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Electromagnetic Fields and Calcium Signaling by the Voltage Dependent Anion Channel

January 2021
Open Journal of Veterinary Medicine 11(01):57-86

DOI:10.4236/ojvm.2021.111004

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CC BY 4.0

Authors:

Volker Ullrich

University of Konstanz

Hans-Jürgen Apell

University of Konstanz

Schematic representation of the different functional states of VDAC in response to EMFs. Left panel: Under physiological conditions the high-capacity anion selective state is prevalent, named "open state". The protein complex consists besides VDAC of several enzymes: plasma membrane oxidoreductase (PMOR) which consists of TSPO and a flavoprotein, hexokinase1 (HK), adenine nucleotide transporter (ANT), and NO-synthase (NOS). The complex is also coupled to an electron-transport system based on ubiquinone, CoQ (Q). The extracellular electron acceptor is a semidehydroascorbate (Asc). Middle panel: Low-capacity cation-selective state, named (misleadingly) "closed" state. The conserved glutamate (E 73) affects a major conformational change to a cation (including Ca 2+ ) selective state and HK dissociation. The transition into this state may be promoted or stabilized by EMFs contrary to the physiological needs of the cell. Right panel: A high capacity cation-selective state, named "mega pore", is formed upon a prolonged unphysiological condition in the cell and allows high Ca 2+ fluxes into the cytosol, which activates subsequently the mitochondrial permeability transition pore (MitoPTP) inducing cell death by apoptosis. Oxidative oligomerization of VDAC under this condition is indicated by "VDAC-S-S-".

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Open Journal of Veterinary Medicine, 2021, 11, 57-86

https://www.scirp.org/journal/ojvm

ISSN Online: 2165-3364

ISSN Print: 2165-3356

DOI:

10.4236/ojvm.2021.111004 Jan. 29, 2021 57 Open Journal of Veterinary Medicine

Electromagnetic Fields and Calcium Signaling

by the Voltage Dependent Anion Channel

Volker Ullrich, Hans-Jürgen Apell

Department of Biology, University of Konstanz, Konstanz, Germany

Abstract

Electromagnetic fields (EMFs) can interact with biological tissues exerting

positive as well as negative effects on cell viability, but the underlying sensing

and signaling mechanisms are largely unknown. So far in excitable cells EMF

exposure was postulated to cause Ca2+ influx through voltage-

dependent Ca

channels (VDCC) leading to cell act

ivation and an antioxidant response.

Upon further activation oxidative stress causing DNA damage or cell death

may follow. Here we report collected evidence from literature that voltage

dependent anion channels (VDAC) located not only in the outer microsom

membrane but also in the cytoplasmic membrane convert to Ca2+

conducting

channels of varying capacities upon subtle changes of the applied EMF even

in non-excitable cells like erythrocytes. Thus, VDAC can be targeted by ex-

ternal EMF in both types of membranes to release Ca2+

into the cytosol. The

role of frequency, pulse modulation or polarization remains to be investigated

in suitable cellular models. VDACs are associated with several other proteins,

among which the 18 kDa translocator (TSPO) is of spec

ific interest since it

was characterized as the central benzodiazepine receptor in neurons. Exhi-

biting structural similarities with magnetoreceptors we propose that TSPO

could sense the magnetic component of the EMF and thus together with

VDAC could trigge

r physiological as well as pathological cellular responses.

Pulsed EMFs in the frequency range of the brain-wave communication net-

work may explain psychic disturbances of electromagnetic hypersensitive

persons. An important support is provided from human p

sychology that

states deficits like insomnia, anxiety or depression can be treated with diaze-

pines that indicates apparent connections between the TSPO/VDAC complex

and organismic responses to EMF.

Keywords

Ca Signaling, VDAC, Benzodiazepine Receptor, Mechanistic Concept,

Pulsed EMFs, Electromagnetic Hypersensitivity, TSPO, Erythrocytes,

How to cite this paper:

Ullrich, V

. and

Apell

, H.-J. (2021)

Electromagnetic Fields

and Calcium Signaling by the Voltage D

pendent Anion Channel

Open Journal of

Veterinary Medicine

, 57-86.

https://doi.org/10.4236/ojvm.2021.111004

Received:

December 12, 2020

Accepted:

January 26, 2021

Published:

January 29, 2021

21 by author(s) and

Scientific

Research Publishing Inc.

This work is licensed under the Creative

Commons

Attribution International

License (CC BY

4.0).

http://creativecommons.org/licenses/by/4.0/

Open Access

V. Ullrich, H.-J. Apell

DOI:

10.4236/ojvm.2021.111004 58 Open Journal of Veterinary Medicine

NADH-Oxidase, Apoptosis, Magnetosensor, Membrane Potential, Oxidative

Stress, Brain Signaling, Autism

1. Introduction

The digital revolution changes life in our modern world dramatically and enables

mankind to solve increasingly complex problems in shorter times. Mankind is

forced to act more and more in networks and relies on mobile communication

that significantly affects our way of life. The technical progress is reflected in ev-

er more rapid developments of wireless communication systems emitting EMFs

from radio frequencies to microwave radiation (e.g. 3G, 4G, Wi-Fi, Bluetooth)

and now 5G. This new ultra-rapid and high capacity network is currently being

installed worldwide, not without increasing warnings on possible health risks for

humans and living organisms in general [1] [2] [3] [4]. There are numerous re-

ports on so-called “electromagnetic hypersensitive” persons who suffer from

headache, insomnia, unrest, deficits in memory and learning, skin sensations or

even depression in possible connection with mobile communication [5] [6].

Meanwhile about 8% of the human population suffer from such obviously

brain-related diseases and together with the rising incidences of the burn-out

syndrome [7]. In the male population a severe reduction of sperm counts [8] and

breast cancer was found to increase in young women carrying their cell phone

close to the breast [9]. Such epidemiological studies on humans are always sub-

ject to criticism and hence health effects by cell phone exposure were mostly

disputed in literature. On this background and in view of the multiple physio-

logical and psychological effects, it became obvious that a broad range of toxico-

logical and mechanistic studies were required to find solid support for potential

risks of microwave exposure from mobile phones and their base stations. Animal

studies were the first choice in risk assessment and long-term exposure at low

dosage was required to eliminate potential thermic effects of the irradiation. In a

study carried out over 20 years and published in 2018 the FDA within the US

National Toxicology Program performed a risk assessment with over 20,000 rats

and mice showing a significant increase in glioma and its precursors in male rats

(emfdata.org). In parallel a similar study by the Ramazzini Institute performed

with 2448 Sprague-Dawley rats the animals developed significantly more heart

Schwannomas in male rats [10]. In such animal studies [11] [12] DNA damage

as well as oxidative stress symptoms were also present which suggests a causal

relationship with tumor formation. Not only tumors but also developmental ha-

zards seem to be a consequence of low-dose microwave exposure as seen in liv-

ers from fertilized chick embryos [13]. Increased mortality and severe malfor-

mations were reported in developing chicken eggs daily exposed for 50 min to

mobile phone irradiation [14]. These authors also refer to 8 other studies with

similar outcome indicating the developing chicken egg to be a reliable model for

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potential teratogenic effects of microwave irradiation. In this context results of

M. Hässig

et al

. [15] must be mentioned which suggested nuclear cataracts in

calves born upon exposure to radiation from antenna base stations during gra-

vidity. In spite of such abundant epidemiological and toxicological data, the offi-

cially tolerable exposure levels to EMFs, designated the specific absorption rate

(SAR), refers only to the thermal effects of microwave irradiation and allow

heating of the skin by 1˚C, and a safety factor which still allows exposure levels

of EMFs that exceed by far the level at which biological effects were reported in a

vast amount of literature. The need for reliable biological tests is obvious and

hence a rational and mechanistic approach is necessary to determine justifiable

EMF-exposure levels. It is the goal of this review to compile the abundant data

available in this field for new prospects on biochemical events triggered by ex-

posure of tissues and cells to electromagnetic fields.

There may be a general agreement on clearly non-thermal effects of EMFs in

biological systems. The amount of energy absorbed in biological tissue provided

by EMFs relies crucially on frequency, intensity and exposure time. Radiation in

the GHz range will interact preferably with electrons and primarily produce

thermal effects. Pulse protocols modulated onto the carrier frequency provide,

however, in their frequency spectrum components which are able to induce res-

ponses in proteins. Such signals and pulse trains with pattern in the range of

milliseconds or longer are able to trigger conformational rearrangements leading

to channel opening or closing. Another important question is the penetration of

tissue. From basic physical principles it is known that electromagnetic radiation

declines exponentially in matter due to absorption. The penetration depth,

which typically is specified by the length at which the intensity at the surface is

reduced to 37% (=1/e), depends on the applied wavelength and the material

through which the radiation has to pass. In the GHz range it was found, the

higher the frequency the higher the absorption coefficient. Besides thermal ab-

sorption it is, however, largely unknown by which mechanisms EMFs interact

with different tissues and cells and above which radiation intensities significant

effects on cellular processes are probable. To understand the molecular and me-

chanistic pathways of cause and effect, insight into the involved cellular compo-

nents has to be gained and it has to be uncovered how energy from EMFs is

transferred to cellular targets. In addition, so far no published research is availa-

ble that quantifies EMF effects with respect to the probability of noxious effects

in dependence of radiation intensity and duration. In biophysical terms we have

to determine the acceptors (and their sensitivities) that trigger biochemical and

functional responses in cells. The identification of the associated biochemical

components and their reactions will allow not only a prediction of the impact

caused by exposure to EMFs but also a proposal of suitable test systems to study

the consequences in different cells, tissues and species. Defined effects can be

quantified by

in vitro

assays for different frequencies, modulation and pulse

techniques. Such an approach will help the society to guide the current, partly

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emotional discussion on the risks of mobile-phone communication back to

scientific reliability.

