholipid molecules and consequently (2) cooperative superdiamagnetism of phospholipid molecules under influence of SMFs could cause displacement of Gd³⁺ ions from the membrane bilayer and thus remove the MscL channel block.     

EVIDENCE FOR MECHANOSENSITIVE TRANSMEMBRANE ION CHANNELS OF SMALL CONDUCTANCE IN MAGNETOTACTIC BACTERIA

To determine whether or not mechanosensitive (MS) ion channels are present in the magnetotactic bacterium Magnetospirillum magnetotacticum, techniques for spheroplast preparation in Escherichia coli were adapted for this bacterium. This resulted in the formation of 2–3-μm spheroplasts, which were used for patch clamp analysis. Ion channel activity in M. magnetotacticum was compared with that of the MS of small conductance (MscS) in E. coli. This comparison reveals the presence of MscS-like channels in M. magnetotacticum and, as this bacterium produces intracellular magnetite (Fe₃O₄) particles similar to those found in the human brain, provides a model for investigation of the effects of magnetic fields on MS ion channels in magnetite-bearing cells.     

Mechanosensitive channel of Thermoplasma, the cell wall-less archaea

By using a functional approach of reconstituting detergent-solubilized membrane proteins into liposomes and following their function in patch-clamp experiments, we identified a novel mechanosensitive (MS) channel in the thermophilic cell wall-less archaeon Thermoplasma volcanium. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the enriched protein fractions revealed a band of approx 15 kDa comparable to MscL, the bacterial MS channel of large conductance. 20 N-terminal residues determined by protein microsequencing, matched the sequence to an unknown open reading frame in the genome of a related species Thermoplasma acidophilum. The protein encoded by the T. acidophilum gene was cloned and expressed in Escherichia coli and reconstituted into liposomes. When examined for function, the reconstituted protein exhibited properties typical of an MS ion channel: 1) activation by negative pressure applied to the patch-clamp pipet, 2) blockage by gadolinium, and 3) activation by the anionic amphipath trinitrophenol. In analogy to the nomenclature used for bacterial MS channels, the MS channel of T. acidophilum was termed MscTA. Secondary structural analysis indicated that similar to MscL, the T. acidophilum MS protein may have two transmembrane domains, suggesting that MS channels of thermophilic Archaea belong to a family of structurally related MscL-like ion channels with two membrane-spanning regions. When the mscTA gene was expressed in the mscL ⁻ knockout strain and the MscTA protein reconstituted into liposomes, the gating of MscTA was charaterized by very brief openings of variable conductance. In contrast, when the mscTA gene was expressed in the wild-type mscL ⁺ strain of E. coli, the gating properties of the channel resembled MscL. However, the channel had reduced conductance and differed from MscL in its kinetics and in the free energy of activation, suggesting that MscTA and MscL can form functional complexes and/or modulate each other activity. Similar to MscL, MscTA exhibited an increase in activity in liposomes made of phospholipids having shorter acyl chain, suggesting a role of hydrophobic mismatch in the function of prokaryotic MS channels.     

EVIDENCE OF SYNCHRONIZATION OF NEURONAL ACTIVITY OF MOLLUSCAN BRAIN GANGLIA INDUCED BY ALTERNATING 50 Hz APPLIED MAGNETIC FIELD

Experimental evidence for the appearance of synchronized bioelectric activity in neurons under applied extremely low frequency (ELF) magnetic fields is shown. We have studied the synchronizing process by recording the intracellular bioelectric activity from pairs of neurons randomly chosen from the brain ganglia of the snail Helix aspersa. The recordings were made in real time under exposure to sinusoidal low frequency (50 Hz) weak (B₀=1–15 mT) magnetic fields. Synchronization was observed in 27% of the pairs tested. A linear dependence of the firing frequency f with the energy density of the applied magnetic field (i.e., f∝B₀²) was presented. The ability of low frequency sinusoidal weak magnetic fields to promote “magnetic synchronization” is exciting and opens new avenues for induced electromagnetic field bioeffects.     

