Chapter 2: How Each Such EMF Effect Is Produced via Voltage-Gated Calcium Channel Activation: Role of the Voltage Sensor in Producing the Extraordinary Sensitivity to EMF Effects

Chapter 2: How Each Such EMF Effect Is Produced via Voltage-Gated Calcium Channel Activation: Role of the Voltage Sensor in Producing the Extraordinary Sensitivity to EMF Effects 

The Pall, 2013 [4] study showed that in 24 different studies (there are now a total of 26 [5]), effects of low-intensity EMFs, both microwave frequency

and also lower frequency EMFs, could be blocked by calcium channel blockers, drugs that are specific for blocking voltage-gated calcium channels (VGCCs). There were 5 different types of calcium channel blockers used in these studies each thought to be highly specific, each structurally distinct and each binding to a different site on the VGCCs. In studies where multiple effects were studied, all studied effects were blocked or greatly lowered by calcium channel blockers. These studies show that EMFs produce diverse non-thermal effects via VGCC activation in many human and animal cells and even in plant cells where some similar calcium channels are involved [6]. Furthermore, many different effects shown to be produced in repeated studies by EMF exposures, including the effects discussed above, can each be produced by downstream effects of VGCC activation, via increased intracellular calcium [Ca2+]i, as discussed below. 

Various EMFs act via VGCC activation, as shown by calcium channel blocker studies. These include microwave frequency EMFs, nanosecond pulse EMFs, intermediate frequency EMFs, extremely low frequency EMFs and even static electrical fields and static magnetic fields. 

It is important to discuss why the VGCCs are so sensitive to activation by these low-intensity EMFs. Each of the VGCCs have a voltage sensor which is made up of 4 alpha helixes, each designated as an S4 helix, in the plasma membrane. Each of these S4 helixes has 5 positive charges on it, for a total of 20 positive charges making up the VGCC voltage sensor [5,8]. Each of these charges is within the lipid bilayer part of the plasma membrane. The electrical forces on the voltage sensor are extraordinarily high for three distinct reasons [5,8]. 1. The 20 charges on the voltage sensor make the forces on voltage sensor 20 times higher than the forces on a single charge. 2. Because these charges are within the lipid bilayer section of the membrane where the dielectric constant is about 1/120th of the dielectric constant of the aqueous parts of the cell, the law of physics called Coulomb’s law, predicts that the forces will be approximately 120 times higher than the forces on charges in the aqueous parts of the cell. 3. Because the plasma membrane has a high electrical resistance whereas the aqueous parts of the cell are highly

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conductive, the electrical gradient across the plasma membrane is estimated to be concentrated about 3000-fold. The combination of these factors means that comparing the forces on the voltage sensor with the forces on singly charged groups in the aqueous parts of the cell, the forces on the voltage sensor are approximately 20 X 120 X 3000 = 7.2 million times higher [5,8]. The physics predicts, therefore, extraordinarily strong forces activating the VGCCs via the voltage sensor. It follows that the biology tells us that the VGCCs are the main target of the EMFs and the physics tells us why they are the main target. Thus the physics and biology are pointing in exactly the same direction. 

We have, then, very strong arguments that the EMFs act directly on the voltage-sensor to activate the VGCCs. There are several other types of evidence, each providing important evidence supporting this view: 

1. In a study published by Pilla [12], it was found that pulsed EMFs produced an “instantaneous” increase in calcium/calmodulin-dependent nitric oxide synthesis in cells in culture. What this study [12] showed was that following EMF exposure, the cells in culture, must have produced a large increase in [Ca2+]i, this in turn produced a large increase in nitric oxide synthesis, the nitric oxide diffused out of the cells and out of the aqueous medium above the cells into the gas phase, where the nitric oxide was detected by a nitric oxide electrode. This entire sequence occurred in less than 5 seconds. This eliminates almost any conceivable indirect effect, except possibly via plasma membrane depolarization. Therefore, it is likely that the pulsed EMFs are acting directly on the voltage sensors of the VGCCs and possibly the voltage-gated sodium channels, to produce the [Ca2+]i increase. 

