ISIS Report 21/09/11
Membrane Potential Rules
An electric property universal to all
cells turns out to determine their vital states, from cell division and pattern
formation to differentiation, regeneration, and cancer; lending support to the
electrodynamics basis of life Dr. Mae-Wan Ho
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The body electric hums again
The membrane potential refers to the
electrical potential difference across the cell membrane: the inside with
respect to the outside. When the cell is ‘at rest’ in a steady state, its ‘resting’
membrane potential averages about -50 mV, with a range of -10 to -100 mV.
Although a great
deal of work had been done on the nerve cell, pioneered by British
physiologists and biophysicists Alan Hodgkin (1914-1998) and Andrew Huxley in
the 1950s , focussing on the action potential - an
event in which the membrane potential rapidly rises and falls - the membrane potential of other cells has been relatively
Within the past decade,
however, the membrane potential of a wide range of
cells has been coming under scrutiny, thanks to voltage-sensitive dyes that
fluoresce or change colour according to electrical potential. Researchers can
now follow electric potential changes both of large populations of cells and of
localized patches of membrane or organelles in a single cell. These
potential changes appear to determine vital states - from cell division and
pattern formation to differentiation, regeneration, and cancer [2-6] - lending considerable
support to the idea that cells and organisms intercommunicate and coordinate
their activities coherently by means of electric and electromagnetic fields
(see  The Rainbow and the
Worm, The Physics of Organisms, ISIS publication). In this article, I shall
concentrate on development and regeneration.
Electric embryos, wound healing, and
Research on bioelectric activities of cells
in general actually goes back at least as far as the 1930s (see ), but never
quite reached mainstream status. In the 1970s, Lionel Jaffe (1918-2011) and
Richard Nuccitelli, then at Purdue University Lafayette, Indiana, in the United
States, pioneered the vibrating probe technique for measuring electrical
currents noninvasively near individual living cells . This led to the discovery
that all developing embryos drive ionic currents through themselves. These
ionic currents are thought to be responsible for electric fields generated
inside the embryos, which had been discovered earlier using microelectrodes.
Electric fields ~20 mV/mm have been measured in chick and frog embryos using
microelectrodes ; and perturbing these electric fields results in
developmental abnormalities. Mammalian skin wounds, similarly, generate large
fields of up to 150 mV/mm right next to the wound, and reducing or augmenting these
fields could delay or accelerate healing.
It has been
known since the 1950s that the membrane potential varies throughout the cell cycle . Cell types with very high resting potentials such
as muscle cells and neurons show little if any tendency to divide, while a
decrease in membrane potential follows malignant transformation, in which cells
multiply out of control. In the 1970s, Clarence D. Cone Jr. induced DNA
synthesis and mitosis in fully differentiated neurons from the central nervous
system using a variety of agents that depolarized the cell membrane (made it
less negative) 
Microelectrode and related
patch clamp techniques are difficult and time consuming, and can only measure the
membrane potential of an extremely local area. Voltage sensitive dyes are much
easier to use and can give information on spatial and temporal variations of membrane
potentials over large areas or populations of cells (though it is difficult to
determine the actual electric potentials without first standardising colour
changes against measurements made with microelectrodes). As a result, new
vistas have opened up for bioelectricity research; especially for scientists at
Tufts University, Medford, Massachusetts in the United States, studying stem
cell growth and differentiation, cancer, and embryonic development.
Stem cells and cancer
Stem cells are the key to regenerative
medicine (see  The Promise
of Induced Pluripotent Stem Cells, SiS 51). However, one major
difficulty is in controlling their growth and differentiation; and it has
become clear that the misregulation of stem cells can lead to cancer . There
is an intimate connection between correct differentiation of stem cells during
development and in tissue replacement. Michael Levin and his team at Tufts University perturbed stem cell differentiation in the developing frog in order to throw
light on what makes stem cells multiply out of control.
