bioelectric signals and membrane ion channels

Posted comment on ´Bioelectrical Signals and Ion Channels in the Modelling of Multicellular Patterns and Cancer Biophysics` by J. Cervera, A. Alcaraz and S. Mafe published online in Nature Science Reports February 4th 2016 6:20403 doi 10.1038/srep20403

SUMMARY

Cervera, Alcaraz and Mafe state in their article that bioelectrical signals and ion channels are central to spatial patterning in cell assemblies (ensembles) and are therefore, important in determining cell positioning and the organisation of multicellular groups. Defects or deficiencies of the bioelectric signal are also implicated in cancer. The research group proposed a model or an approach to a bioelectrical network of non-neural cells where the biochemical coupling of the assembly is brought about by the action of an ion channel blocker. Their approach was based on three concepts: that a cell`s electrical state was characterised by the membrane potential of that cell which was regulated by voltage gated channels that had depolarising and hyperpolarising capabilities; that the long-range electrical coupling between neighbouring cells of multicellular assemblies was brought about by gap junctions; and that the electrical state of the whole multicellular assembly could be changed by the administration of a biochemical agent locally in the cell`s microenvironment and this could alter the conductivity potential of the membrane`s ion channels. Using their model, Cervera, Alcaraz and Mafe investigated the electrical effects of small neuronal assemblies in spatial patterning, the role of ion channels in cancer biophysics and the distribution of charged nanoparticles over neuronal assemblies. They found that spatial patterns arising from their model are characterised by a map of cell potentials which are ultimately regulated by the voltage gated channels on each cell. The spatiotemporal patterns could normalise areas where there were abnormal cell electrical states ie. from the administration of charged nanoparticles and these findings indicated that bioelectric signals of multicellular groupings provide a new insight into the biophysics of cancer.

Cervera, Alcaraz and Mafe began their article by describing the connectivity between cells of multicellular groupings as not just biochemical in nature, but also bioelectrical, a fact they said is often disregarded. They cited examples of where tissue morphology and morphostatic fields are important in building and maintaining cell group architecture and where cancer results from the destruction of this architecture. Cancer cells have been observed to have an overexpression of specific ion channels (ie. an up-regulation of sodium ion channels and a down-regulation of potassium ion channels) and so tend to exhibit sustained depolarisation (ie. have a low membrane potential in absolute value). According to Cervera, Alcaraz and Mafe, depolarisation of the cell leads to a significant level of spatial distribution of negatively charged lipids and a significant effect on their interactions with positively charged proteins which may lead to the activation of intracellular biochemical pathways that result in cell proliferation.

The authors continued in their article with a description of the cell electrical state. They said that the electrical state of the cell is described by the membrane potential (Vmem) being less than zero and this is defined as the potential difference between the cell cytoplasm and the extracellular microenvironment under zero current conditions. This potential difference regulates the entry of sodium, potassium, calcium ions and other biologically-relevant molecules into the cell. It is a dynamic system transitioning between low (depolarized, abnormal) and high (hyperpolarized, normal) electrical states. Cervera, Alcaraz and Mafe said that the cells of their model exhibit bioelectric bistability (observed with for example neural cells) and others have demonstrated it also in their experiments. Hence, there are 3 values for Vmem: one for the hyperpolarised state, one for the depolarised state and one for the intermediate unstable transition state. Transitions are induced by modifying the equilibrium potential (Ein) which is dependent on ionic concentrations within the cell. It may also be associated with changes in biological parameters such as the pH and the ionic concentrations of the salt solution regulating conductance ratio (Gout/Gin). Other ion channels aside from those of sodium and potassium are also likely to be involved.