SNAIL NEURON BIOELECTRIC ACTIVITY INDUCED UNDER STATIC OR SINUSOIDAL MAGNETIC FIELDS REPRODUCES MAMMAL NEURON RESPONSES UNDER TRANSCRANIAL MAGNETIC STIMULATION

We have applied static (SMF) or alternating magnetic fields (AMF) to snail (Helix aspersa) single-unit neurons, in the range of those applied in magnetic stimulation (MS)/transcranial magnetic stimulation (TMS). From the experiments we have performed during the past 10 years, we have collected a blind selection of neurons and their responses to either SMF or AMF. Blind selection means that we do not know the nature of neurons. We do not know whether they are sensitive, motor, secretory, pacemaker, or inter-neurons. We have seen that the behavior of single-unit neurons under SMF/AMF exposure (SMF range: 3 mT–0.7 T; AMF range: 1–15 mT) fits well with the electrophysiologic activity described for mammals and human whole brain under MS/TMS (pulsed magnetic field range: 0.3 mT–2.4 T). The neuron experiments shown here have been aleatorily selected from a collection of about 200 neurons studied. Our results could explain some of the effects described induced in mammal neurons under MS/TMS for clinical purposes.     

Enhancement of the hydrolysis activity of F0F1‐ATPases using 60 Hz magnetic fields

The effects of extremely low frequency (ELF) magnetic fields on membrane F₀F₁‐ATPase activity have been studied. When the F₀F₁‐ATPase was exposed to 60 Hz magnetic fields of different magnetic intensities, 0.3 and 0.5 mT magnetic fields enhanced the hydrolysis activity, whereas 0.1 mT exposure caused no significant changes. Even if the F₀F₁‐ATPase was inhibited by N,N‐dicyclohexylcarbodiimide, its hydrolysis activity was enhanced by a 0.5 mT 60 Hz magnetic field. Moreover, when the chromatophores which were labeled with F‐DHPE were exposed to a 0.5 mT, 60 Hz magnetic field, it was found that the pH of the outer membrane of the chromatophore was unchanged, which suggested that the magnetic fields used in this work did not affect the activity of F0. Taken together, our results show that the effects of magnetic fields on the hydrolysis activity of the membrane F₀F₁‐ATPases were dependent on magnetic intensity and the threshold intensity is between 0.1 and 0.3 mT, and suggested that the F1 part of F₀F₁‐ATPase may be an end‐point affected by magnetic fields. Bioelectromagnetics 30:663–668, 2009. © 2009 Wiley‐Liss, Inc.     

Dynamic properties of Lednev's parametric resonance mechanism

This paper presents a further development of the mechanism for the detection of weak magnetic fields proposed by [Lednev (1991): Bioelectromagnetics 12:71–75]. The fraction of excited oscillator states of an unhydrated ion is studied in a dynamic model driven by the predicted (time-varying) transition probability in the presence of thermal noise and an unspecified excitation mechanism. The main results of Lednev are confirmed. In addition, I conclude that ultraharmonic and ultrasubharmonic resonances may also be observed, provided that the response time of the dynamic system is similar to the period of the oscillating magnetic field. I discuss the time scales involved in the mechanism and present theoretical constraints on these parameters. The crucial requirement for the theory's applicability is that the lifetime of the excited states of the affected ion oscillator exceeds the period of the applied magnetic field. Numerical solutions of the dynamic system are given and are shown to correspond well to theoretical expectations. The main discrepancy between the theories of Lednev and of Blanchard and Blackman [Blanchard and Blackman (1994): Bioelectromagnetics 15:217–238] appears to be due to an inconsistency in the latter paper. The general problem of robust analysis of experimental data is discussed, and I suggest a test of compliance with the Lednev model that is independent of all parameters except for the ratio of oscillating and static field strength (B₁/B₀) for many resonance conditions and experimental models. © 1996 Wiley-Liss, Inc.     

Electric-field ion cyclotron resonance

We consider the possibility that DC magnetic fields can interact in a resonant manner with endogenous AC electric fields in biological systems. Intrinsic electric-field ion cyclotron resonance (ICR) interactions would be more physically credible than models based on external AC magnetic fields and might be expected as an evolutionary response to the long-term constancy of the geomagnetic field. Bioelectromagnetics 17:85–87, 1997. © 1997 Wiley-Liss, Inc.     

Experimental search for combined AC and DC magnetic field effects on ion channels

The hypothesis that specific combinations of DC and low frequency AC magnetic fields at so-called cyclotron-resonance conditions could affect the transport of ions through ion channels, or alter the kinetics of ion channels (opening and closing rates), has been tested. As a model system, the ion channels formed by gramicidin A incorporated in lipid bilayer membranes were studied. No significant changes in channel conductance, average lifetime, or formation rate as a function of applied fields could be detected over a wide range of frequencies and field strengths. Experiments were carried out to measure the time-resolved single-channel events and the average conductances of many-channel events in the presence of K⁺ and H⁺ ions. The channel blocking effect of Ca⁺⁺ was also studied. © 1993 Wiley-Liss. Inc.     