A broad variety of scientific reports substantiate that EMFs have been applied

since decades in medicine to treat pain and enforce healing of bone fractures

using EMFs with frequencies between 10 and 30 Hz [16] [17] [18] [19]. Growth

of tumor cells could be arrested by application of alternating electric fields [20],

at which Ca2+ influx through Cav1.2 channels seems to be involved. Early studies

by Blackman [21] suggested Ca2+ to be involved in EMFs action on brain tissue.

Furthermore, detailed studies are available that stimulation of white fast muscle

fibers with 10 Hz caused a conversion into red slow muscle fibers due to an en-

forced rate of mitochondrial biogenesis [22]. Stimulation for time periods longer

than certain thresholds caused significant oxidative stress in the cells which was

detected and emphasized by nitration of tyrosine residues in various cytoplasmic

proteins [23]. It is well established that nitrated proteins are a fingerprint of pe-

roxynitrite (ONOO−) formation resulting from nitric oxide (NO•) synthesis and

superoxide radical (

−

) generation [24]. On the other hand, peroxynitrite en-

hances also significantly Ca2+ levels in cells by inhibition of the Ca-ATPase as

was measured also in such “overstimulated” muscle fibers [25]. Increased Ca2+

levels and oxidative stress are known to be indicators of severe cell damage and

lead to cellular dysfunction and even apoptosis [26]. Overall, positive as well as

negative effects of EMFs were reported, most of them being of non-thermal na-

ture.

Our present scrutiny of the available literature on appropriate cellular meta-

bolic processes and malfunctions can provide new insights into EMFs-related

processes and will allow an advanced mechanistic approach to understand the

interaction of EMFs and biological systems. Our study indicated unexpectedly

the involvement of a plasma membrane voltage dependent anion channel

(VDAC) in electrically non-excitable cells as a common target of EMF actions,

and VDAC associated sensors as mediators. This establishes new aspects in the

field of research on those cells and even on the biophysical and biochemical

network that controls brain functions.

2. Results and Discussion

2.1. Current Concepts of EMFs Action on Cells

An abundance of papers on the effects of EMFs on biological systems can be

found in the literature, and hundreds of them refer to biochemical findings with

cells. Martin L. Pall [27], proposed a specific characteristic of the effect of EMFs

in excitable cells, namely an increase of the cytoplasmic Ca2+ concentration

through voltage-dependent Ca channels (VDCC). Ca2+ is a second messenger in

cells and it is well known that the concentration of free Ca2+ is tightly regulated

under physiological conditions, to maintain the cellular metabolism intact. Sur-

plus Ca2+ is immediately stored away in intracellular repositories or pumped at a

limited rate actively out of the cell. If the storage capacity or cellular energy is

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exhausted, the cytoplasmic Ca2+ concentration will be continuously enhanced.

Pathological effects of such a Ca2+ overload have been investigated since many

decades, and its stimulation of DNA damage, tumor formation, mitochondrial

dysfunction, and apoptosis is beyond any doubt [28] [29]. For Review see [30].

Upon the EMF-induced increase of the cellular Ca2+ concentration a chain of ac-

tions may be triggered that cause deleterious and adverse effects, as has been

uncovered by analysis of the mechanistic background provided by a series of

publications which all centered on the increase of intracellular Ca2+ as disclosed

in the literature cited e.g. in [31].

In numerous papers the authors described that EMFs triggered Ca2+ influx

mostly occurs through VDCC [27], and considered positive charges in ami-

no-acid side chains as sensors of EMFs in such channels proteins within the lipid

environment of the plasma membrane, and it was concluded that changes by

EMFs could trigger an activation of the voltage sensor which is known to control

the opening of these channels. This mechanistic proposal is represented in the

left scheme of Figure 1. By EMF-induced conformational modification such

channels switch to the open state and enable Ca2+ to flow along the large con-

centration gradient from the outside into the cell. Based on the selectivity and

efficacy of various pharmacological inhibitors, most of the analyzed channels

were identified as L-type VDCC. The resulting brief elevations of intracellular

Ca2+ levels were sufficient to elicit a cell-specific signaling response. Typically,

the Ca2+ signal appeared as concentration spike and reached the basal Ca2+ con-

centration before the process was evoked again. An innovative finding of Pilla

[32] was that NO• formation, which was measured electrochemically by an NO•

electrode, could be detected as early as 5 - 10 s after beginning of the EMF irrad-

iation. Therefore he postulated the activation of nitric oxide synthase (NOS) as a

secondary event since both constitutive NOSs are Ca-dependent. The released

NO• activated in turn the guanylyl cyclase, and cGMP kinases followed with

Nfr2 as final target [33]. The activation of antioxidant enzymes could be induced

by this pathway as appropriate response to counteract oxidative stress which is

caused by peroxynitrite as product of the recombination of NO• with superoxide.

As mentioned above, peroxynitrite is able to block oxidatively the thiol groups of

the Ca-ATPase. This process inhibits the ion pump and leads to a further in-

crease of intracellular Ca2+ and eventually to pathological effects via mitochon-

drial dysfunction. According to Pall the postulated activation of the VDCC vol-

tage sensor is based on the assumption of a millionfold enforcement of the elec-

tric charge effects in the membrane environment. In view of the very low electric

fields acting on cells this assumption may be a point of criticism on the proposed

mechanism. Nevertheless, the commonly detected EMF-induced Ca2+ influx re-

mains as established fact.

A paper published by Friedman

et al

. [34] disagreed with the hypothesis of

Pall who did not address the source of the required superoxide or the reactive

oxygen species (ROS) derived therefrom. This question was assessed experi-

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mentally by Friedman and collaborators, who established that a chain of bio-

chemical events led to an activation of extracellular receptor kinases (ERK) by

phosphorylation. A corresponding mechanistic proposal is represented in the

right panel of Figure 1. Using pharmacological and chemical inhibitors these

authors held a plasma-membrane associated NADH oxidoreductase (PMOR)

responsible for the ROS formation. In HeLa cells, which they used in their expe-

riments, it was suggested to be H2O2, since it was shown that NADH is oxidized

aerobically to NAD+ by enzymes localized in the plasma membrane. As a control

it was demonstrated that levels of 100 - 200 µM H2O2 were able to initiate ERK

phosphorylation. It was argued that H2O2 would activate a matrix metallopro-

teinase that cleaves the Hb-epidermal growth factor (EGF). This process has a

stimulating effect on the EGF receptor and subsequently on ERK [35]. These

data are well documented, and the frequencies, intensities and exposure times

chosen were close to typical mobile-communication exposure and demonstrated

Figure 1. Comparison on two proposed mechanistic concepts that describe the effects of

EMFs on cell membranes. Left panel: Model of Pall [27]. The voltage-dependent Ca

channel (VDCC) is assumed to be the sensor of EMFs. Two different pathways of res-

ponses are expected: 1) an antioxidant defense by which Ca2+ entry causes NO• produc-

tion which in turn triggers a chain of enzyme activities including cyclic guanosine mo-

nophosphate release (cGMP), cGMP-dependent protein kinase (PKG), and NFE2-related

factor 2 (Nfr2). 2) Pathological effects generated by the initial NO• release causing the

production of further radicals. Right panel: Model of Friedman

et al

. [34]. EMFs affect the

NADH-oxidase (NADH-OX) which was in case of their experiments a tumor associated

NADH-OX (tNOX). This leads to the production of reactive oxygen species (ROS),

probably H2O2, that activates the matrix metalloproteinase (MMP) and subsequently the

heparin-binding epidermal growth factor (HbEGF), the epidermal growth factor receptor

(EGF-R) and eventually the extracellular-signal regulated kinase (ERK), thus provoking

cellular defense mechanisms.

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that the two investigated tumor cell lines responded in a non-thermal way by de-

fined mechanisms, albeit the authors missed to include an EMF sensor that trig-

gered the NADH oxidase as a initiating mechanism for the experimentally well

documented phosphorylation cascade. No pathology was associated with the

proposed ERK pathway in [34].

Taken together, both mechanistic approaches to explain EMFs-induced res-

ponses refer to two different signaling pathways that are able to allow cells to

cope with an oxidative challenge that has been postulated as main event follow-

ing exposure to EMFs. Cellular repair pathways originate either from ERK sig-

naling or NO• synthesis, and both constitute a physiological response which is

not hazardous to cells. Instead, cells and organs may become more resistant to

further challenges and could respond favorably towards continuing oxidative

stress conditions. This would be a beneficial consequence of EMFs exposure

which is in line with the long-known healing effects of low-frequency irradiation

as mentioned above.