Cervera, Alcaraz and Mafe went on in their article to describe the case of cancer cells with relation to membrane potential and conductance ratios. In the case of cancer, anomalous inward-rectifying potassium channels were found in tumour cell lines giving values of Vmem different to those found in normal cells. It was also hypothesised that voltage-gated sodium channels are associated with depolarized values for Vmem in cancer cells. Abnormally low absolute values of Vmem correspond to plastic (not yet differentiated) cells while high absolute values of Vmem are found in terminally differentiated cells. Therefore, it was concluded that there is in the case of cancer cells simultaneous up-regulation of sodium ion channels (and hence, increased sodium ion inward currents) and down-regulation of potassium ion channels (leading to a decreased potassium ion outward current) giving a conductance ratio for Gout to Gin greater than 1. This implies that the membrane potential Vmem decouples from the normal hyperpolarized value Ein, bistability and cell depolarization regime. Therefore, depolarization is seen as a characteristic of abnormal cells that promotes the initiation of biochemical signal cascades. The addition to the external microenvironment of an ion channel blocker which would act on the outward rectifying channel in these circumstances could then decrease the channel conductance ratio Gout to Gin to less than 1 and have an effect on the existing bioelectrical state of the cell.

The authors then went on to extend their model (using long-range electrical coupling simulated by effective conductance ratios (G) and capacitances (C) arranged in parallel) to describe electrical states within multicellular groupings. This topic was of interest to the authors since abnormal tissues appear to have defective intercellular communication. Using their model, Cervera, Alcaraz and Mafe described the bioelectrical states of the cells in terms of protein channels acting as gap junctions between neighbouring cells of the cellular assembly. External ionic concentrations were ignored in the model with potential changes by the cell only occurring because of the gap junctions present.

Having described the role of non-functional junctions and defective intercellular communication in uncontrolled growth regulation, Cervera, Alcaraz and Mafe went on to report on the anticipated normalisation of small regions of the cell`s membrane of abnormal, depolarised cells brought about by modulation of the ion channels present. This would have the potential for restoring long-range gap junctions occurring in cell patterning and result in the removal of the conditions associated with tumorigenesis. The authors hypothesised that it would be possible to do this if the conductance ratios of coupling cells were high enough. The non-uniform distribution of gap junctions was suggested as allowing the coexistence of spatial regions of cells having increased intercellular communication (ie. having high G values – strong coupling and hyperpolarised state) acting as an electrical buffer with other cellular regions having decreased communication (low G values – depolarised state and abnormal). However, this was suggested as not being possible if there were a high number of abnormal cells within the multicellular assembly. Another reason given for the unlikelihood of this scenario was that low values of G produce cell isolation with inhibitory electrical signals characteristic of hyperpolarised potential neighbouring cells. This could be modulated however and reversed by the administration of particular agents in the external cell microenvironment. The authors did suggest that this was unlikely in this scenario because of the slow speed of diffusion of the agent and the biochemical reactions that may result.

A second scenario discussed in the article was the effect of the upregulation of the ion channels which leads to abnormal cell functioning and stimulates uncontrolled proliferation. In some cases, this causes persistent depolarisation and modifies the spatial distribution of negatively charged lipids in the cell membrane leading to the clustering of signalling proteins with positive residues around them. This can result in the initiation of biochemical pathways promoting cell proliferation. Cervera, Alcaraz and Mafe gave the example of upregulation of the outward rectifying channel by increasing conductance Gout to Gin thus promoting depolarisation. They said that this condition could not be normalised, but instead the region could expand and invade the normal cell region. Blocking of the outward rectifying channels by the administration of an external ion channel blocker would have the opposite effect with the conductance ratio Gout to Gin decreasing locally. The authors described this effect as immediate and coupling between the cells means that the effect on a single cell would spread to the multicellular assembly as a whole. Therefore, there would be a gradual transition from the depolarised abnormal state to the normal hyperpolarised cell state. This scenario would, according to the authors, require specific blockers for specific ion channels. If instead channels associated with membrane hyperpolarisation were blocked then the depolarised area would be extended and the authors gave as an example the situation in CHO and HeLa cells where the blocking of the potassium ion channels that are responsible for maintaining the membrane potential by cation nanoparticles would lead to significant cell depolarization.