2. There are also additional findings pointing to the voltage sensor as the direct target of the EMFs. In addition to the VGCCs, there are also voltage-gated sodium, potassium and chloride channels, with each of these having a voltage sensor similar to those found in the VGCCs. Lu et al [13] reported that voltage gated sodium channels, in addition to the VGCCs were activated by EMFs. Tabor et al [14] found that Mauthner cells, specialized neurons with special roles in triggering rapid escape mechanisms in fish, were almost instantaneously activated by electrical pulses, which acted via voltage-gated sodium channel activation to subsequently produce large [Ca2+]i increases. Zhang et al [15] reported that in addition to the VGCCs, potassium and chloride channels were each activated by EMFs, although these other voltage-gated ion channels had relatively modest roles, compared with the VGCCs, in producing biological effects. Each of these three studies [13-15] used specific blockers for these other voltage-gated ion channels to determine their roles. The Tabor et al [14] study also used genetic probing to determine the role of the voltage-gated sodium channels. Lu et al [13] also used whole cell patch clamp measurements to measure the rapid influx of both sodium and calcium into the cell via the voltage-gated channels following EMF exposure. Sodium influx, particularly in electrically active cells, acts in the normal physiology to depolarize the plasma membrane, leading to VGCC activation such that the voltage-gated sodium channels may act primarily via indirect activation of the VGCCs. In summary then, we have evidence that in animal including human cells, seven distinct classes of voltage-gated ion channels are each activated by EMF exposures: From Ref. [4], four classes of voltage-gated ion channels were shown from calcium channel blocker studies, to be activated by EMFs, L-type, T-type, N-type and P/Q –type VGCCs. In this paragraph we have evidence that three other channels are also activated, voltage-gated sodium channels, voltage-gated potassium channels and voltage-gated chloride channels. Furthermore the plant studies strongly suggest that the so called TPC channels, which contain a similar voltage sensor, are activated in plants allowing calcium influx into plants to produce similar EMF-induced responses [6]. In summary, then we have evidence for eight different ion channels being activated by EMF exposure, four classes of VGCCs, one class each of voltage-gated sodium,

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potassium and chloride channels and also one class of plant channel, with each of these channels having a similar voltage-sensor regulating its opening. One can put those observations together with the powerful findings from the physics, that the electrical forces on the voltage-sensor are stunningly strong, something like 7.2 million times stronger than the forces on the singly charged groups in the aqueous phases of the cell. Now you have a stunningly powerful argument that the voltage sensor is the predominant direct target of the EMFs. 

3. The most important study on this subject, was published by Tekieh et al [16]. It showed that microwave frequency EMFs directly activate the VGCCs in isolated membranes. A variety of microwave frequencies were used in these studies and each such frequency produced VGCC activation in a completely cell-free system. This study clearly shows that the EMF activation of the VGCCs is direct and not due to some indirect regulatory effect. 

How then does the estimated sensitivity of the voltage-sensor, about 7.2 million times greater forces than the forces on singly charged groups, compare with previous estimates of levels of EMF exposure needed to produce biological effects? The ICNIRP 2009 [17] safety guidelines allowed for 2 to 10 W/m2 exposure, depending upon frequency. In contrast, the Bioinitiative Working Group 2007 [18] proposed a precautionary target level of 3 to 6 ìW/m2 or about a million-fold lower, using a safety factor of 10. If one uses a more commonly used safety factor of 50 to 100, then the 7.2 million-fold sensitivity of the voltage-sensor, predicted by the physics, falls right in the middle of the Bioinitiative Working Group 2007 calculations. So again, it can be argued that the physics and the biology are pointing in the same direction, in this case pointing to the same approximate range of sensitivity. 

You may be wondering why I am spending so much time and space going through each of these studies. The answer is that a well over a trillion dollar (or trillion euro) set of industries, the telecommunications industry, has been putting out propaganda for over two decades, arguing that there cannot be a mechanism of action of these non-thermal EMFs to produce biological effects; and that these EMFs are too weak to do anything and that only thermal effects are documented. It is essential to dot every i and cross every t with regard to the main mechanism of action of non- thermal effects. That is exactly what has been done here. 

How Can the Diverse Effects of Such EMF Exposures Be Produced by VGCC Activation?