Criticism was expressed regarding the sequence of reactions proposed by

Friedman

et al

. because of the unspecificity of diphenylene iodonium (DPI) as

inhibitor for NADH oxidase [27] [36]. Otherwise Morré [37] described the

NADPH oxidase of the plasma membrane as a specific target of DPI and this ac-

tivity is also part of the plasma membrane electron transport system. Related to

the hypothesis of Friedman

et al

. a main issue of concern is that the nature of a

required EMF sensor was not addressed. The NADH oxidase would have to act

as a EMFs sensor but it has not been explained with this function. Moreover,

H2O2 concentrations of at least 100 µM, which are necessary to activate the ma-

trix metalloproteinase (MMP), appear to be too high under physiological condi-

tions and probably will not be built up by the NADH oxidase activity available in

normal cells. According to the literature MMP-2 is effectively activated by pe-

roxynitrite [38]. It is noteworthy to mention that in tumor cells, as used in

Friedman’s study, the process of “reprogramming” affects glucose metabolism

and redox equilibria in a dominant way as pointed out later. It is specific to tu-

mor cells such as HeLa cells to exhibit PMOR activities higher than in normal

cells which are under control of growth hormones [39]. As will be shown below,

the PMOR may indeed play a role, but by a different mechanism.

The positive and beneficial effects of EMFs are lost, however, upon prolonged

challenge that causes enhanced Ca2+ influx and subsequently stimulated NO•

formation. There are several sources from which superoxide could be provided

under conditions of repetitive EMFs challenge. One is the exhaustion of the re-

ductant tetrahydrobiopterin that elicits an oxidase function of the NOS [40].

Second, NO• is known to block Complex IV of the respiratory chain in mito-

chondria, and the resulting electron overflow is redirected to oxygen and yields

in the formation of

−

[41]. Upon a steady increase in

−

, it will combine

with NO• and the resulting product ONOO− reacts with an excess of NO• and

gives rise first to a nitrosating species (like NO+) until equal amounts of NO• and

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−

radicals are generated and ONOO− formation remains at a steady state level

[42]. It is important to recall that the critical oxidant peroxynitrite will prevail

only as long as NO• is not present in excess [43].

In summary, when EMFs are applied only for short exposure periods, the in-

duced Ca2+ influx leads to NO• radical production that will be even beneficial

since it counteracts Ca-induced cellular activation [44]. Prolonged exposure

leads, however, to oxidative-stress conditions which can be compensated by the

cellular antioxidant defense systems only up to a certain level, whereas high and

continuing EMF applications are able to provoke deleterious effects such as

DNA damage, lipid peroxidation, mitochondrial dysfunction and apoptosis.

This noxious outcome is depicted in Figure 1 (left panel) in which the dual

pathways of Ca2+ influxes are visualized that may result in positive and negative

effects on cell survival.

2.2. VDAC as a New Origin of EMF-Induced Effects

Our survey of the available literature data was assigned to provide new insight

into the interaction of EMFs with cells and will allow a better understanding of

the molecular mechanism triggered by EMFs in biological systems. The promi-

nent result is an unexpected involvement of voltage-dependent anion channels

(VDACs) in the plasma membrane and their functional association with sensor

proteins as mediators of EMFs.

As outlined above, EMF effects through Ca2+ entry via VDCC seem to be well

established for excitable cells. The question is, however, whether this entry

pathway for Ca2+ is true also in non-excitable cells. In the experiments of Fried-

man

et al

. [34] with isolated HeLa cell plasma membranes an irradiation resulted

in a more than twofold amount of NADH oxidation compared to control cells.

Considering that NADH oxidase could be a target of EMFs as well, we scruti-

nized the literature on NADH oxidase and PMOR activity. The result was at first

a complex and even confusing pattern of redox-active components and sug-

gested the involvement of activating metabolic pathways [45] [46]. Several fla-

voproteins are engaged in the transfer of hydrogen from NADH and NADPH

[47] [48]. One of these proteins uses ubiquinone (CoQ) as acceptor and gene-

rates a pool of ubihydroquinone within the plasma membrane. This compound

seems to be linked to the generation of a proton gradient by expelling protons to

the outside of the plasma membrane upon oxidation. Potential electron accep-

tors for reduced CoQ could be dioxygen as postulated by Friedman in the phos-

phorylation cascade that leads to the formation of H2O2. A second system was

reported to reduce a b-type cytochrome but also a 32 kDa copper protein with

sequence identity to VDAC of the outer mitochondrial membrane [49], where it

has an important function for cytochrome c release by formation of “megapores”

as a consequence of mitochondrial dysfunction and apoptosis [50]. The fact that

VDAC is expressed in the plasma membrane is known for a long time [45] [51].

There it is part of the PMOR system and has the ability to reduce ferricyanide

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that was provided as artificial electron acceptor [52]. The extra-mitochondrial

location was verified by detection of a leader sequence for transport into the

plasma membrane [53] and by isolation as part of caveolae [54]. Physiologically

it is able to reduce diferric transferrin and releases ferrous iron to be taken up in

the cell where it is reoxidized and stored in ferritin [52] [55]. Semidehydroas-

corbate is another acceptor that is reduced by one-electron reduction to ascor-

bate, and thus can be recycled as antioxidant [56] [57]. Several isoforms of

VDACs were identified that have different reactive sulfhydryl groups [58], but at

least two sulfhydryl groups are conserved in all isoforms and may serve in

thiol-disulfide exchange [59]. Surprisingly, the redox activity of VDAC is con-

trolled by growth hormones [60], and even more interesting, transformed tumor

cells have expressed a different NADH oxidase (tNOX) and a different VDAC

(VDAC2) [61]. For this reason HeLa cells as used by Friedman

et al

. may not be

representative for NOX/VDAC mediated activities. In summary, HeLa cells, like

most cancer cells, are clearly distinct with regard to the plasma membrane elec-

tron-transport system, which may contribute to their autonomous growth capa-

bility.

In view of such complex functional properties, the common isoform, mito-

chondrial VDAC1, was cloned and expressed for detailed biophysical and bio-

chemical investigations. It was characterized as a beta barrel membrane protein

with six membrane spanning domains [62]. High resolution NMR and molecu-

lar dynamics calculations discovered that glutamate 73 is a very flexible and mo-

bile residue, which is exposed in lipid micelles to the fatty-acid moiety and

showed a fast protonation/deprotonation equilibrium with dynamics in the µs to

ms time range [63]. This was considered to be important since this glutamate re-

sidue was found to be crucial for binding of hexokinase-1 in the deprotonated

state and for a condition that provided stability to the anion-selective resting

state of VDAC. In the case of the mitochondrial VDAC an attachment of hex-

okinase-1 was found and associated with an augmented supply of NADH in the

glycolytic pathway [64]. The same applies for NADPH formation by the pen-

tose-phosphate cycle needed for NO• synthesis. Interestingly, also NOS was as-

sociated with VDAC [65].

Investigations of the electrophysiological behavior of VDACs [66] provided

intriguing new aspects on an EMF-induced Ca2+ influx into cells. It is known

that VDAC in the cell membrane exists in a high-conductance anion selective

state in which it is able to transfer ATP out of the cell [67] [68], which is its ma-

jor physiological function as channel. At membrane voltages < −20 mV a “mul-

tiple states” condition exists, in which “open” (Cl− conducting) and “closed”

(Ca2+ conducting) states existed simultaneously [66]. Studies of its voltage de-

pendence in the range of −40 to +40 mV reported transitions between different

low-conductance substates with selectivity for small cations and a further transi-

tion to a cation-selective state of high conductance [69] [70]. Upon a monotonic

voltage increase from −40 to +40 mV it was found that at −10 mV the low con-

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ductance cation selective state of VDAC could be transformed to a large con-

ductance anion selective state. When the voltage was increased above +20 mV,

another transition was observed to a selective state of large cation conductance.

Other conductance changes were found between −30 to −40 mV. Studies of the

channel properties of VDAC in membranes from mitochondria as well as from

plasma membrane and in reconstituted planar lipid bilayers revealed by electro-

physiological experiments that transitions between at least three different states

occurred between an anion transporter, a Ca2+ channel with low conductance

and even a megapore that allowed massive Ca2+ and possibly also Cl- influxes

[71]. Under electrophysiologically undisturbed conditions,

i.e

. at the resting po-

tential, the preferential functional state of VDAC is an ATP transporter with an

outward-directed flux.

In excitable cells action potentials will evoke short Ca2+ influx spikes through

VDAC only if it is in the Ca2+-conducting so-called “closed” state. Upon persist-

ing depolarizing conditions (several ms) VDAC promotes Ca2+ influxes, and

those depolarizations may cause pathologic Ca2+ influxes and eventually Ca2+

overload of the cell [72] [73] [74]. This causes in turn oxidative stress as depicted

in the left scheme of Figure 1.

VDAC in the outer mitochondrial membrane shows an analogous behavior

which is well documented [75] [76]. Especially the transition to the megapore,

known as permeability transition, marks a crucial step towards apoptosis [77],

when cytochrome c is released through mitochondrial aggregated VDAC into

the cytosol and activates the apoptosis-inducing factor (AIF) and caspases [78].

In contrast to the cell membrane, however, changes of the membrane potential

of the outer mitochondrial membrane are considered of minor relevance, and

thus have found only limited attention in the published literature [79]. There-

fore, our focus was set on the literature on VDAC in the plasma membrane and

on the question whether and how EMFs are able to trigger a mechanism that

promotes transitions into states which are able to explain the observed graded

Ca2+ influx.