In vertebrates, the ‘neural
crest’ is a population of embryonic stem cells that form many structures
including smooth muscle cells, peripheral neurons and glia, cartilage and bone
in the head and face, as well as endocrine and pigment cells. Neural crest
misregulation gives rise to an important class of birth defects in humans. Neural
crest cells not only reveal dynamics of cell migration, but are important for
understanding melanoma, a malignant cancer of melanin-producing cells.
Armed with the knowledge that
depolarization of the membrane potential leads to cell proliferation, and that
depolarization of the neural crest cells can be achieved by targeting the
glycine-receptor chloride channels (GlyCl), which has been previously achieved in
vitro, the team carried out the experiment in vivo on developing
frog embryos .
a drug commonly used as an anti-parasite agent and known to specifically open
GlyCl. Keeping chloride channels open leaks Cl- into the medium,
thereby depolarizing the cell (making it less negative).
(African clawed frog) embryos develop a characteristic pattern of pigmentation
after a fraction of the neural crest cells in the early embryo become
melanocytes during the later neurula and somite stages, and begin to produce
melanin during the still later tail bud stages. The pigmented cells are largely
confined to the mid region in the head and trunk of the young tadpole (Figure
1, top). In contrast, embryos treated with ivermectin showed extensive
hyperpigmentation, the melanocytes migrating to regions normally devoid of
pigment cells (see Fig. 1, middle). This happened to 98 percent of the embryos
treated. The treated embryos were paralyzed as tadpoles, as expected from the
depolarizing effect. Development was otherwise normal, and no other organ or
tissue derived from neural crest cells was affected. Thus, opening the glycine
chloride channels in embryos specifically resulted in hyperpigmentation.
hyperpigmentation also involved inappropriate cell migration, as blocking
melanocyte movement with metalloprotease inhibitor NSC-84093 resulted in
tadpoles hyperpigmented only in the mid regions (Fig. 1 bottom). Otherwise, the
migration of the abnormal melanocytes was extensive; they colonized not only
the lumen of the neural tube, but also penetrated the dense neural tissues, and
sent long projections from the edges of the somite (blocks destined to become
trunk muscles) into the neural tube. They are reminiscent of metastasis
(spreading of cancer cells).
To prove that
the ivermectin effect was due to opening the GlyCl, the embryos were exposed to
the normal binding partner of GlyCl, glycine, which binds to the protein at a
different location from ivermectin. Treatment with glycine induced the same
hyperpigmentation as ivermectin.
which cells expressed the ivermectin target, in situ hybridization with
an antisense probe for the mRNA of GlyCl a-subunit (to block its
expression) confirmed that the GlyCl was expressed exclusively in the
neural-crest area in the early embryo, and later in a sparse peppered pattern
throughout the embryo, but not expressed in the melanocytes themselves.
1 Depolarization of membrane potential and hyperpigmentation
To show that the effect of ivermectin
was due to depolarization of the membrane potential rather than some other
alteration of GlyCl function, chloride concentration in the external medium was
increased from 10 mM to 30, 60 and 90 mM. As the internal chloride
concentration can be as high as 60 mM, increasing the chloride in the external
medium will reverse the depolarization. Indeed, the hyperpigmentation effects
were partially suppressed at 60 mM and completely inhibited at 90 mM chloride
in the external medium.
depolarization resulting in hyperpigmentation could be achieved by blocking
other proteins, for example, by injecting the non-functional mutant of the
ductin subunit of V-ATPase pump, which hyperpolarizes cells.
Thus, it is a
change in membrane potential as such that causes the effect, a result that’s
repeated for many other functions investigated by the researchers.
The ‘face’ of frog in membrane potential
The most exciting serendipitous new discovery
of the Tufts University researchers is the ‘face’ of the frog roughed out in
membrane potential differences very early in development when the embryo is
still a shapeless ball of cells with very few anatomical features .