Cervera, Alcaraz and Mafe then discussed in their article the association between bioelectric cell status and cellular patterning. They said that the coordinated action of external effects along defined spatial directions could produce cellular patterning providing it was a fast channel blocking action and the spatial map of the electric potentials closely followed the concentration of the blocking agent. In the example they gave in their article, the patterning required no anisotropic electrical coupling between the neighbouring cells and so they stated that the positional information from the individual cell could be determined by its electrical potential value plus individual cell properties such as its ion channels and the charged lipids and proteins in the cell membrane. The multicellular assembly was regarded as a bioelectrically coupled network of cells supported by intercellular gap junctions allowing the transmission of bioelectric signals. Interactions among the cells were crucial for positional information in addition to the presence of specific morphogens.

This led the authors to discuss the distribution of charged nanoparticles on multicellular assemblies. With the number of particles scaled to an exponential function of local electrical potential, they found that positive nanoparticles concentrated around negatively charged cells. Therefore, the spatial map of potentials would in their opinion influence the local uptake of charged nanoparticles over the tissue as a whole. They also discovered that the binding of the nanoparticles could also disrupt the cell membrane potential leading to modified intercellular communication. This indicated to them that the administration of nanoparticles would be a method for altering maps of potentials for multicellular groups by external means.

Cervera, Alcaraz and Mafe concluded their article by summarising the importance of bioelectrical signals in cancer. They suggested that the cancer cell`s microenvironment may show long-range bioelectrical signals and gap junctional cell insulation which may be involved in tumorigenesis and that the membrane depolarization caused may trigger gene transcriptional changes that regulate morphogen transport. Changes in single cell membrane potential and polarization states can occur when specific ion channels are up-regulated, down-regulated, or physically blocked. These modulations change the activity of those particular ion channels and possibly lead on to a promotion of depolarisation and normalisation. However, modification of the bioelectric state can also be brought about by modifying the gap junction conductance ratio by shifting the balance between the outward- and inward-rectifying channels. This can be achieved by blocking specific ion channels. Although the role of ion channels in cancer biophysics is already known, the authors reminded their readers that studying them in vivo in humans with ion channel blocking agents has problems since for example, these agents can cause cardiac arrhythmias. The authors also concluded that the bioelectrical characteristics associated with the spatial distribution of external morphogens over the multicellular assembly can be of significance for cellular patterning. The map of bioelectric potentials resulting locally from cell coupling regulates the experimental uptake of charged nanoparticles over the tissues themselves. These nanoparticles can disrupt the cell membrane potential and modify intercellular communication which is suspected of having a role in the biophysics of cancer.

COMMENT

What makes this article interesting is that it links together cell outer membrane potential (transmembrane potential, or Vmem), bioelectrical cell status and cell connectivity within multicellular groupings and describes dysfunctioning of these mechanisms in terms of ´limiting causes` for problems of cell proliferation, cell organisation and patterning and possibly to the disease, cancer.  Cervera, Alcaraz and Mafe stated that what they called the bioelectric status and bioelectric signaling of the cell are often disregarded in research on diseases with emphasis usually on physiology and biochemical systems reactions. This is probably not completely correct since the source of the topic being considered, that of the change in electric potential of cells through membrane channel functioning, is an enormously important topic in neurochemistry and forms the basis of neuronal firing mechanisms, neuronal firing patterns and cell connectivity and these are associated with cognitive functioning and mental health. Although this type of research may be centered on brain tissue, bioelectric signals or the movement of charged particles whether simple ions, charged molecules, protons or electrons underpin a huge number of biochemical reactions in the whole body and are always in consideration when looking at cell functioning.

That said we must consider what the authors are saying in their article. They concluded from their simplified model of bioelectric signals relating to outer cell membranes that the bioelectric status of the cell in question comes about by the presence and functioning of cell membrane ion channels. Through an imbalance of concentration of charge across a membrane due to their action then a voltage difference across the membrane would exist (just like that seen in neurons) called the transmembrane potential (TMP). The passage of ions through these ion channels, according to Cervera, Alcaraz and Mafe, could lead to cellular depolarisation (inward flow of sodium ions and up-regulation of channels leading to abnormal cell functioning) or hyperpolarisation (outward flow of potassium ions and down-regulation of channels leading to normalisation of function). The authors also concluded that connectivity between multiple cells within a group could be achieved by shared bioelectric signals through gap junctions that require functioning ion channels between neighbouring cells and this connectivity could lead to cell positioning and organisation within the cell`s grouping. In addition, another conclusion made was a logical extension of the two and that was that modulation of ion channels by a number of different measures (eg. administration of ion channel blockers, surface distribution of externally administered nanoparticles) could lead to changes in individual cell functioning and multicellular group functioning some of which could be detrimental to the organism, for example in the case of cancer.