Electrophysiological experiments on channel properties of VDAC in mito-

chondrial and plasma membranes or reconstituted in black lipid membranes

were performed to study the three different ion-conducting states (Figure 2). In

the “open” conformation the channel is anion selective. In the “closed” state

larger anions are excluded but cations are well permeable, and a distinct con-

ductance for Ca2+ was detected. Due to its role as second messenger, the basal

Ca2+ concentration in the cytosol is about 100 nM, but outside the cell in the or-

der of millimolar and in the mitochondrial matrix between 20 µM (physiological

condition) and 500 µM (Ca2+ load) [80] [81]. Across the VDAC-containing mi-

tochondrial and plasma membrane the electrochemical potential gradients are

always directed towards the cytosol and facilitate in the Ca-conducting state a

Ca2+ influx into the cytosol. Studies of the voltage dependence of VDAC revealed

that the open probability decreases from about 1 at a membrane potential of zero

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to about 0.5 at typical membrane potentials of −40 mV to −60 mV in the case of

non-excitable cells [66] [82] [83]. In the open and preferably anion-transporting

state of VDAC the Ca2+ conductance is less than 4% of the ion flux. In the

so-called “closed” state at (positive or negative) membrane potentials the anion

conductance is significantly decreased while the Ca2+ conductance is amplified

(especially at negative potentials) by a factor of 4 to 10. Extrapolating from expe-

rimental results [66] and accounting the probability of the Ca-conducting state,

under an assumption of 1 mM extracellular Ca2+ about 20.000 Ca2+ ions would

flow per VDAC and per s into the cytosol. To balance this influx in order to

maintain the physiologically required low basal concentration, the cell possesses

active transporters such as the plasma membrane Ca-ATPase and the Na, Ca

exchanger. If their outward transport capacity is not sufficient in the case of

so-called Ca2+ spikes, finally mitochondria will act as buffer storage compart-

ments to secure rapid recovery of the basal Ca2+ concentration in the cytosol. If,

however, an elevated Ca2+ influx persists, the mitochondrial storage capacity

runs out, the high Ca2+ concentration inside the mitochondria induces VDAC

closure of the anionic state and converts to the cationic substate, which prepares

the way to apoptosis [66].

Figure 2. Schematic representation of the different functional states of VDAC in response to EMFs. Left panel: Under physiologi-

cal conditions the high-capacity anion selective state is prevalent, named “open state”. The protein complex consists besides

VDAC of several enzymes: plasma membrane oxidoreductase (PMOR) which consists of TSPO and a flavoprotein, hexokinase1

(HK), adenine nucleotide transporter (ANT), and NO-synthase (NOS). The complex is also coupled to an electron-transport sys-

tem based on ubiquinone, CoQ (Q). The extracellular electron acceptor is a semidehydroascorbate (Asc). Middle panel: Low-capacity

cation-selective state, named (misleadingly) “closed” state. The conserved glutamate (E 73) affects a major conformational change

to a cation (including Ca2+) selective state and HK dissociation. The transition into this state may be promoted or stabilized by

EMFs contrary to the physiological needs of the cell. Right panel: A high capacity cation-selective state, named “mega pore”, is

formed upon a prolonged unphysiological condition in the cell and allows high Ca2+ fluxes into the cytosol, which activates sub-

sequently the mitochondrial permeability transition pore (MitoPTP) inducing cell death by apoptosis. Oxidative oligomerization

of VDAC under this condition is indicated by “VDAC-S-S-”.

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Another important detail of VDAC is the presence of a conserved negatively

charged amino acid. According to NMR data glutamate 73 is buried in the hy-

drophobic core of the phospholipid bilayer and calculations on the flexibility

within the beta strands 2 to 7 increased with the negative charge on the gluta-

mate 73 [67]. Even a perturbation of the membrane polar head groups was post-

ulated from MD simulations pointing to a local thinning of the membrane [67].

Ca2+ influxes into the cell, permitted not only by VDAC but also by other

VDCC, which are carefully balanced by active export, may be enhanced due to

various reasons, be it mechanical injuries, noxious chemical compounds and al-

so EMFs. As already mentioned above, a common initial event observed in cells

exposed to pulsed EMFs is an elevated cytoplasmic Ca2+ level in the cytosol.

Based on the experimental findings presented above, VDAC is an appropriate

candidate as primary target being affected by pulsed EMFs. Since its distribution

between open and closed states can be modulated by the membrane voltage (and

therefore named “voltage-dependent anion channel”), the protein must possess a

voltage sensor, although it has not yet been identified conclusively so far [84].

Basic physical properties require, however, inevitably the existence of electric

charges that interact with the electric field and transmit changes of the electric

field into functional responses. At appropriate frequencies even minor changes

of the electric field seem to be able to affect the charge distribution within the pro-

tein and thus modify the pattern between the open and closed state of VDAC.

It is evident that electromagnetic waves in the GHz range are too fast by or-

ders of magnitudes to evoke motions of amino-acid side chains or protein do-

mains. Pulsed EMFs with various frequency patterns up to 1000 Hz are, howev-

er, suitable candidates, which are able to transfer energy absorbed by charged

structural components in the protein that may be amplified, when provided in a

fitting (resonance) frequency window, and thus evoke transitions into specific

conformational states. A proposal of a possible molecular mechanism that causes

enhanced Ca2+ influxes through VDAC is based on the assumption that suitable

frequencies in the pattern of pulsed EMFs stimulate the transition to and/or

cause a stabilization of a Ca-conducting state of the channel.

Therefore, we recommend to perform experimental studies with various cell

species to analyze the resulting cytoplasmic Ca2+ concentrations as function of

the pulse frequencies in the (commercially used) range between 6 and 1600 Hz.

An effect of pulsed EMFs on the behavior of the membrane inserted VDAC may

be assessed experimentally by patch clamp technique with cells or model mem-

branes. Another option would be the use isolated cells or cultured cells together

with intracellular fluorescent indicator dyes to detect in real time Ca2+ uptake

upon exposure to user-defined EMF protocols. The results of such studies may

answer the pertinent question of whether and how exposure to EMFs is able to

affect the function of VDAC. This may occur either via modifications of the

membrane potential that affects the whole protein or by interaction with specific

protein domains or single amino-acid side chains such as the negatively charged

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glutamate 73. Even a rise or collapse of a proton gradient across the membrane

could influence the ionization state of this critical residue. Interactions with

proteins in functional contact with VDAC may contribute in addition (Figure 2)

as will be discussed in the following paragraph. In essence, VDAC possesses all

properties required to act as sensor of electromagnetic signals.

2.3. VDAC and the 18 kDa Translocator Protein

One of the proteins that interacts closely with VDAC is known as the 18 kDa

translocator protein (TSPO), earlier named benzodiazepine receptor (BR) since

it binds psychoactive drugs of this group with high affinity [85]. Benzodiaze-

pines are among the most extensively studied drugs because of their antidepres-

sant, anxiolytic and anticonvulsive properties. Detailed Reviews are [86] [87].

TSPO is primarily located in the outer mitochondrial membrane of many peri-

pheral tissues and has been named PBR to discern it from the central benzodia-

zepine receptor, CBR, found preferentially in neurons [88]. Both proteins are

similar in sequence and possess five membrane-spanning domains but differ in

their binding affinities to a variety of pharmacological ligands with psychoactive

impact [86]. Surprisingly, their physiological mode of action is not well defined

although tight binding of cholesterol and porphyrins indicated a role in steroid

and heme biosynthesis until observations with knock-out mice have challenged

an essential role in both pathways [89]. PBR was found to be associated with ac-

tivated glia cells and microglia in brain, and by

in vivo

use of diagnostic ligands

this fact is now used to locate inflamed areas caused by brain tumors or neuro-

pathological events [90].

An early report on CBR location on the plasma membrane of neurons stirred

our attention [91]. The finding that CBR might occur in connection with VDAC

and form a functional entity as part of the PMOR complex fueled our hypothesis

that VDAC is involved in this complex as Ca channel. With respect to the fact

that in neurons CBR in the PMOR complex could be the target of psychoactive

drugs is intriguing since the symptoms of electromagnetic hypersensitive per-

sons can be associated with dysfunctionalization of neurons and the well-known

relieving effects of benzodiazepines.

A mechanistic link became apparent when the sequence of benzodiazepine

receptors was analyzed and compared with other TSPO-like proteins. They fell

into the category of “tryptophan-rich proteins” which are characterized by a tri-

ad of Trp residues that also occur in photolyases [92] [93] [94] [95] and cryp-

tochromes [92] [96]. These enzymes have blue-light receptors that use light to

trigger an internal redox reaction in which a triplet state converts into two radi-

cals that form by electron hopping across two Trp-residues a tryptophanyl radi-

cal on the last one of the triad [93]. The resulting spatial separation of two un-

paired spins prevents a short term relaxation to the ground state and allows dis-

tinct chemical reactions, e.g. with oxygen. Such reactions can be affected by a

magnetic field which affects the product pattern. This behavior imparts such

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enzymes properties of a magnetosensor [97] if different products result in the

presence and absence of magnetic fields such as superoxide or hydrogen perox-

ide. Reports in the literature assign orientation of birds in the geomagnetic field

of about 50 µT to the presence of cryptochromes, in which a flavin absorbs blue

light and enters a triplet state. In presence of a neighboring Trp residue this state

converts to a tryptophanyl-flavin-radical pair [98]. Electron/proton tunneling

allocates the electron on the last Trp of the triad and this spatial charge separa-

tion leads to an increased lifetime of the two radicals. Their subsequent decay

can be affected by a magnetic field and possibly leads to different chemical decay

pathways [99]. This mechanism was considered as basis for orientation control

of migrating birds in the geomagnetic field [92].

