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Ion transport and electrochemical gradients under DC and AC signals
Alexander Walls, Qurat-ul-Ann Mirza, Vannara Chhim
(Mentors: Horia I. Petrache, Merrell A. Johnson, Bruce D. Ray, Elliott D. Rosen,
Yogesh N. Joglekar)
The plasma membrane of a human cell remains much an enigma as far as its behavior
with the extracellular environment is involved. The plasma membrane is a phospholipid bilayer
composed of molecules that are polar on one side and nonpolar on other, thus they are
amphipathic. The cell must be able to exchange materials with its surrounding environment in
order to survive. Transport across the cell membrane can be accomplished in two complementary
ways: passive and active. Active transport utilizes specialized membrane proteins such as the
Na+/K+ pump and requires the expenditure of energy in the form of ATP or an applied potential
like that of the proton gradient established by a mitochondrion. Here we investigate passive
transport, which relies only on an electrochemical gradient across a lipid membrane. The gradual
change in the concentration of a compound over a distance is its chemical concentration gradient.
Passive diffusion occurs naturally in the direction of lower concentration. Charged molecules
however, also flow in the direction of electrical gradients. These two types of gradients combine
to form a net electrochemical gradient dictating how charged molecules should move. In the lab,
we synthesize isolated lipid membranes in ionic solutions and insert the antibiotic protein
gramicidin which is permeable to positive ions such as sodium and potassium. The energy
required for ion transport is supplied by an applied external potential (voltage). The objective of
our study is primarily centered on the electrical properties of biological materials under DC and
AC applied signals.
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Experimentation with cell ion channels helps to reinforce other fields that indirectly
depend on cell ion channel methods and techniques. For example, Hewlett Packard has finally
proven that they can build devices that use memristors, instead of the transistors that enable all
current computer chips. Memristors can store and process data simultaneously and function at
much smaller sizes than a transistor, this advance could increase the power and memory of
computers and cellphones to astronomical proportions within only a couple of years. Not only
that, but memristors could be the first step toward the creation of artificial intelligence and
artificial brains that work like real biological brains. Therefore, the study and application of
biological materials under an applied voltage ties in well with the theory on memristors.
The behavior, function, and cell-to-cell communication within the body more closely
resembles the actual work done in the lab. As stated previously, cell communication is crucial for
the survival of the cell. For example, in order for a neuron at its resting potential to generate an
action potential, there must be a net movement of ions into and out of the cell. The rapid influx
of ions must occur within a matter of milliseconds using specific protein channels that are
imbedded in the plasma membrane of the neuronal cell (Takazawa et al., 2012). Not only that,
but nutrients are found outside of the cell, making protein receptors on the surface of the
membrane the main way for cells taking in nutrients. A most notable example is the regulation of
blood glucose levels by the liver. If the blood glucose level is too high the liver will secrete
insulin. Insulin is a water soluble peptide hormone that diffuses into the blood and attaches to
receptor proteins on the surface of the cell membrane. Due to the polarity of insulin, it cannot
simply diffuse through the cell membrane like that of the lipid soluble steroid hormones (Wilson,
2013). The net effect of insulin is to signal cells to take up glucose, thereby decreasing blood
glucose levels. Some individuals with diabetes do not synthesize enough surface proteins for
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insulin or glucagon, causing in poor cell communication that can be life threatening. As a result
blood glucose levels go unregulated because the cells are unable to metabolize the glucose in the
interstitium (Wilson, 2013).
The research conducted in the lab applies to the types of lipids that compose the
membrane as well. In order for macrophages to know which cell to kill, there must be a signaling
method involved. This time, the signaling method is done by the externalization of
phosphatidylserine (PS) lipids rather than a surface protein receptor (Justice 2013). PS lipids
were commonly used in the lab to perform lipid membrane experiments using the protein
gramicidin.
Five academic peer reviewed articles were chosen based on how well they related to
explaining the concept and the work done in the lab. In their 2013 peer reviewed article, the
Effects of Lipid Interactions on Model Vesicle Engulfment by Alveolar Macrophages Matthew J.
Justice et al. claim that the role of phosphatidylserine and of ceramide in phagocytosis is
dependent on lipid-mediated modification of membrane properties. Their article allows for an in
depth understanding of interactions that involve both lipids and proteins.
In their 2007 peer reviewed academic article, Interfacial Polar Interactions affect
Gramicidin Channel Kinetics Tatiana Rostovtseva, et al. examine the role of lipid headgroup
interactions by using gramicidin A channels in planar bilayers as a probe. This paper allowed for
good predictions of what happens when DOPC lipids interact with gramicidin A ion channels, as
these were the main lipid and proteins used in the lab. It was important to have an in depth
understanding of the chemicals being worked with on a daily basis.
In their 2012 academic peer reviewed article, Maturation of Spinal Motor Neurons
Derived from Human Embryonic Stem Cells, Takazawa research motor neuron function in
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human postmortem tissue in order to model motor neuron function. One of the main features of a
neuronal cell are is the Na+/K+ pump made of a membrane protein that aids in the movement of
ions through the plasma membrane much like that of the gramicidin A protein channels used in
the lab. In the 2013 peer reviewed article Type 2 Diabetes: An Epidemic in Children Valerie
Wilson gives a simplified example on the importance of cell communication within the body and
how this is crucial to the survival the cell and ultimately the person. This article makes it possible
to stress the importance of why cell ion channel research is significant to problems in healthcare.