The role of voltage-gated Ca2+ channels in neurite growth of cultured chromaffin cells induced by extremely low frequency (ELF) magnetic field stimulation

The ion Ca²⁺ has been shown to play an important role in a wide variety of cellular functions, one of them being related to cell differentiation in which nerve growth factor (NGF) is involved. Chromaffin cells obtained from adrenals of 2- to 3-day-old rats were cultured for 7 days. During this time, these cells were subjected to the application of either NGF or extremely low frequency magnetic fields (ELF MF). Since this induced cell differentiation toward neuronal-like cells, the mechanism by which this occurred was studied. When the L-Ca²⁺ channel blocker nifedipine was applied simultaneously with ELF MF, this differentiation did not take place, but it did when an N-Ca²⁺ channel blocker was used. In contrast, none of the Ca²⁺ channel blockers prevented differentiation in the presence of NGF. In addition, Bay K-8644, an L-Ca²⁺ channel agonist, increased both the percentage of differentiated cells and neurite length in the presence of ELF MF. This effect was much weaker in the presence of NGF. [³H]-noradrenaline release was reduced by nifedipine, suggesting an important role for L-Ca²⁺ channels in neurotransmitter release. Total high voltage Ca²⁺ currents were significantly increased in ELF MF-treated cells with NGF, but these currents in ELF MF-treated cells were more sensitive to nifedipine. Amperometric analysis of catecholamine release revealed that the KCl-induced activity of cells stimulated to differentiate by ELF MF is highly sensitive to L-type Ca²⁺ channel blockers. A possible mechanism to explain the way in which the application of magnetic fields can induce differentation of chromaffin cells into neuronal-like cells is proposed.     

FTIR and UV spectroscopy in real-time monitoring of S. cerevisiae cell culture

A combination of FTIR and UV spectroscopy is proposed as a novel technique for integrated real-time monitoring of metabolic activity and growth rates of cell cultures, required for systematic studies of cellular low-frequency (LF) electric and magnetic field (EMF) effects. As an example, we investigated simultaneous influence of periodic LF 3D EMFs on a culture of Saccharomyces cerevisiae (baker's yeast) cells. Amplitudes, frequencies and phases of the field components were the variable parameters. Electromagnetic fields were found to efficiently control the activity of the yeast cells, with the resulting CO₂ production rates, as monitored by FTIR spectroscopy, varying by at least one order of magnitude due to the field action. Additionally, population dynamics of the yeast cells was monitored by UV absorption of the yeast culture at λ_prob = 320 nm, and compared to the CO₂ production rates. The detected physiologically active frequencies are all below 1 kHz, namely, 800 Hz excitation was effective in reducing the metabolic rates and arresting cell proliferation, whereas 200 Hz excitation was active in accelerating both cell proliferation and overall metabolic rates. The proposed methods produce objective, reliable and quantitative real-time results within minutes and may be used in various tasks that could benefit from a rapid feedback they provide in the form of metabolic and growth rates. Amplitude and frequency dependences of the LF EMF effects from individual field components with different polarizations were recorded and qualitatively interpreted based on a simple model, describing ion diffusion through a membrane channel.     

Static Magnetic Fields Inhibit Proliferation and Disperse Subcellular Localization of Gamma Complex Protein3 in Cultured C2C12 Myoblast Cells

Magnetic fields may delay the rate of cell cycle progression, and there are reports that magnetic fields induce neurite outgrowth in cultured neuronal cells. To demonstrate whether magnetic field also effects on myoblast cells in cell growth, C2C12 cell lines were cultured and 2000G static magnetic field was applied. After 48 h of incubation, both the WST-1 assay (0.01 < P < 0.025, t-test) and direct cell counting (P < 0.0005, t-test) showed that static magnetic fields inhibit the proliferation of cultured C2C12 cells. Immunocytochemistry for alpha and tubulin gamma complex protein (TUBA and GCP3) was made and applying a static magnetic field-dispersed tubulin GCP3 formation, a intracellular apparatus for tubulin structuring in cell division. This protein expression was not altered by western blot. This study indicates that applying a static magnetic field alters the subcellular localizing of GCP3, and may delay the cell growth in cultured C2C12 myoblast cells.     