Cervera and colleagues first conclusion ie. that the bioelectric status of the cell relies on cell membrane ion channels and the passage of ions through them results in the electrical potential of the cell (designated Vmem) or the voltage of the cell, is supported by neurochemical studies and is well-known for neurons and neuronal firing. It is also known in the case of non-neural cells in the brain, eg. glial cells where neurotransmitter binding can cause the opening and closing of associated ion channels. The authors link their ion channel functioning to the electrical status of the cell in non-neural cells and formulated a two-channel model. One type of channel was the voltage gated sodium ion channel which was associated with the inward flow of the ion and subsequent depolarisation of the cell. An upregulation of this type of ion channel and resulting persistent depolarisation was proposed by the authors and others to be linked to cell dysfunction observed in cancer. The other type of channel was a voltage gated potassium ion channel which was associated with the outward flow of potassium ions and hyperpolarisation leading to the ´normalisation` of cell functioning in the case of, according to the authors, cancer cells. If we look at these conclusions, we can say that there is support for the ´limiting cause` of cancer being at the level of the membrane since essentially if there is going to be a root cause then the cell outer membrane is the logical first step, the first encounter, the protector of cell functioning from external influences. The ion channels also span from outside to inside (or vice versa) of the cell and therefore, provide a route between external causal event and internal effect (or vice versa). Describing the effect as a signal is also supported since the mechanism relies on the transfer of charged particles eg. electrons from one side of the cell to another thus promoting a change in the voltage across the membrane and this acts as the message/signal being transmitted from external sources to internal mechanisms (or vice versa). It should be noted however, that according to Levin and colleagues the Vmem of non neural cells is itself important and not how it is achieved, ie. ion transfer must occur but not linked to any type of channel in particular. This is not the case of neural cells where specific cell surface receptors and channels are involved in signal transfer. However, the idea that the voltage difference across a membrane is a signal is further supported by evolutionary evidence linking change in bioelectrical status with cell movement as with amoeba, cell functioning as with paramecium, regeneration as with frogs and salamanders, and voltage differences observed between organs as in the so-called life fields in humans, plus on a smaller scale in correct muscle functioning and bone repair.

However, a change in bioelectric status is only a signal. It may lead to a change in proteins or molecules at the membrane level possibly leading to conformational changes that result in a change in reactions or function of the cell internally. To keep this comment relating to cancer, we should look at what effects this bioelectric signal could have on cell differentiation and proliferation, both systems appearing to be defective/abnormal in cancer. If we take a look at the observations made in life fields research we see that the bioelectric signal is believed to be associated with the health and well-being of an organism and is thought to be associated with growth and repair of an organism. In the case of new bone growth an application of an electric current is reported to lead to new growth around the negative electrode implying that the bioelectric signal was negative on the outside of the cell and positive on the inside. (This is opposite to what is known about the resting potential of the neuron which has a negative charge inside the cell relative to the positive charge on the outside.) The observation was explained by looking at bone stress and the so-called pizoelectric effect where the stressed bone acts as a matrix biphasic semiconductor with positive apatite outside (P) and collagen inside (N negative). With the PN junction and P to N current flow then the current rectifies the charge on the compressed internal side leaving it with a negative signal that stimulates cells to grow to form new bone.