By understanding more about the plasma membrane and its components we can better treat
diseases like diabetes mellitus.
Our summer MURI research involved preparing lipid membranes, such as DOPC and
using Gramicidin A protein as ion channels. In the beginning, research was conducted solely on
the membrane without gramicidin or any protein. This was done to better understand the basic
set up of the equipment. Later, experiments were performed to learn how the membrane
interacted in an aqueous ionic solution without protein. Midway through the 9 week program,
experiments with gramicidin proteins were to be taken more seriously, not that the ones without
gramicidin were not important. As a matter of fact, comparison of a 2mg/mL DOPC lipid
membrane in 1M KCl/ NaCl to that with gramicidin allowed for observation and analysis of
differences when 5𝜇L of gramicidin were added to each side of the ion chamber.
Originally experiments were conducted under the traditional direct current (DC) applied
signal. A DC signal is a continuous flow of positive charges from anode to cathode. Toward the
end of the program, we also performed experiments with alternating currents (AC). An AC
signal applies an alternating voltage causing the flow of charges to alternate directions. The
simplest AC signal is sinusoidal written as ∆𝑉𝑚𝑎𝑥 sin 𝜔𝑡, where the sine value represents some
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fraction of the maximum voltage experienced at the peaks of the sine wave depicted in figure 1
below:
One of the main results of the research was the passive diffusion experiment where 1M and
0.5M KCl were placed on opposite sides of the ion chamber and diffusion was allowed to take
place. The initial high current shown in Figure 2 below is due to the large difference of ion
concentrations between the two sides of the membrane. As salt ions move, concentrations
equalized and the current approaches zero. The results of this experiment demonstrated that ions
move toward areas that afford the highest entropy and lowest free energy. Ions will move down
their concentration gradient toward areas of lower concentration as shown below in figure 2:
Figure 1
Figure 2
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One of the first DC experiments using 2mg/mL DOPC and 1M NaCl demonstrated that
as you increase the concentration of protein you will also tend to increase the number of protein
channels that form in the membrane. Below figure 3 represents 5µL gramicidin and figure 4
represents 20µL of gramicidin. The rectangular formations in the graphs show the opening and
closing of ion channels in the membrane. Notice there are much more in figure 4 than there are
in figure 3 below:
Figure 3
Figure 4
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Finally, figure 5 depicts an AC signal being applied to a 2mg/mL lipid membrane without
gramicidin protein. If this was a DC signal it would be a straight line across the graph, instead an
AC signal oscillates from a maximum to a minimum. This wave is not a sine wave, but a triangle
wave in which the y-axis is current and the x-axis is time (min):
The current status of the research is well defined in the areas that apply to DC signaling,
however AC signaling across lipid membranes is still underdeveloped and more experimentation
needs to be done. More experiments with gramicidin should also be conducted for AC purposes.
AC experimentation started late in the research and mimicked that of the DC research in the
sense that the first experiments were carried out using only DOPC lipid solutions to understand
how the membrane itself functioned in an AC environment. Also, much more experimentation
involving dilute lithium ion solutions need to be carried out. Lithium destabilized the lipid
membrane, but it is not known why this happens. Of all the ionic solutions used potassium
chloride works the best under both conditions and ensures a stable membrane.
The overall implications of the experiment demonstrated that channel proteins are crucial
for the survival of the cell which depends on ionic flows across membranes. Experiments also
Figure 5
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reveal that certain salt solutions can destabilize lipid membranes and eventually break it. The
next steps in experimentation of cell ion channels should be to go much further in depth with
experiments involving AC signaling. DC signaling is a good application to how cell membranes
really function, but AC is still underdeveloped. More experiments with organic salts should be
carried under both DC and AC applied signaling. No experiments with organic salts were carried
out over the nine weeks, due to their toxicity. Given the right conditions and necessary
precautions successful trials can be done using organic salts. Analysis of how the lipid
membrane reacts to such an aqueous environment can provide insight on how to treat toxin that
enter the body.
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Works Cited
Chua, L. O., & Kang, S. M. (1976). Memristive devices and systems. Proceedings of the
IEEE, 64(2), 209-223.
Justice, J. Matthew, Petrusca, N. Daniela, et al. (2009). “Effects of Lipid Interactions on Model
Vesicle Engulfment By Alveolar Macrophages.” 1-7.
Takazawa, T., Croft, G. F., Amoroso, M. W., Studer, L., Wichterle, H., & MacDermott, A. B.
(2012). Maturation of Spinal Motor Neurons Derived from Human Embryonic Stem Cells. Plos
ONE, 7(7), 1-9.
Rostovtseva, K. Tatiana, Petrache, I. Horia, et al. (2007). “Interfacial Polar Interactions Affect
Gramicidin Channel Kinetics.“L-23-L25.
Wilson, V. (2013). TYPE 2 DIABETES: AN EPIDEMIC IN CHILDREN. Nursing Children &
Young People, 25(2), 14-17.