A team led by Dany Adams used a
combination of voltage and pH sensitive dyes to follow the development of Xenopus
embryos under a microscope fitted with a time-lapse camera. It recorded “never-before-seen”
dynamic patterns of membrane potentials on the outermost cell layer evolving in
roughly three sequences (see Figure 2). These are clear signs of electrodynamical
processes determining structures that appear much later on.
The first (Fig. 2A,
Course I) is a wave of hyperpolarization (more negative membrane potential)
sweeping across the entire embryo in about 15 minutes at stage 13, when the
embryo is a gastrula, a double layer ball of cells.
2 Dynamic patterns of membrane potentials in the developing Xenopus embryo
dorsal, V, ventral)
The second (Fig
2B, Course II) following the first, coincides with the closure of the neural
tube (the future spinal cord and central nervous system) in which a bright line
of hyperpolarization coming from the median ectoderm (green arrows) gets
occluded as the folds close over it, while somewhat dimmer patches appear at
the lateral ectoderm (orange arrows). As the neural tube closure ends, distinct
bright spots and lines of hyperpolarization appear in anterior areas that
subsequently invaginate (sink inside the embryo). These hyperpolarized regions
mark out the future mouth area (yellow arrows), the cement glands (brown
arrows) the nose area (lavender arrows) and the first pharyngeal fold (brown
arrows), the eye field (blue arrows) and the future ear (magenta arrow). At
some point, the patterns do take on an eerie semblance of a face (stages 18 and
19), though except for the mouth and the pharyngeal folds, the other facial
features are not mapped out in their normal positions of what one would
recognize as the ‘face’.
sequence of bioelectric activities (Fig. 2C) takes place after the closure of
the neural tube and is coincident with the elongation of the embryo (along the
anterior posterior axis). It consists of an embryo-wide series of localized hyperpolarizations,
less orderly than the first two, forming and spreading in small areas that
sometimes overlapped with the regions established during the second sequence.
the same report, the researchers showed that the development of head features depends
on the proton transporter protein H+-V-ATPase, which exports
protons (H+) from the cell using ATP. Many V-ATPases are found on
intracellular vesicles but the ones involved in head development are found
embedded in the cell membrane. By pumping H+ out, it hyperpolarizes
the cytoplasm, making it more negative. Disrupting this enzyme by chemicals or
injecting mRNA that makes faulty H+-V-ATPase led to many head and
was identified as the result of a hierarchical screening procedure with drugs
that inhibit progressively more specific ion transporters that block the
requisite change in membrane potential for the function under investigation.
The Tufts group refers to this specialty as “chemical genetics” [3, 4], which
enables them to make good use of genomics information.
to other agents that altered membrane potential by preventing hyperpolarization
or preventing the cytoplasm becoming less acid (from loss of H+) gave
the same abnormalities; while another proton pump that normally sits in the
cell membrane can compensate for the loss of H+-V-ATPase function.
These findings again indicate that it is membrane potential as such that
appears to determine head and face formation at a critical time, upstream of
many of the genes identified as crucial for head and face formation.
Young Xenopus tadpoles
readily regenerate their tails when cut off. That too, depends on the proton
pumping activity of the H+-V-ATPase , but only through the change
in membrane voltage, “an early mechanism necessary and sufficient to induce
tail regeneration .
Head or tail
not alone in depending on membrane potential for embryonic development and
The planarian flatworm
is a favourite model organism for studying regeneration, even more so than Xenopus.