Continual cell proliferation relating to the bioelectric signal and life fields was described in the case of the frog and its ´current of injury` reported with leg amputation and limb regeneration. Early ideas of ´electric currents coming from a wound` (Galvani) developed into ideas about changes in transmembrane potentials across the cell membrane dictating repair. In the frog it was reported that a stream of electrons (negative current) came from the wound from an amputated limb as a result of cell damage. With the transmembrane potential ions leaking out this was said to lead to the reversal of the polarity of the cell to positive. This only occurred on the wound surface and the current of injury was found to be proportional to the amount of nerve present. The change in current was associated with regrowth. In the case of the salamander a similar experiment was reported where a limb was amputated and the wound washed to remove blood clots. An immediate fall of membrane potential from minus 10 to plus 22 was observed. However, after about 3 days there was no membrane potential and no blastema (a mass of undifferentiated cells capable of proliferation and differentiation). After 6-10 days the Vmem rose to negative 30 and a blastema formed. This indicates that an external negative charge promoted cell proliferation. However, the potential was recorded as changing relative to regrowth since the external negative charge dropped to around minus 10 up to 22 days and the limb regenerated over that time. The view that cell proliferation is linked to bioelectric signals was further supported by studies where an external application of an electric current (AC current) or field was applied to tissues or tissue samples and this caused them to divide in a manner similar to cancer.

The implication from the observations of life fields researchers and cancer is that cell differentiation and cell proliferation mechanisms involve in some form bioelectric signals relating to the external membrane (ie. via the transmembrane potential). They may have an indirect effect through influencing gene transcription and the release of factors in response to signal changes. We know that differentiation is guided by transcription factors and morphogens and that differentiation is changed by a bioelectric signal. Therefore, we should ask whether the bioelectric signal could act as a ´transcription factor`. This is unlikely since the conditions arising from the signal are more like a ´state` rather than a specific factor. Transcription factors bring about DNA methylation and modification of the histone structure, but the bioelectric signal instigates changes in molecules to bring about these reactions and therefore, cannot itself be described as a transcription factor, but as a factor that modulates indirectly transcription via another party.  More likely is the consideration of it as a ´morphogen` with the signal arising from its own cell and/or from others and beginning at the level of the outer membrane (ie. differentiation`s  ´limiting cause`). Since a morphogen usually acts by a general mechanism of ligand binding to a receptor (possible bioelectric signal involvement here through conformational changes of the receptor induced by the presence of ions) leading to active ligand-receptor complexes which result in the catalysation of reactions through phosphorylation type reactions (processes requiring proton exchanges and conformational changes of molecules involving ionic charge changes also indicating bioelectric signal involvement). These can lead to the activation of dormant transcription factors (the direct intervention) or cytoskeletal protein changes (indirect intervention via the action of actin and myosin in microfilament and microtubule functioning – also involving the bioelectric signal in their mechanism and direction of movement) that result in changes to the differentiation process. Alternatively, cell differentiation can be affected by matrix elasticity which is also dependent on an actin-based intracellular microstructure reacting to the tugging motion of the cell`s contents during the cell`s division. This acts as a signal and hence, the bioelectric signal is again involved through molecular motor molecules and their reliance on the bioelectric signal for their correct functioning.

Therefore, Cervera, Alcaraz and Mafe`s model that bioelectric signals relating to outer cell membranes may play a role in cell differentiation and dysfunctioning of that system in the form of cancer may be valid. However, although there is support, there are also factors that count against such an easy and plausible cause. We must consider that in the case of neuronal depolarisation, persistent depolarisation arising from only a change in membrane function is unlikely to occur since under normal circumstances there are feedback mechanisms that switch off the mechanism, eg. the refractory period for neuronal cell recovery after sustained firing. Also, nothing is ever simple regarding cell functioning. In neuronal cells there are multiple receptors in play and multiple stages in any mechanism with multiple areas for feedback mechanisms to possibly right any wrong.  For example, it is unlikely that any cell has just two types of membrane bound ion channel and their actions are often linked to the performance of a third (eg. calcium channels with depolarisation or GABA channels with hyperpolarisation) or changes in intracellular conditions eg. pH changes counteract enzymatic mechanisms. Therefore, the bioelectric status of cells is likely to be highly controlled and to override this control there must be continual gene transcription changes and possibly involving many cells. This is seen in the case of cancer cells where transcription changes induce continual division of the cells against normal instructions.