The influence of static magnetic fields on mechanosensitive ion channel activity in artificial liposomes

The influence of static magnetic fields (SMFs) on the activity of recombinant mechanosensitive ion channels (the bacterial mechanosensitive ion channel of large conductance—MscL) following reconstitution into artificial liposomes has been investigated. Preliminary findings suggest that exposure to 80-mT SMFs does not induce spontaneous MscL activation in the absence of mechanical stimulation. However, SMFs do appear to influence the open probability and single channel kinetics of MscL exposed to negative pipette pressure. Typical responses include an overall reduction in channel activity or an increased likelihood of channels becoming trapped open in sub-conducting states following exposure to SMFs. There is a delay in the onset of this effect and it is maintained throughout exposure. Generally, channel activity showed slow or limited recovery following removal of the magnetic field and responses to the magnetic were often reduced or abolished upon subsequent exposures. Pre-exposure of the liposomes to SMFs resulted in reduced sensitivity of MscL to negative pipette pressure, with higher pressures required to activate the channels. Although the mechanisms of this effect are not clear, our initial observations appear to support previous work showing that the effects of SMFs on ion channels may be mediated by changes in membrane properties due to anisotropic diamagnetism of lipid molecules.     

Correlation of magnetic AC field on cardiac myocyte Ca2+ transients at different magnetic DC levels

The purpose of this study was to determine the effect of extremely low frequency and weak magnetic fields (WMF) on cardiac myocyte Ca²⁺ transients, and to explore the involvement of potassium channels under the WMF effect. In addition, we aimed to find a physical explanation for the effect of WMF on cardiac myocyte Ca²⁺ transients. Indo‐1 loaded cells, which were exposed to a WMF at 16 Hz and 40 nT, demonstrated a 75 ± 4% reduction in cytosolic Ca²⁺ transients versus control. Treatment with the K_ATP channel blocker, glibenclamide, followed by WMF at 16 Hz exposure, blocked the reduction in cytosolic calcium transients while treatment with pinacidil, a K_ATP channel opener, or chromanol 293B, a selective potassium channel blocker of the delayed rectifier K⁺ channels, did not inhibit the effect. Based on these finding and the ion cyclotron resonance frequency theory, we further investigated the effect of WMF by changing the direct current (DC) magnetic field (B₀). When operating different DC magnetic fields we showed that the WMF value changed correspondingly: for B₀ = 44.5 µT, the effect was observed at 17.05 Hz; for B₀ = 46.5 µT, the effect was observed at 18.15 Hz; and for B₀ = 49 µT the effect was observed at 19.1 Hz. We can conclude that the effect of WMF on Ca²⁺ transients depends on the DC magnetic field level. Bioelectromagnetics 33:634–640, 2012. © 2012 Wiley Periodicals, Inc.     

Influence of magnetic field on activity of given anaerobic sludge

Two modes of magnetic fields were applied in the Cr⁶⁺ removal sludge reactors containing two predominated strains—Bacillus sp. and Brevibacillus sp., respectively. The magnetic field mode I* of 0–4.5 or 0–14 mT between pieces was obtained by setting the magnetic pieces with the surface magnetic density of 0–6 or 0–20 mT into the reactor, and the magnetic field mode II* of 6, 20, or 40 mT on the return line was obtained by controlling the working distance of the permanent magnet outside the sludge return line. The effects of different magnetic fields on the activity of the given anaerobic sludge were studied by comparing with the control (absent of magnetic field). The results showed that the magnetic field of 0–4 mT improved the activity of given sludge most effectively, U_{max·\textCH4} U_{{\max \cdot {\text{CH}}_{4} }} (the peak methane-producing rate) and the methane producing volume per gCOD_Cr reached 64.3 mlCH₄/gVSS.d and 124 mlCH₄/gCOD_Cr, which increased by 20.6 and 70.7%, respectively, compared with the control. And the magnetic field of 20 mT took second place. It could be concluded that the input of some magnetic field could improve the activity of anaerobic sludge by increasing the transformation efficiency of COD_Cr matters to methane, and the total organic wastage did not increase.     

Action of combined magnetic fields on aqueous solution of glutamic acid: the further development of investigations

In the present work the results of the known investigation of the influence of combined static (40 μT) and alternating (amplitude of 40 nT) parallel magnetic fields on the current through the aqueous solution of glutamic acid, were successfully replicated. Fourteen experiments were carried out by the application of the combined magnetic fields to the solution placed into a Plexiglas reaction vessel at application of static voltage to golden electrodes placed into the solution. Six experiments were carried out by the application of the combined magnetic fields to the solution placed in a Plexiglas reaction vessel, without electrodes, within an electric field, generated by means of a capacitor at the voltage of 27 mV. The frequency of the alternating field was scanned within the bounds of 1.0 Hz including the cyclotron frequency corresponding to a glutamic acid ion and to the applied static magnetic field. In this study the prominent peaks with half-width of ~0.5 Hz and with different heights (till 80 nA) were registered at the alternating magnetic field frequency equal to the cyclotron frequency (4.2 Hz). The general reproducibility of the investigated effects was 70% among the all solutions studied by us and they arose usually after 40–60 min. after preparation of the solution. In some made-up solutions the appearance of instability in the registered current was noted in 30–45 min after the solution preparation. This instability endured for 20–40 min. At the end of such instability period the effects of combined fields action appeared practically every time. The possible mechanisms of revealed effects were discussed on the basis of modern quantum electrodynamics.     