When it is cut in half, the part that has the head regenerates the tail, and
the tail half regenerates the head. And even when it is chopped up into more
pieces so that some of the pieces have neither head nor tail, the fragments can
still remember which side was nearer to the head and which nearer the tail, and
regenerate both head and tail in the correct orientation.
planarian Dugesia japonica has adult stem cells (neoblasts) for replacing
differentiated tissues lost during normal turnover of cells. When the adult is amputated,
these neoblasts proliferate and migrate to restore the missing parts by forming
a regenerative mass, the blastema, which eventually differentiates into the
missing structures. Previous research indicates that crucial determinations at
the site of injury take place during the first day of regeneration and involve
signals from both local and distant tissue. Among proteins already implicated
in the correct specifications of structures during regeneration are gap
junction channel proteins located in the cell membrane and involved in direct
literature dating back to the 1950s and 1960s suggests that the nervous system
is an essential component of regeneration in both vertebrates and
invertebrates, but the mechanism remains poorly understood.
team began by screening for gap junction (GJ) blockers . In planarians,
treatment with known GJ blockers heptanol or octanol, but not hexanol (which
does not block GJ) led to consistent alteration of anterior/posterior (AP)
polarity during regeneration. The researchers optimized the dose of octanol so
it induced consistent AP polarity alterations without blocking all gap
junctions simultaneously, which would otherwise poison the organism and the
A series of
transverse cuts were made to give fragments cut at both ends along the AP axis;
the fragments were then treated with octanol or left untreated. Both the
untreated controls and the octanol-treated fragments developed normal anterior
blastemas that gave rise to heads. However, fragments treated with octanol
often formed anterior blastemas at the posterior-facing ends that developed
into heads, resulting in animals with heads at both ends, an abnormality never found
in untreated animals. The tendency to developing heads at both ends (bipolar
head) gradually increased as the plane of amputation moved towards the
posterior end, starting from the head, reaching maximum (~100 percent) just
behind the pharynx (see Figure 3).
3 Double headed regeneration after octanol treatment
previous work showed that planarian GJ proteins, innexins, are important for stem cell function and blastema
formation, it remains unclear which, or how many of the dozen innexins are
actually involved in AP patterning during regeneration. Michael Levin’s team
used RNA interference, injecting short stretches of double stranded RNA
to target genes in a sequence-specific manner, to silence the genes in the
adult planarians before they were amputated. They targeted up to four innexins
simultaneously by injecting more than 600 animals with the iRNA combinations,
and narrowed down to three, which when blocked simultaneously resulted in
abnormalities similar to those caused by octanol: Dj-Inx-5, Dj-Inx -13,
and Dj-Inx-12. These genes are expressed in the central nervous system
as well as in sub-epithelial cell populations throughout the animal, and are
up-regulated within the regenerating blastema. Injecting the iRNAs of these
genes into intact animals caused behavioural changes as well as inversions of
the AP polarity, with extra pharynxes growing at inappropriate places.
Central nervous system also prevents
The planarian brain can prevent regeneration
of secondary heads within the same animal. Thus, the head fragment produces the
fewest double headed animals. This inhibition can act at long range. As any
fragment containing the head, however far away the head is from the cut end,
will rarely give rise to double headed animals when treated with octanol.
suspected that the ventral nerve cord was responsible for inhibiting head
formation in the presence of octanol. To test the idea, they made cuts that did
or did not disrupt the ventral nerve cord (VNC) along the AP axis. All treated
and untreated animals formed blastemas, but those with interrupted VNC treated
with octanol developed multiple AP axes, with heads, pharynxes and protrusions
in the wrong places (see Figure 4).
4 Abnormal AP axis with octanol treatment and VNC disruption
Do the central
nervous system and the GJ signals also affect healing or regeneration of wounds
on the sides of the body? To answer this question, the researchers made lateral
cuts in post-pharyngeal fragments treated with octanol that had the VNC disrupted,
or not. All cuts resulted in blastema formation; and to their surprise, all
exhibited behavioural changes as if they are being pulled in different
directions simultaneously, and photoreceptor pigments (normally present in the
eyes) appeared in two, three or four different blastemas within the same
fragment, suggestive of rudimentary heads. And three or four heads did
eventually form in their fragments (see Figure 5). Thus, the CNS and GJ provide
signals to both posterior and lateral wounds to inhibit head formation.