Cervera, Alcaraz and Mafe also made a hypothesis in their article about the bioelectric signal and physical connectivity between multiple cells in a functional grouping. Their view is that connectivity occurs through one type of cell signaling mechanism, that of gap junctions and that disruptions of that connectivity can have catastrophic effects on the cell, the grouping or even the organism itself. Support for connectivity between cells of a group is clear, eg. from neuronal cell assemblies in memory, consciousness (connectivity supported by the observations of  brain wave synchronicity between participating cells), and cancer with connectivity between tumour cells. There is also support for connectivity between long-range parts of a total organism, for example: Planarium and organism polarity; cell positioning and growth alignment (eg. matrix arrangements of Planarium, hydra, salamander and frog); ´current of injury` in regeneration (frogs and salamanders); and more controversially humans and the so-called chi meridians. Although support is there the situation is more complicated than just having coupling via gap junctions since some examples exhibit long-range connectivity and the gap junction is a close-quarter communication mechanism with connexon proteins spanning physical connections between neighbouring cells. Membrane ion channels effects may explain the bioelectric state of the cell at the level of a single cell, but it must apply to a whole number of cells to get to the large scale connectivity required to explain positioning, polarity etc. A possible way of approaching this is to view connectivity more like Jenga or dominos ie. a multitude of single pieces making up the shape of the whole or the shape is made by the hole that is left (template vs stencil). In this way, maybe only certain cells in close quarters need to have gap junctions as the signaling mechanism and these can produce other signals that act long-range by alternative transmission mechanisms, eg. by exuding neurotransmitters and/or hormones. However, either way assumes that each single cell has a property that makes it special from one that does not experience the ´switched on/active` bioelectric state and the whole takes on that ´quality`. (Salt describes this as the ´E` quality meaning ´energised state`.) This property must be apparent for other cells within the group and guesses could be made as to what the cell with the ´switched on` bioelectric state  does eg. does it vibrate, shine, hum etc whereas the non-active cell remain indistinguishable from other non-active cells. This question still remains to be answered. However, continuing with the view that the multitude of cells within a grouping (Cervera and colleagues` ensemble or in the case of memory, the neuronal cell assembly) has a particular electrical status then the authors` view that a quantity of abnormal cells in the multitude grouping determines the overall status of the group is also plausible. When the number of abnormal (or non-switched on) cells is low then the ´quality` of the grouping is likely to be minimally effected, but the effect would increase in some manner (a threshold and then total dysfunction or a sliding scale effect for example) as the number of abnormal cells rises.

Observations on life fields lend support to bioelectric signals and electric fields having a role in connectivity between cells. The overall view is that they act as a signal for new growth as described above and then as a mould or matrix for that cell growth and in the organisation and maintenance of positioning. For example, life field studies have shown that specific voltage potentials reflect the arrangement of the nervous system of worms and fish. One area of positive potential in the brain is reported to match the one major nerve ganglion and in the case of the salamander there is a report of strong positive charges in three areas (the brain, brachial plexus and lumbar nerve ganglions) and negative charges (8-10millivolts) towards the extremities and tail. Organisation based on voltage potentials was also proposed for humans with voltage potential differences shown to be just like with the salamander (head and spinal cord, brachial plexus between the shoulder blades, lumbar arrangement at the base of spine dictated as positive and hands and feet as negative). There are also reports of the human brain having direct current coming from the reticular activating system and flowing from the back to the front of the brain through the middle with the olfactory lobes (positioned at the front) several millivolts more negative than the occipital lobe located at the rear. Current was assumed to flow up from the brain stem. Naturally, the automatic arrangement of the neurons with its polarised positive end at the input dendrite end and negative at the axon output end lent support to the idea of the bioelectric signal being used to organise cells and connect them.