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1212

Cytochrome mitochondrial energy metabolism, steroid hormone synthesis

Microwave/Lower VGCC

[Ca2+]i Nitric
activation Oxide (NO) NO

protein Nrf2 kinase G
Therapeutic effects 

The mechanisms by which various effects can be generated by VGCC activation are outlined in Fig. 1. Going across the top of Fig. 1, it can be seen that increased intracellular calcium [Ca2+]i can increase nitric oxide (NO) synthesis, stimulating the NO signaling pathway (going to the right from top, center), to produce therapeutic effects. NO (very top) can also bind to cytochromes and inhibit their activity. NO binding to the terminal oxidase in the mitochondria inhibits energy metabolism and lowers, therefore, ATP. NO binding to cytochrome P450s, lowers synthesis of steroid hormones, including estrogen, progesterone and testosterone. The P450 lowering also lowers detoxification and vitamin D activity. Most of the pathophysiological effects are produced by the peroxynitrite/free radical/oxidative stress pathway center to lower right (Fig. 1) and also by excessive calcium signaling pathway (slightly left of center, Fig. 1). Some of the ways these are thought to produce various well-established EMF effects are outlined in Table 1. 

Table 1. How Eight Established Effects of EMFs Can Be Produced by VGCC Activation

Frequency EMFs

signaling (cGMP)

Calcium signaling

Super- oxide
Peroxynitrite +/-CO2 (ONOO-)

+
Free
radicals

O

Oxidative stress

Fig. 1 How EMFs Act via VGCC Activation to Produce Various Effects

Pathophysiological effects

NF-kappaB Inflammation

EMF effect	Probable mechanism(s)
Oxidative stress	Produced by elevated levels of peroxynitrite and the free radical breakdown products of peroxynitrite and its CO2 adduct. Four studies of EMF exposure, cited in [4] showed that oxidative stress following exposure was associated with major elevation of 3- nitrotyrosine, a marker of peroxynitrite, thus confirming this interpretation. Two other studies each found 3-nitrotyrosine elevation, both following 35 GHz exposures [19,20].
Lowered male/female fertility, elevated spontaneous abortion, lowered libido	Both the lowered male fertility and lowered female fertility are associated with and presumably caused by the oxidative stress in the male and female reproductive organs. Spontaneous abortion is often caused by chromosomal mutations, so the germ line mutations may have a causal role. Lowered libido may be caused by lowered estrogen, progesterone and testosterone levels. It seems likely that

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	these explanations may be oversimplified. One additional mechanism that may be important in producing lowered fertility is that VGCC activation and consequent high [Ca2+]i levels is known to have a key role in avoiding polyspermy. Consequently, if this response is triggered before any fertilization of an egg has occurred, it may prevent any sperm from fertilizing and egg.
Neurological/ neuropsychiatric effects	Of all cells in the body, the neurons have the highest densities of VGCCs, due in part to the VGCC role and [Ca2+]i role in the release of every neurotransmitter in the nervous system. Calcium signaling regulates synaptic structure and function in 5 different ways, each likely to be involved here. Oxidative stress and apoptosis are both thought to have important roles. Lowered sleep and increased fatigue are likely to involve lowered nocturnal melatonin and increased nocturnal norepinephrine.
Apoptosis	Apoptosis can be produced by excessive Ca2+ levels in the mitochondria and by double strand breaks in cellular DNA; it seems likely that both of these mechanisms are involved following EMF exposure. A third mechanism for triggering apopotosis, endoplasmic reticulum stress (see bottom row in this Table), may also be involved.
Cellular DNA damage	Cellular DNA damage is produced by the free radical breakdown products of peroxynitrite directly attacking the DNA [7].
Changes in non-steroid hormone levels	The release of non-steroid hormones is produced by VGCC activation and [Ca2+]i elevation. The immediate effects of EMF exposures is to increase hormone release and to raise, therefore, hormone levels. However many hormone systems become “exhausted” as a consequence of chronic EMF exposures. The mechanism of exhaustion is still uncertain, but it may involve oxidative stress and inflammation.
Lowered steroid hormone	Steroid hormones are synthesized through the action of cytochrome P450 enzymes; activity of these hormones is inhibited by binding of high levels of nitric oxide (NO) leading to lowered hormone synthesis.
Calcium overload	Produced by excessive activity of the VGCCs; secondary calcium overload is produced by oxidative stress activation of TRPV1, TRPM2 and possibly some other TRP receptors, opening the calcium channel of these receptors.
Heat shock protein induction	There is a large literature showing that excessive [Ca2+]i induces very large increases in heat shock proteins. This is thought to be produced by complex calcium signaling changes involving the endoplasmic reticulum, mitochondria and the cytosol and also involving excessive [Ca2+]i producing increasing protein misfolding [21-23]. It should be noted that some calcium is essential for proper protein folding in the endoplasmic reticulum such that only excessive calcium leads to misfolding and consequent endoplasmic reticulum stress.