5 Multiple heads from lateral wounds treated with octanol
Correct specification of AP polarity
occurs at the earliest times
To find out when the central nervous system
and the GJ proteins are needed for correct specification of the AP polarity,
the researchers carried out treatment at different times after amputation.
Fragments without heads treated with octanol (GJ inhibitor) gave the most
significant effects within the first 3-6 hours after amputation, dropping to
less than 60 percent for treatment beginning more than 12 hours after
amputation. For VNC disruption, a dramatic, 4-fold decrease in the incidence of
AP defects occurred after 3 hours post-amputation. These findings suggest that
both GJ proteins and the central nervous system act early during regeneration
to correctly specify the missing structures.
Inheritance of acquired heads
Amazingly, animals with two, three or four
heads regardless, not only survive, but can regenerate the same acquired
pattern on further amputations in the absence of octanol. It
appears that a single treatment is enough to reset the AP axis, and the memory of
that can now be perpetuated indefinitely. This is a genuine inheritance of acquired
characters, and does not involve any change in the genetic material; the amount
of octanol used in the original experiment was not mutagenic. It is reminiscent
of a ’morphogenetic field effect (see later).
Membrane potential rules
In a follow-up study, the researchers
uncovered, once again, that membrane depolarization, mediated by H+, K+-ATPase,
is essential for anterior gene expression and brain induction  (Figure 6).
As in Xenopus, independent manipulation of the membrane potential with
ivermectin confirms that depolarization drives head formation, even at
6 Depolarization means heads
Morphogenesis and pattern formation
remains as mysterious as ever
Decades of painstaking molecular genetic
analysis followed by genomics have succeeded in mapping out detailed pathways
of gene induction and repression in the formation of different body structures
during development. Despite that, there is little progress in our understanding
of how patterns and forms are generated, starting with an almost
entirely featureless egg or cell mass. The patterns of specific gene
expressions follow in the wake of pattern determination processes, which
invariably include a change in membrane potential.
identifying membrane potential changes is only a start, for one must also ask
how a localized membrane potential change arises in the first place. And
following that, how more complicated patterns, such as repeated body segments,
limbs, shoots and roots are determined. We shall examine all these questions
next ([17-19] Genes don’t
Generate Body Patterns, Liquid
Crystalline Morphogenetic Field, and Electronic
Induction Animates the Cell, SiS
52), in order to flesh out the electrodynamical nature of life.
There are 4 comments on this article so far. Add your comment
|Brigitte Hansmann Comment left 21st September 2011 22:10:09|
Dear Mae Wan, thank you very much for your work! It is most valuable for my work as a clinician in dfa somatic pattern recognition. I am looking forward to the three articles you announce.
|Kaviraj Comment left 21st September 2011 22:10:00|
It is in .this context interesting to compare the work of G.W. Crile in 1926, when his seminal book on this very subject was published. "A Bi-polar Theory of Living Processes" was its title. Well reasoned out and with clear illustrations in colour and black and white, it make a strong case for the electromagnetic basis of vital-force=defence-system and its implications for all sciences.
In homoeopathy we already use electromagnetic means of cure. Boyd of Glasgow (1946) and Abrams of the US (1930's) built machines that could read the level and provide the matching remedy.
Glad to see it making a comeback in more mainstream thinking.
|Todd Millions Comment left 22nd September 2011 09:09:15|
The field map of electrical potential(normally),Becker(Robert O-see;Body Electric)described was(if I'm remembering correctly)-Positive at centre,and negitive charge at the extremities.This was I seem to remember re enforced in vertabrates by the diode peizo electric currents generated by (living) bone.
Would this orientate this feild?
|Rory Short Comment left 22nd September 2011 10:10:37|
I have never studied the life sciences but have always been fascinated by life approaching it at a more macro level I suppose. Despite my lack of knowledge at this more micro level I found this article very exciting. It is great that knowledge gleaned at this micro level is beginning to provide a hard science foundation for previous understanding that was in a sense arrived at more intuitively.