Highly selective enrichment of phosphopeptides by on‐chip indium oxide functionalized magnetic nanoparticles coupled with MALDI‐TOF MS

Selective and efficient preconcentration is indispensable for low concentration of phosphopeptides in phosphorylated protein‐related samples prior to MS‐based analysis. Herein, an on‐chip system coupled magnetic SPE with MALDI‐TOF MS was designed. A metal oxide affinity chromatography material, indium oxide, was coated on the surface of Fe₃O₄ magnetic nanoparticles to prepare the adsorbent, spatially confined with an applied magnetic field. The adsorbent exhibited high selectivity for phosphopeptides in tryptic digests of the mixture of β‐casein and BSA (1:1000) and the mixture of β‐casein, BSA, and ovalbumin (1:100:100). Thanking to the enrichment ability and specificity for phosphopeptides with the adsorbent, the on‐chip magnetic SPE‐MALDI‐TOF MS approach showed high sensitivity with a low detection limit of 4 fmol. In addition, the developed approach was used to analyze phosphopetides in non‐fat milk digests and human serum successfully.     

Voltage-independent Adaptation of Mechanosensitive Channels in Escherichia coli Protoplasts

Mechanosensitive (MS) ion channels, with 560 pS conductance, opened transiently by rapid application of suction pulses to patches of E. coli protoplast membrane. The adaptation phase of the response was voltage-independent. Application of strong suction pulses, which were sufficient to cause saturation of the MS current, did not abolish the adaptation. Multiple-pulse experimental protocols revealed that once MS channels had fully adapted, they could be reactivated by a second suction pulse of similar amplitude, providing the time between pulses was long enough and suction had been released between pulses. Limited proteolysis (0.2 mg/ml pronase applied to the cytoplasmic side of the membrane patch) reduced the number of open channels without affecting the adaptation. Exposing patches to higher levels of pronase (1 mg/ml) removed responsiveness of the channel to suction and abolished adaptation consistent with disruption of the tension transmission mechanism responsible for activating the MS channel. Based on these data we discuss a mechanism for mechanosensitivity mediated by a cytoplasmic domain of the MS channel molecule or associated protein. Received: 29 January 1998/Revised: 16 April 1998     

ENHANCEMENT OF INVERTASE PRODUCTION BY Aspergillus niger OZ-3 USING LOW-INTENSITY STATIC MAGNETIC FIELDS

The aim of this study is to investigate the effect of low-intensity static magnetic fields (SMFs) on invertase activity and growth on different newly identified molds. The most positive effect of SMFs on invertase activity and growth was observed for Aspergillus niger OZ-3. The submerged production of invertase was performed with the spores obtained at the different exposure times (120, 144, 168, and 196 hr) and magnetic field intensities (0.45, 3, 5, 7, and 9 mT). The normal magnetic field of the laboratory was assayed as 0.45 mT (control). Optimization of magnetic field intensity and exposure time significantly increased biomass production and invertase activity compared to 0.45 mT. The maximum invertase activity (51.14 U/mL) and biomass concentration (4.36 g/L) were achieved with the spores obtained at the 144 hr exposure time and 5 mT magnetic field intensity. The effect of low-intensity static magnetic fields (SMFs) on invertase activities of molds was investigated for the first time in the present study. As an additional contribution, a new hyper-invertase-producing mold strain was isolated.

Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.     

Myosin Light Chain Modification Depending on Magnetic Fields: II. Experimental

Recent experiments have revealed that Ca²⁺ -calmodulin dependent myosin light chain phosphorylation in a cell-free preparation exhibits unexpectedly high sensitivity to weak magnetic fields. This enzyme system is a well-studied biochemical system, which appears to depend upon ion binding. A previous article in this journal discussed the theoretical background of myosin phosphorylation and the ion-dependent interactions of EMF with soft tissues. Because of the electromagnetic field (EMF) sensitivity of this cell-free system, experiments were designed to test the effect of a pulsed radio frequency (PRF) field, pulsating magnetic fields (TEMF), gradient magnetic fields (Magnabloc), and homogeneous static magnetic fields (in Helmholtz arrangement) designed for clinical application. It is concluded that these various magnetic fields affect this cell-free enzyme system by modulating ion–protein interactions.