As indicated by Cervera, Alcaraz and Mafe, the bioelectric status of a multicellular grouping associated with connectivity achieved through gap junctions dictates the functioning capability of that grouping. Supporters of the life fields and bioelectric signal views suggest that the bioelectric signal is linked to correct functioning and conversely abnormal changes in fields or bioelectric status can be an indicator of dysfunction and possibly disease eg. voltage differences have been observed between certain organs with cancer. The bioelectric signal and field exhibits normal changes according to the function assigned to that individual cell or grouping of cells, eg. changes of voltage potential in salamanders is associated with sleep status. In this case, it was reported that negative potentials are linked to frontal regions of the salamander brain and the periphery of the nervous system and these potentials are associated with wakefulness, sensory stimuli and muscle movements. The greater the shift to the negative, the greater the activity recorded whereas a shift to positive charge was found to be associated with rest and sleep. Studies on anaesthesia revealed that the negative charge vanished from the extremities and a reversing of the voltage occurred with the limbs and tails becoming positive and the brain and spinal cord negative. The administration of minute currents from the front of the salamander`s brain to the back was found to render the animal unconscious. An examination of brain waves during the process showed the presence of delta brain waves with the unconscious salamander and these increased in size as the current grew bigger. This was plausible since delta brain waves are believed to coordinate brain area connectivity during slow wave sleep. As the anaesthesia wore off, it was found that the normal potential differences returned to normal. A similar pattern was observed in humans with the brain becoming more negative during physical activity, declining during sleep and reversing to positive under general anaesthesia. Therefore, the association between bioelectric status of individual cells linked to others within the grouping via gap junctions (or as suggested by other means) and overall function appears plausible and may give an alternative approach to explaining how certain cognitive functions that rely on connectivity of large areas, eg. consciousness and memory, may occur. Applying knowledge obtained from studies on cell growth (eg. proliferation, organisation and positioning) of non-neural cells and cancer relating to the bioelectric status of a cell achieved by its ´limiting cause` the ion channels present at the cell membrane, the effect of this on neighbouring cells and the corresponding connectivity of those cells of the groupings may give an indication of how these cognitive functions could occur.

The third area explored by Cervera, Alcaraz and Mafe leads on from the other two points that they made, and that is that deliberate modulation of the outer membrane ion channels and hence, modification of the bioelectric signal, leads to changes in bioelectric status and changes in cell functioning. This can be positive for the organism as in for example the life field`s observed ´current of injury` and regeneration of a missing limb, or they can be negative as in cancer where there is a upregulation of sodium ion channels resulting in unwanted changes in cell proliferation, differentiation and organisation for example. We can assume that modulation is achieved in the same way as that for neurons at the first level (´limiting cause`) at the outer membrane stage, eg. by changing the number of  cell membrane receptors or ion channels, or by changing the secondary messenger systems (eg. by a change in conformation of molecules). In their article, Cervera, Alcaraz and Mafe describe direct methods for changing the bioelectric signal at the outer membrane level. This was achieved by the administration of blockers that act directly at the ion channels or receptors or nanoparticles that produce positive or negative changes in the microenvironment of the membrane where the relevant ion channels/receptor molecules are located. Other methods observed to have effects at this level and known to influence the bioelectric status of a cell is the administration of silver (eg. silver electrodes are shown to bring about the destruction of bacteria in close vicinity) and bleach or a weak acid. It is thought that these methods produce a change in the phospholipid basis of the cell`s membrane physiology and hence, affect the functioning of the ion channels and other membrane proteins by changing the conformation of the proteins within their phospholipid microdomains. With all methods the likely result of such a modulation is a detrimental end-effect on gene transcription, which could have positive or negative consequences for the cell. For example, stripping the outer cell membrane of its bioelectric charge using diluted bleach leads to changes in cell growth and proliferation and as reported here a change in transcription rate and cancer. Similarly, leaving cells bathed in diluted bleach takes electrons off a molecule that activates NF- Kb transcription factor resulting in an inhibition of the inflammatory pathway and faster healing of burns and increased skin cell production. Studies have also shown that a weak acid bath can lead to the reprogramming (de-differentiation) of lymphocytes to STAP cells.