Each of the seven established EMF effects, discussed above, can be generated through the mechanisms outlined in Fig. 1, as shown by Table 1. An eighth, heat shock protein induction can also be so explained (Table 1). Several other such effects, including EMF causation of

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cataracts, breakdown of the blood-brain barrier, lowered nocturnal melatonin are also so explained, as discussed earlier [5]. The primary mechanism for therapeutic effects was discussed in [4,24,25] and was also shown to be generated via such VGCC downstream effects. Fifteen mechanisms for EMF cancer causation are described in ref [7]; these are far too complex to describe in this document so the reader is referred to ref [7]. 

It can be seen, in summary, that we are far beyond the issue whether there are non-thermal EMF effects. Rather many researchers have identified many established effects of EMF exposure. The main direct targets of non-thermal EMF exposure, the VGCCs have also been identified and how these get activated by EMF exposure acting on the VGCC voltage-sensor has also been determined. And finally we have identified how a wide variety of these effects can be generated via downstream effects produced by such VGCC activation. 

Our current safety guidelines are based only on heating (thermal) effects. Heating is produced predominantly by forces on singly charged groups in the aqueous phases of the cell but the forces on the voltage sensor are approximately 7.2 million times higher. Therefore, our current safety guidelines are allowing us to be exposed to EMFs that are approximately 7.2 million times too strong. That 7.2 million figure is somewhat similar to the estimate given by the Bioinitiative Report and by the Building Biologists, based on completely different considerations. 

It should be obvious, that non-thermal EMFs:

1.    Attack our nervous systems including our brains leading to widespread neuropsychiatric
effects and possibly many other effects. This nervous system attack is of great concern.

2.    Attack our endocrine (that is hormonal) systems. In this context, the main things that
make us functionally different from single celled creatures are our nervous system and our endocrine systems – even a simple planaria worm needs both of these. Thus the consequences of the disruption of these two regulatory systems is immense, such that it is a travesty to ignore these findings.

3.    Produce oxidative stress and free radical damage, which have central roles in all common chronic diseases.

4.    Attack the DNA of our cells, producing single strand and double strand breaks in cellular DNA and oxidized bases in our cellular DNA. These in turn produce both cancer and mutations in germ line cells with germ line mutations producing mutations impacting future generations.

5.    Produce elevated levels of apoptosis (programmed cell death), events especially important in causing both neurodegenerative diseases and infertility.

6.    Lower male and female fertility, lowered sex hormones, lowered libido, increased levels of spontaneous abortion and, as already stated, attacks on the DNA in sperm cells.

7.    Produce excessive intracellular calcium [Ca2+]i and increased calcium signaling.

8.    Act in the cells of our bodies via 15 different mechanisms to cause cancer.

By attacking all of these important systems in the body, EMFs attack everything we care about including our health (in many ways), our reproductive systems, the integrity of our genomes and our ability to produce healthy offspring.

There are 79 different reviews listed at the end of Chapter 1, with each documenting the existence of one or more of these various non-thermal EMF effects. What, then, do the two organization reports that the EU authorities and U.S. authorities rely upon, ICNIRP and SCENIHR 2015, have to say about these independent reviews. The answer is absolutely nothing! Neither one of them

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uses any of these independent reviews to assess EMF effects. This whole area is discussed in much more detail in Chapter 5, below.

Chapter 3. Strong Evidence for Cumulative and Irreversible EMF Effects