Applying this concept to the TSPO proteins with their similar structural com-

ponents and allows the suggestion of an interaction with electromagnetic fields.

Radical formation would not be facilitated by light but by NADH oxidation with

oxygen as acceptor which is a potent source of energy and redox activity. Radical

intermediates could be easily formed and be part of a magnetoreceptive radical

system comprising two electrons or even three as recently proposed [97].

In conclusion, additionally to the presence of the sensor of the electric field by

VDAC, the TSPO/CBR complex may provide in a concerted way a supporting

and synergistic role as magnetosensor responding to the magnetic field compo-

nent of EMFs. In such a combined effort even low-intensity EMFs could be mo-

nitored by the TSPO/VDAC complex due to the combined absorbed energies

from electric and magnetic fields and could amplify a promotion or stabilization

of the Ca-conducting substate of VDAC.

Such a device could even act as transformer of information from do-

main-overlapping brain waves to Ca2+ pulses in single cells. To our knowledge

the molecular mechanism for converting the information (

i.e

. energy) of such

electromagnetic waves into chemical signals still awaits elucidation.

2.4. The PMOR/TSPO/VDAC System in Erythrocytes

In numerous published studies the function of the plasma membrane oxidore-

ductase could not be well separated from the various activities of the redox

components in the mitochondrial membrane due to the used experimental ap-

proach. Investigations with erythrocytes are less complicated since they are de-

void of mitochondria, and hence electron transport in the plasma membrane can

be studied without distortive interference. In addition, red blood cells (RBC) do

not grow, and therefore, are unaffected by growth factors that normally control

part of the PMOR activity [60].

Although erythrocytes appear to be glycolysis-driven empty membrane enve-

lopes, their plasma membrane is highly complex. It contains all components of

the PMOR system such as flavoproteins, ubiquinone pool, TSPO and even three

kinds of VDACs [100] [101] [102]. Electron acceptors are ferricyanide, oxidized

ascorbate, and with low activity oxygen. In combination with ANT, VDAC is

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able to export ATP. This process was found to be mediated by prostacyclin in a

PKA-dependent pathway, and the released ATP constitutes a physiological re-

laxation of smooth muscle for better oxygen supply in the blood vessels [103].

These facts point to the presence of a pronounced anion-selective VDAC activity.

Erythrocytes are non-excitable cells with a resting potential of −10 mV [104],

suggesting that transitions into the Ca-selective substates of VDAC are possible.

Since the VDAC protein in the RBC is identical to the well-studied VDAC1 in

mitochondria, this has to be expected and was indeed observed [105]. By stimu-

lation with the TSPO ligand Ro5-4864 the Ca2+ influx was enhanced about four-

fold, and the increase was blocked by the VDAC inhibitor Bcl-XLBH4 [106]. A

hundred channels per cell were probably enough to produce a Ca2+ influx suffi-

cient to modify the properties of the cell membrane which led to the characteris-

tic aggregation behavior of erythrocytes [107] as it was observed upon exposure

to EMFs. This phenomenon is called “rouleaux” or coin-roll formation and con-

stitutes the physiologically needed stacking of RBC which helps the cells to move

through narrow capillaries for oxygen supply in peripheral blood vessels [108].

The enzyme lipid scramblase has distinct affinity for Ca2+ and is able to flip

phosphatidyl lipids from the inner surface of RBC to the outside which may

promote the agglutination during coin-roll formation [109].

Coin-roll formation was observed already early with mobile phone users and

was proposed even as a simple test for EMFs effects on living cells [110]. It

would be worthwhile to investigate experimentally the sequence of events post-

ulated above and possibly re-establish RBC agglutination as a sensitive test for

non-thermal EMFs interactions. For quantitation this effect might have restrictions

since the process is reversible and may be enforced by the activity of the Ca channels,

Piezo1/2, which contribute with pressure-induced and membrane-potential depen-

dence Ca2+ influxes [111].

There are more pathways of Ca2+ influx into RBC that lead eventually to Ca2+

overload [104] [111]. While other cells would respond with apoptosis, this can-

not occur in RBC due to the lack of mitochondria. In RBC, however, an equiva-

lent to apoptosis, called eryptosis, was observed which is characterized by

Ca-induced activation of the Gardos channel that allows the extrusion of K+ and

Cl− and results in shrinking and collapse of the erythrocyte structure as is seen in

clot formation [108]. Aggregation of VDAC and TSPO to corresponding dimers

or higher polymers was observed in RBCs under such stress conditions. This

suggests a similar role for the VDAC/TSPO complex as in mitochondrial mega-

pore formation during apoptosis [112].

Erythrocytes used as a model system to study EMF interactions with living

cells may more definitely proof the important role of NO• production as one of

the first impacts of Ca2+ influx. The presence of NOS in the plasma membrane of

RBCs has been established even though NO• released to the cytosol may be

trapped as nitrate upon reaction with oxyhemoglobin [113]. When released to

the outside NO• acts as a potent vessel relaxant through stimulation of G-cyclase

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in the cells of the vascular wall. Under hypoxic conditions deoxyhemoglobin will

be present that converts nitrite to NO• with the same dilation-inducing effect on

vessels, which leads in turn to improved oxygen supply. Such positive features of

NO• will be eliminated by superoxide since it promotes formation of peroxyni-

trite which was held responsible for oxidative stress and the accompanying neg-

ative impact of Ca2+ overflow as well as inducing the pathway to apoptosis. Since

mitochondria are absent in RBCs the source for superoxide should be related to

the PMOR system in the cell membrane using NADH or NADPH for oxygen

reduction to

−

. (In this respect it is noteworthy to mention that in mitochon-

dria NO• is inhibitory to the formation of the megapore [44] and triggering

apoptosis. This property could be noted as another positive effect of NO• that is

abrogated by superoxide. The always neglected source of this radical in the

PMOR system could be highly significant in redox regulation.)

2.5. Psychological Disorders and Autism

Finally an interesting aspect of human psychology should be mentioned by con-

sideration of the TSPO/VDAC complex as mechanistic interface between EMFs

and neuronal responses. Based on medically well accepted psychological disord-

ers of electromagnetic hypersensitive persons in form of insomnia, unrest,

burn-outs, or cognitive and memory deficits, possible explanations will involve

necessarily the complex subject of brain physiology. This starts with the bio-

physics of brain waves and ends in the biochemistry of NMDA or GABA recep-

tors. Significant advances in understanding were achieved by studies on the me-

chanisms of sleep control in

Drosophila

where a complex network from different

brain waves controls the activity of interwoven receptors [114]. To gain insight

into kinesic behavior a neuronal oscillation code could be established in which

coupling of Ca2+ spiking with gamma and theta oscillations plays a dominant

role [115]. Brain information systems seem to be built up by microcircuits of

electrical and magnetic impulses, and it became challenging to find out whether

external EMFs are able to disturb the internal communication. On the back-

ground that on one hand diazepines bind to CBR with high affinities and on the

other hand diazepines are able to repair deficits in psychiatric brain disorders, a

connection between both findings may be considered. A discussion of more de-

tails which were published in the pharmacological literature is, however, outside

the scope of this contribution.

Based on considerations of the molecular components known to be affected

by EMFs, an impact of EMFs on the development of autism in childhood cannot

be excluded [116]. A possible hint on such a connection may be deduced from

statistical data reflecting the observation of an exponential increase of reported

cases of autism in children during the recent thirty years [117].

Clinical findings were reported that antibodies against VDAC and hexokinase

were frequently identified in autistic children [118]. Both antibodies inhibit the

function of VDAC and hexokinase. Additionally, it is known that VDAC anti-

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bodies impair growth and induce apoptosis in neuroblastoma cells in culture

[119] [120]. Thus, a kind of autoimmune effect may develop against VDAC and

result eventually in brain activity affected in the grown-up organism. In autistic

children enhanced NO• metabolites and increased glutathione peroxidase activi-

ty were detected, and these observations point to a moderate chronic inflamma-

tion [121]. Therefore, a primary effect of EMFs on the function of VDAC in

embryonic tissue may be a disturbance of the equilibrium of differentiation and

apoptosis that controls embryonic cell development. Another connection of the

TPSO-VDAC system and autism was reported in a study by Crane

et al

. [122]

that highlighted the significance of its upstream redox partners, ubiquinones.