In the context of voltage, bioelectric signal and transmission of charged molecules other researchers report the effect of external electromagnetic fields whether electric, magnetic, geomagnetic, or EMF on cell functioning. Cell alignment and cell positioning changes have been reported for applied EMF as well as changes in cell-to-cell communication, membrane physiology and change in cell division for magnetic fields. If such influences on gene transcription rates should occur as the authors suggest through the starting point of the electrical voltage differences at the inner and outer membrane surfaces of the cell, then administration of electric currents or magnetic fields can have probably effects at the single cell level. A problem may arise in explaining how this can influence the whole organism since there are multiple cells present. Again the hypothesis that the bioelectric status of one cell can translate to an effect on a multitude of cells through the connectivity mechanism of gap junctions may provide an answer, but the situation is made more complex by an organism being made up of many different materials within close vicinity to each other all having different physical characteristics eg. fluids, gases, bone.

To summarise, the bioelectric status of a cell through voltage differences across a the outer membrane dependent on two ion channels acting in opposite polarising directions as proposed by Cervera, Alcaraz and Mafe is likely to be an over-simplification of what is actually occurring, but the knowledge obtained from the studies can be applied to the areas of cognitive function like consciousness and memory which have yet to be fully explained. From what is known it can be said that bioelectric signaling is likely to be the first step (the ´limiting cause`) in the action of many cell types. It can also be said that this type of mechanism can provide a means of signaling from the external environment to the inner workings of the cell and hence, is a mechanism of cell-to-cell communication providing connectivity between a multitude of cells. It is unlikely that gap junctions are the sole mechanism of such connectivity as suggested by Cervera and colleagues since this method in neural cells is intended only for close neighbouring cells. Instead a mixed system may be more plausible with gap-junctions and a longer ranging signaling system eg. exudation of another transmitter or signaling molecule such as oestrogen. Cervera, Alacaraz and Mafe suggest that the bioelectric status of a cell from the membrane voltage differences attributed to ion channels present may provide a link to cancer and since it has been reported that bioelectric signals attributed to life field observations are linked with cell proliferation, cell organisation and cell positioning from other species such as the frog and salamander this may be plausible. It definitely needs further investigation, but in the case of humans, the mechanism may not be so applicable. It may be the case early on in development and later on in some areas where pluripotent differentiation and growth occurs normally eg. hippocampus and neurogenesis, skin cells and skin repair, but it may not be applicable in all organs and in all situations. The area needs more research especially in its application to explain the mechanisms behind memory and consciousness and also to the fields of specific targeting of electrical ´charge-busting` molecules on to cell membranes in the hope of normalising cell dysfunction, but maybe more emphasis should be placed on looking at whole organisms or larger groupings of cells rather than single cells in culture.

Since we`re talking about the topic……………………………

…..calcium ion channels have been implicated in the growth of some fish cells. What effect on the bioelectric status of the cell is there if calcium blockers are used? Would the use of specific calcium blockers at each stage of the intracellular calcium signaling mechanism show a dependency for growth on the calcium ion channel at the outer membrane and hence a link to bioelectric status?

……does stripping cells of their external electrical charge with diluted bleach cause a change in structure and performance of the sodium ion channel or potassium ion channel? Could this be observed with fluorescent imaging techniques?

…..would the cooling of cells or the use of specific blockers of phospholipids definitively show that microdomains of the cell outer membrane and particular phospholipids are important in ion channel functioning and hence, bioelectric status of the cell?

….reversine is said to de-differentiate cells by inhibiting the phosphorylation of a histone and preventing the activation of a specific cell differentiation kinase. Can we assume that the dual administration of a targeting cell membrane sodium ion channel blocker and reversine would have no greater effect on cell regeneration than the former alone because the signal for cell growth would have been prevented at the level of the cell membrane and not at the level of gene transcription?

…would the use of voltage sensitive fluorescent dyes to track non-invasively ionic gradients allow the changes in Vmem to be measured for each cell type and a comparison to cell function and cell division and proliferation be made?

 

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