Reduced ubiquinone serves as reductant for VDAC with its copper center and

reactive thiolate residues. When in control experiments ubiquinone was ex-

tracted with organic solvents, the reduction of ferricyanide, used as artificial

electron acceptor, was diminished for the most part but was restored by addition

of ubiquinone. In a clinical trial the authors supplied reduced ubiquinone to au-

tistic children at a daily dose of 50 mg over a three-month period and found sig-

nificant improvements in verbal communication, playing games, sleeping and

reduced food rejection. Plasma CoQ levels increased during the time of treat-

ment by almost a factor of five. The sensitivity of NADH-ferricyanide reductase

against mercurial compounds is high and a connection of mercury with autism

has been repeatedly postulated, which is another joint link between NADH oxi-

dase and autism [123]. In VDAC the thiolate groups are extremely sensitive to

mercurials and this property could affect the activities of VDAC. Such effects are

independent from the genetic disposition as contributing factor in autism.

In summary we like to propose from such observations that an undisturbed

function of the VDAC/TSPO/hexokinase complex in the membrane of neural

cells is crucial for the developing brain. When this protein complex becomes a

target of antibodies or environmental factors such as toxic agents or EMFs above

a crucial threshold, the complex network of neuronal communication within

and between different areas of the brain will become increasingly disturbed with

the consequence that autism may emerge in the developing brain. In autistic pa-

tients smaller sizes of the cerebellum vermis were reported [124]. In adult rats

prolonged treatment with clonazepam changed the pattern of brain receptors,

especially the subunits of the NMDA receptors, which became reversible after

discontinuation of the drug [125]. However, in newborn rats similar treatments

led to drug-induced changes in the developing brain that were conserved in

adulthood, and increased apoptosis and suppressed neurogenesis were reported.

The authors concluded the appearance of possible deficits in behavior later in life.

Based on such mechanistic background it can be proposed that mental dis-

orders may originate from disturbances of the brain electromagnetic network

caused by disadvantageous effects on the associated receptors in cell membranes.

TSPO in its CBR form certainly plays a major role in this process, since many of

the disorders like insomnia or depression can be treated with diazepines of the

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clonazepam type which interact with this enzyme complex. In view of the

alarming increase of autism and the well-funded research to study this pheno-

menon, we consider it is justified to propose that the possible role of CBR in

mediating internal and external EMF effects in brain should become subject of

future research. Biochemical and histological tests to verify or dismiss a connec-

tion are within experimental reach.

3. Conclusions

Numerous studies on effects of EMFs in biological systems revealed a multitude

of biochemical changes with positive or negative physiological outcome in cells,

tissues or the whole organism. Most of such actions can be explained by an in-

flux of Ca2+ into cells as initial event. For electrically excitable cells VDCCs were

suggested as possible pathways. For non-excitable cells we found intriguing evi-

dence that VDAC fulfills all criteria of an EMF-controlled Ca channel. This sur-

prising result found its explanation by structural and functional modifications

associated with conformational changes of the membrane-bound VDAC. In the

outer mitochondrial membrane with a potential around zero the channel trans-

ports preferentially anions. VDAC is also found in smaller amounts in cell

membranes and this leads to the pertinent and crucial question of VDAC func-

tion in the two very different locations.

It is known that VDAC in mitochondria facilitates under physiological condi-

tions mainly ATP release. When Ca2+ overload occurs and/or the proton-motive

force ceases and no more ATP is produced, VDAC switches to the so-called

closed state in which it releases Ca2+ to the cytoplasm. By further oxidative mod-

ifications VDAC is able to form expanded pores by oligomerization. These lead

eventually to a permeability transition allowing cytochrome c release as the

committing step towards apoptosis.

The role of VDAC in the plasma membrane is elucidated by its role in RBC,

which do not possess mitochondria. There the channel is part of the PMOR sys-

tem, a complex with several other redox proteins, especially TSPO. It catalyzes

the reduction of extracellular iron complexes and controls ATP release from

RBCs in a protein A kinase dependent way.

Interestingly, RBCs responds to mobile phone radiation by rouleaux forma-

tion, and this intercellular attachment is likely to be due to a moderate Ca2+ in-

flux. Although only about 100 VDAC/TSPO channels are present per RBC the

Ca2+ influx provides enough Ca2+ in the sub-plasma membrane space to promote

aggregation. VDAC is a likely candidate for such a Ca2+ influx, but so far the

mechanosensitive Piezo 1 channel cannot be ruled out since it displays also vol-

tage sensitivity [111].

As a summary of the first part of our quest to identify potential targets of

EMFs we conclude that VDAC as part of highly sophisticated “transduceosome”

has to be considered to be a potential Ca2+ channel with various substates that

are formed in response to subtle changes of the membrane potential either in the

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outer mitochondrial or plasma membrane. Physiologically this applies for situa-

tions of energy deprivation leading to pro-apoptotic conditions. Under regular

conditions the VDAC complex controls substrate supply to mitochondria and

ATP export to the cytosol. Ca2+ overload, provoked by longer-lasting elevated

Ca2+ in the cytosol, is compensated first by mitochondrial uptake on the expense

of the membrane potential and the proton gradient, until VDAC converts to a

Ca2+ channel that levels the excess of Ca2+.

Exposure to EMFs causes an additional influx of Ca2+ by enhancing the Ca

channel activity of VDAC either in the plasma membrane or the outer mito-

chondrial. Resulting elevated Ca2+ levels will be lowered by the various extrusion

mechanisms on the expense of energy. Even short pulses may produce Ca2+

spikes that lead to accumulation of Ca2+ in the ER and eventually in mitochon-

dria. Subsequent emergence of oxidative stress relies on secondary events such as

NO• formation, which are well described in the literature. In case of the erythro-

cyte membrane we consider VDAC to be the EMF-sensitive channel facilitating

sufficient uptake of Ca2+ that leads to a membrane modification with rouleaux

formation. Thus, RBCs could become a suitable system to study various aspects

of EMF effects. So far no studies on the EMF impact on VDAC in mitochondria

were reported in the literature. Since under normal metabolic conditions Ca2+

levels in the cytosol and matrix are low, one would not expect a significant

change of the Ca2+ gradient if the mitochondrial VDAC changes to the Ca2+

conductance. Whether ATP or metabolite transport is affected by EMFs remains

to be investigated.

In the second part of our study we became aware of the role of TSPO, a pro-

tein closely associated with VDAC. It seemed to be more than a carrier for heme

and cholesterol as it is suggested in literature, especially since its amino acid se-

quence contained a Trp triad that is characteristic for the function of magneto-

sensors in migrating birds. The sensing mechanism requires oxidation of Trp

and stabilization of at least two radicals which decay to different products in de-

pendence of the magnetic field. Interestingly, VDAC possesses NADH oxidase

activity and would be able to perform such oxidations in the vicinity of TSPO.

The hypothesis of TSPO as a magnetosensor is under investigation.

Published properties of TSPO, which is known to be the benzodiazepine re-

ceptor in its peripheral mitochondrial (PBR) and central form (CBR) in neuron-

al tissue, introduced an interesting new functional aspect. Highly effective

pharmacological ligands of CBR are clinically used to treat neurological deficits

such as insomnia, burnout or depression, which were observed as syndromes in

electromagnetic hypersensitive persons. We have outlined that such characteris-

tic neuronal deficits and their treatment with TSPO receptor ligands suggest a

mechanistic connection originating in a participation of TSPO in a disturbed

signal transduction by brain waves. This hypothesis can be subject to straight

forward experimental verification with a suitable hardware for EMF generation

and modification of frequencies, intensities and pulsations. A second piece of

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evidence that VDAC/TSPO may play a crucial role as interface between psychic

disorders and EMF exposure became obvious by abundant reports on autism.

Pediatric patients exhibited in their blood significant amounts of antibodies in-

hibiting VDAC and hexokinase1. Accordingly it was already suggested that aut-

ism may be classified as autoimmune disease.

Summarizing the second part of our literature search we propose the hypo-

thesis that TSPO acts also as a magnetosensor. In conjunction with VDAC as the

sensor for the electric field the specificity and sensitivity of the complex to mon-

itor EMFs would be enhanced, possibly to the extent that brain waves could be-

come detectable by the VDAC/TSPO complex in neuronal plasma membranes.

Even this assumption can be tested under the influence including effects of ex-

ternally applied EMFs.

In essence we suggest that in brain the plasma membrane VDAC/TSPO com-

plex is able to sense brain-derived EMFs but would also be subject of external

fields. Their effect would be a strengthening of the oxidative defense systems,

under overstimulation, however, a consequence could be a disturbance of in-

formation processing especially in developing organisms. This is summarized

and depicted in Figure 3.

Figure 3. Mechanistic concept proposed in this paper. Ca2+ entry is promoted by the

TSPO/VDAC complex in the plasma membrane of cells triggered by EMFs (TSPO: 18

kDa translocator protein, VDAC: voltage-dependent anion channel). Single EMF pulses

(or consecutive pulse with a low frequency that allows recovery of the cytoplasmic Ca2+

concentration in between) evoke an antioxidant defense mechanism identical to the model

of Pall (Figure 1, left scheme) or induces cell-specific responses. By the Ca-dependent

protein kinase C (PKC) phospholipase A2 (PLA2) is activated that in turn releases arachi-

donic acid (AA) from lipid cleavage. Repetitive pulses with higher frequencies that lead to

continuously enhanced Ca2+ concentration generated by the activity of the Ca and NADPH

dependent NADH-oxidase (NOX5) and the nitric oxide synthase (NOS) high enough

concentrations of

−

and NO• which both react to form peroxynitrite (ONOO−). This

highly reactive molecule leads together with the high Ca2+ concentration to mitochondrial

dysfunction and eventually to apoptosis or by protonation yielding OH• and NO2 radicals

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with subsequent potential for DNA damage and possible tumor formation.

Taken together, there are clearly non-thermal effects of electromagnetic fields

on biological systems. We found confirmation of earlier assumptions and ob-

tained additional evidence that Ca2+ influx into cells constitutes a primary event

which leads to cell activation. The resulting activity is cell-specific and may

comprise diverse outcomes such as enhanced cell motility favoring healing

processes or could cause oxidative stress conditions even with DNA damage or

cell destruction. We here propose the voltage dependent anion channel as a Ca2+

influx channel under membrane-depolarizing conditions. Considering the asso-

ciation of the 18 kDa transport protein as the brain benzodiazepine receptor

with VDAC there may be even a mechanistic connection to the various symp-

toms of electromagnetic hypersensitive persons.

In the present state the influence of EMFs on biological systems is no longer a

topic of theoretical interest but needs systematic experimental investigations to

understand mechanisms and effects.

Acknowledgements

The authors thank Dr. Stefan Zbornik for valuable and competent discussion

and comments on issues concerning telecommunication and related literature

research.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this pa-

per.

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Problems in evaluating the health impacts of radio frequency radiation

Article

Dec 2022
ENVIRON RES

In an effort to clarify the nature of causal evidence regarding the potential impacts of RFR on biological systems, this paper relies on a well-established framework for considering causation expanded from that of Bradford Hill, that combines experimental and epidemiological evidence on carcinogenesis of RFR. The Precautionary Principle, while not perfect, has been the effective lodestone for establishing public policy to guard the safety of the general public from potentially harmful materials, practices or technologies. Yet, when considering the exposure of the public to anthropogenic electromagnetic fields, especially those arising from mobile communications and their infrastructure, it seems to be ignored. The current exposure standards recommended by the Federal Communications Commission (FCC) and International Commission on Non-Ionizing Radiation Protection (ICNIRP) consider only thermal effects (tissue heating) as potentially harmful. However, there is mounting evidence of non-thermal effects of exposure to electromagnetic radiation in biological systems and human populations. We review the latest literature on in vitro and in vivo studies, on clinical studies on electromagnetic hypersensitivity, as well as the epidemiological evidence for cancer due to the action of mobile based radiation exposure. We question whether the current regulatory atmosphere truly serves the public good when considered in terms of the Precautionary Principle and the principles for deducing causation established by Bradford Hill. We conclude that there is substantial scientific evidence that RFR causes cancer, endocrinological, neurological and other adverse health effects. In light of this evidence the primary mission of public bodies, such as the FCC to protect public health has not been fulfilled. Rather, we find that industry convenience is being prioritized and thereby subjecting the public to avoidable risks.

Effect of 40 Hz Magnetic Field Application in Posttraumatic Muscular Atrophy Development on Muscle Mass and Contractions in Rats

Article

Dec 2022

Muscle atrophy refers to the deterioration of muscle tissue due to a long‐term decrease in muscle function. In the present study, we simulated rectus femoris muscle atrophy experimentally and investigated the effect of pulsed electromagnetic field (PEMF) application on the atrophy development through muscle mass, maximal contraction force, and contraction–relaxation time. A quadriceps tendon rupture with a total tenotomy was created on the rats’ hind limbs, inhibiting knee extension for 6 weeks, and this restriction of the movement led to the development of disuse atrophy, while the control group underwent no surgery. The operated and control groups were divided into subgroups according to PEMF application (1.5 mT for 45 days) or no PEMF. All groups were sacrificed after 6 weeks and had their entire rectus femoris removed. To measure the contraction force, the muscles were placed in an organ bath connected to a transducer. As a result of the atrophy, muscle mass and strength were reduced in the operated group, while no muscle mass loss was observed in the operated PEMF group. Furthermore, measurements of single, incomplete and full tetanic contraction force and contraction time (CT) did not change significantly in the operated group that received the PEMF application. The PEMF application prevented atrophy resulting from 6 weeks of immobility, according to the contraction parameters. The effects of PEMF on contraction force and CT provide a basis for further studies in which PEMF is investigated as a noninvasive therapy for disuse atrophy development.

Network-Specific Synchronization of Electrical Slow-Wave Oscillations Regulates Sleep Drive in Drosophila

Article

Full-text available

Oct 2019
CURR BIOL

Slow-wave rhythms characteristic of deep sleep oscillate in the delta band (0.5-4 Hz) and can be found across various brain regions in vertebrates. Across phyla, however, an understanding of the mechanisms underlying oscillations and how these link to behavior remains limited. Here, we discover compound delta oscillations in the sleep-regulating R5 network of Drosophila. We find that the power of these slow-wave oscillations increases with sleep need and is subject to diurnal variation. Optical multi-unit voltage recordings reveal that single R5 neurons get synchronized by activating circadian input pathways. We show that this synchronization depends on NMDA receptor (NMDAR) coincidence detector function, and that an interplay of cholinergic and glutamatergic inputs regulates oscillatory frequency. Genetically targeting the coincidence detector function of NMDARs in R5, and thus the uncovered mechanism underlying synchronization, abolished network-specific compound slow-wave oscillations. It also disrupted sleep and facilitated light-induced wakening, establishing a role for slow-wave oscillations in regulating sleep and sensory gating. We therefore propose that the synchronization-based increase in oscillatory power likely represents an evolutionarily conserved, potentially "optimal," strategy for constructing sleep-regulating sensory gates.

Neonatal Clonazepam Administration Induces Long-Lasting Changes in Glutamate Receptors

Article

Full-text available

Oct 2018

γ-aminobutyric acid (GABA) pathways play an important role in neuronal circuitry formation during early postnatal development. Our previous studies revealed an increased risk for adverse neurodevelopmental consequences in animals exposed to benzodiazepines, which enhance GABA inhibition via GABAA receptors. We reported that administration of the benzodiazepine clonazepam (CZP) during postnatal days 7–11 resulted in permanent behavioral alterations. However, the mechanisms underlying these changes are unknown. We hypothesized that early CZP exposure modifies development of glutamatergic receptors and their composition due to the tight developmental link between GABAergic functions and maturation of glutamatergic signaling. These changes may alter excitatory synapses, as well as neuronal connectivity and function of the neural network. We used quantitative real-time PCR and quantitative autoradiography to examine changes in NMDA and AMPA receptor composition and binding in response to CZP (1 mg/kg/day) administration for five consecutive days, beginning on P7. Brains were collected 48 h, 1 week, or 60 days after treatment cessation, and mRNA subunit expression was assessed in the hippocampus and sensorimotor cortex. A separate group of animals was used to determine binding to NMDA in different brain regions. Patterns of CZP-induced alterations in subunit mRNA expression were dependent on brain structure, interval after CZP cessation, and receptor subunit type. In the hippocampus, upregulation of GluN1, GluN3, and GluR2 subunit mRNA was observed at the 48-h interval, and GluN2A and GluR1 mRNA expression levels were higher 1 week after CZP cessation compared to controls, while GluN2B was downregulated. CZP exposure increased GluN3 and GluR2 subunit mRNA expression levels in the sensorimotor cortex 48 h after treatment cessation. GluA3 was higher 1 week after the CZP exposure, and GluN2A and GluA4 mRNA were significantly upregulated 2 months later. Expression of other subunits was not significantly different from that of the controls. NMDA receptor binding increased 1 week after the end of exposure in most hippocampal and cortical areas, including the sensorimotor cortex at the 48-h interval. CZP exposure decreased NMDA receptor binding in most evaluated hippocampal and cortical areas 2 months after the end of administration. Overall, early CZP exposure likely results in long-term glutamatergic receptor modulation that may affect synaptic development and function, potentially causing behavioral impairment.

Human erythrocytes release ATP by a novel pathway involving VDAC oligomerization independent of pannexin-1

Article

Full-text available

Jul 2018

We previously demonstrated that the translocase protein TSPO2 together with the voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT) were involved in a membrane transport complex in human red blood cells (RBCs). Because VDAC was proposed as a channel mediating ATP release in RBCs, we used TSPO ligands together with VDAC and ANT inhibitors to test this hypothesis. ATP release was activated by TSPO ligands, and blocked by inhibitors of VDAC and ANT, while it was insensitive to pannexin-1 blockers. TSPO ligand increased extracellular ATP (ATPe) concentration by 24-59% over the basal values, displaying an acute increase in [ATPe] to a maximal value, which remained constant thereafter. ATPe kinetics were compatible with VDAC mediating a fast but transient ATP efflux. ATP release was strongly inhibited by PKC and PKA inhibitors as well as by depleting intracellular cAMP or extracellular Ca2+, suggesting a mechanism involving protein kinases. TSPO ligands favoured VDAC polymerization yielding significantly higher densities of oligomeric bands than in unstimulated cells. Polymerization was partially inhibited by decreasing Ca2+ and cAMP contents. The present results show that TSPO ligands induce polymerization of VDAC, coupled to activation of ATP release by a supramolecular complex involving VDAC, TSPO2 and ANT.

Voltage-Activated Ion Channels in Non-excitable Cells—A Viewpoint Regarding Their Physiological Justification

Article

Full-text available

Apr 2018

An updated view of translocator protein (TSPO)

Article

Full-text available

Dec 2017
INT J MOL SCI

Decades of study on the role of mitochondria in living cells have evidenced the importance of the 18 kDa mitochondrial translocator protein (TSPO), first discovered in the 1977 as an alternative binding site for the benzodiazepine diazepam in the kidneys. This protein participates in a variety of cellular functions, including cholesterol transport, steroid hormone synthesis, mitochondrial respiration, permeability transition pore opening, apoptosis, and cell proliferation. Thus, TSPO has become an extremely attractive subcellular target for the early detection of disease states that involve the overexpression of this protein and the selective mitochondrial drug delivery. This special issue was programmed with the aim of summarizing the latest findings about the role of TSPO in eukaryotic cells and as a potential subcellular target of diagnostics or therapeutics. A total of 9 papers have been accepted for publication in this issue, in particular, 2 reviews and 7 primary data manuscripts, overall describing the main advances in this field.

Red Blood Cell Passage of Small Capillaries Is Associated with Transient Ca-mediated Adaptations

Article

Full-text available

Dec 2017

When red blood cells (RBCs) pass constrictions or small capillaries they need to pass apertures falling well below their own cross section size. We used different means of mechanical stimulations (hypoosmotic swelling, local mechanical stimulation, passing through microfluidic constrictions) to observe cellular responses of human RBCs in terms of intracellular Ca²⁺-signaling by confocal microscopy of Fluo-4 loaded RBCs. We were able to confirm our in vitro results in a mouse dorsal skinfold chamber model showing a transiently increased intracellular Ca²⁺ when RBCs were passing through small capillaries in vivo. Furthermore, we performed the above-mentioned in vitro experiments as well as measurements of RBCs filterability under various pharmacological manipulations (GsMTx-4, TRAM-34) to explore the molecular mechanism of the Ca²⁺-signaling. Based on these experiments we conclude that mechanical stimulation of RBCs activates mechano-sensitive channels most likely Piezo1. This channel activity allows Ca²⁺ to enter the cell, leading to a transient activation of the Gardos-channel associated with K⁺, Cl⁻, and water loss, i.e., with a transient volume adaptation facilitating the passage of the RBCs through the constriction.

Calcium Channels and Calcium-Regulated Channels in Human Red Blood Cells

Chapter

Jan 2020
ADV EXP MED BIOL

Free Calcium (Ca²⁺) is an important and universal signalling entity in all cells, red blood cells included. Although mature mammalian red blood cells are believed to not contain organelles as Ca²⁺ stores such as the endoplasmic reticulum or mitochondria, a 20,000-fold gradient based on a intracellular Ca²⁺ concentration of approximately 60 nM vs. an extracellular concentration of 1.2 mM makes Ca²⁺-permeable channels a major signalling tool of red blood cells. However, the internal Ca²⁺ concentration is tightly controlled, regulated and maintained primarily by the Ca²⁺ pumps PMCA1 and PMCA4. Within the last two decades it became evident that an increased intracellular Ca²⁺ is associated with red blood cell clearance in the spleen and promotes red blood cell aggregability and clot formation. In contrast to this rather uncontrolled deadly Ca²⁺ signals only recently it became evident, that a temporal increase in intracellular Ca²⁺ can also have positive effects such as the modulation of the red blood cells O2 binding properties or even be vital for brief transient cellular volume adaptation when passing constrictions like small capillaries or slits in the spleen. Here we give an overview of Ca²⁺ channels and Ca²⁺-regulated channels in red blood cells, namely the Gárdos channel, the non-selective voltage dependent cation channel, Piezo1, the NMDA receptor, VDAC, TRPC channels, CaV2.1, a Ca²⁺-inhibited channel novel to red blood cells and i.a. relate these channels to the molecular unknown sickle cell disease conductance Psickle. Particular attention is given to correlation of functional measurements with molecular entities as well as the physiological and pathophysiological function of these channels. This view is in constant progress and in particular the understanding of the interaction of several ion channels in a physiological context just started. This includes on the one hand channelopathies, where a mutation of the ion channel is the direct cause of the disease, like Hereditary Xerocytosis and the Gárdos Channelopathy. On the other hand it applies to red blood cell related diseases where an altered channel activity is a secondary effect like in sickle cell disease or thalassemia. Also these secondary effects should receive medical and pharmacologic attention because they can be crucial when it comes to the life-threatening symptoms of the disease.

Red blood cell aggregation

Book

Sep 2011

Red blood cells in humans-and most other mammals-have a tendency to form aggregates with a characteristic face-to-face morphology, similar to a stack of coins. Known as rouleaux, these aggregates are a normally occurring phenomenon and have a major impact on blood rheology. What is the underlying mechanism that produces this pattern? Does this really happen in blood circulation? And do these rouleaux formations have a useful function? The first book to offer a comprehensive review of the subject, Red Blood Cell Aggregation tackles these and other questions related to red blood cell (RBC) aggregates. The book covers basic, clinical, and physiological aspects of this important biophysical phenomenon and integrates these areas with concepts in bioengineering. It brings together state-of-the-art research on the determinants, mechanisms, and measurement and effects of RBC aggregation as well as on variations and comparative aspects. After an introductory overview, the book outlines factors and conditions that affect RBC aggregation. It presents the two hypotheses-the bridging model and the depletion model-that provide potential mechanisms for the adhesive forces that lead to the regular packing of the cells in rouleaux formations. The book also reviews the methods used to quantify RBC aggregation in vitro, focusing on their importance in clinical practice. Chapters discuss the effect of RBC aggregation on the in vitro rheology of blood as well as on tube flow. The book also looks at what happens in the circulation when red blood cells aggregate and examines variations due to physiological and pathophysiological challenges. The concluding chapter explores the formation of red blood cell aggregates in other mammals. Written by leading researchers in the field, this is an invaluable resource for basic science, medical, and clinical researchers; graduate students; and clinicians interested in mammalian red blood cells.

Report of final results regarding brain and heart tumors in Sprague-Dawley rats exposed from prenatal life until natural death to mobile phone radiofrequency field representative of a 1.8 GHz GSM base station environmental emission

Article

Mar 2018

Background: In 2011, IARC classified radiofrequency radiation (RFR) as possible human carcinogen (Group 2B). According to IARC, animals studies, as well as epidemiological ones, showed limited evidence of carcinogenicity. In 2016, the NTP published the first results of its long-term bioassays on near field RFR, reporting increased incidence of malignant glial tumors of the brain and heart Schwannoma in rats exposed to GSM - and CDMA - modulated cell phone RFR. The tumors observed in the NTP study are of the type similar to the ones observed in some epidemiological studies of cell phone users. Objectives: The Ramazzini Institute (RI) performed a life-span carcinogenic study on Sprague-Dawley rats to evaluate the carcinogenic effects of RFR in the situation of far field, reproducing the environmental exposure to RFR generated by 1.8 GHz GSM antenna of the radio base stations of mobile phone. This is the largest long-term study ever performed in rats on the health effects of RFR, including 2448 animals. In this article, we reported the final results regarding brain and heart tumors. Methods: Male and female Sprague-Dawley rats were exposed from prenatal life until natural death to a 1.8 GHz GSM far field of 0, 5, 25, 50 V/m with a whole-body exposure for 19 h/day. Results: A statistically significant increase in the incidence of heart Schwannomas was observed in treated male rats at the highest dose (50 V/m). Furthermore, an increase in the incidence of heart Schwann cells hyperplasia was observed in treated male and female rats at the highest dose (50 V/m), although this was not statistically significant. An increase in the incidence of malignant glial tumors was observed in treated female rats at the highest dose (50 V/m), although not statistically significant. Conclusions: The RI findings on far field exposure to RFR are consistent with and reinforce the results of the NTP study on near field exposure, as both reported an increase in the incidence of tumors of the brain and heart in RFR-exposed Sprague-Dawley rats. These tumors are of the same histotype of those observed in some epidemiological studies on cell phone users. These experimental studies provide sufficient evidence to call for the re-evaluation of IARC conclusions regarding the carcinogenic potential of RFR in humans.

The translocator protein (18 kDa) and its role in neuropsychiatric disorders

Article

Oct 2017
NEUROSCI BIOBEHAV R

Translocator protein (TSPO) is an 18kDa translocator membrane protein expressed in the outer mitochondrial membrane of steroid-synthesizing cells in the central and peripheral nervous systems. TSPO is involved in cellular functions, including the regulation of cell proliferation, transport of cholesterol to the inner mitochondrial membranes of glial cells, regulation of mitochondrial quality control, and haem synthesis. In the brain, TSPO has been extensively used as a biomarker of injury and inflammation. Indeed, TSPO was up-regulated in several inflammatory and neurodegenerative diseases. In contrast, the expression of TSPO was decreased in peripheral blood from psychiatric patients. Since TSPO is involved in several mechanisms related to mitochondrial function and inflammatory alterations, therapeutic approaches focusing on the regulation of TSPO may provide a new avenue for the treatment of neuropsychiatric disorders. Based on the involvement of mitochondrial alterations in the neurobiology of neuropsychiatric disorders, this review will focus on the functions and physiological roles of TSPO and the potential of TSPO ligands as therapeutic strategies for the treatment of neuropsychiatric disorders.

Electromagnetic Fields and Calcium Signaling by the Voltage Dependent Anion Channel

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