MMED 2933: Neural Circuits

Lecture notes: lecture 3. Membrane Bioelectricity. Simon Brookes

Human beings have vast numbers of nerve cells in their brains – between 1010 and 1011 nerve cells
(ie: ten billion to one hundred billion). The glial cells that surround them are even more numerous ,
although glia do not participate directly in the moment-to-moment electrical activity of the neural
circuits. To understand how neural circuits work, we must have an appreciation of the properties of
the building blocks of the circuits. These are the neurons (neuron=nerve cell). The nerve cell
membrane (=plasmalemma, =cell membrane) that forms the outside of each neuron plays a key role
in determining the electrical activity of the cell and hence the functioning of circuits. During the
next few lectures, we will conceptually “build” a pair of connected neurons, to understand exactly
how these cells work. Please remember throughout this exercise the neurons are just “dumb cells”
– they have no innate intelligence. They don’t “try” to do anything – they don’t “know” what is the
right way to respond. Intelligence arises from the interactions of billions of these dumb cells that
have been organised into circuits by evolution and modified by learning….Effectively, neurons act
a bit like tiny switches or oscillators....when they're organised in just the right way, adaptive
behaviour results. If you can understand how 2 neurons interact, in detail, then you will understand
the cellular basis for how the entire nervous system works (however, you should be aware that the
central nervous system has multiple layers of organisation above the level of the single cells).

Cell membranes
Cell membranes are a feature of all living cells. Indeed, the ability to separate the inside of the cell,
with all of its delicate, complex biochemical machinery, from the outside environment, must have
been a critical development in the origin of life. All types of animal cells are surrounded by a cell
membrane, which consists of a phospholipid bilayer, together with a number of other constituents,
which vary in proportion in various types of cells. There are 4 types of constituents of cell
membranes to consider: phospholipids, cholesterol, glycolipids and membrane proteins.

The primary ingredients of the cell membrane are “phospholipids”. These consist of a glycerol
backbone (a 3 carbon chain), bonded to three groups: 2 long chain fatty acids and a phosphate
group with an attached small polar molecule (eg: choline or inosine). Long chain fatty acids are
non-polar: which means that electrical charge is evenly distributed over the entire molecule which
is made up largely of carbons and hydrogens. This means that they are insoluble in water (because
water molecules are quite polar). In fact, long chain fatty acids will not mix with water and will
tend to form clumps or globules when added to a watery solution. However, the other bit of the
phospholipid is made up of a phosphate group and attached polar group (usually choline,
ethanolamine, inositol or serine). This bit is strongly charged and highly polar. It mixes well with
water. This means that phospholipids, in an aqueous environment, tend to align themselves so that
their non-polar (fatty acid) ends cluster together, where they exclude water. In contrast, the polar
ends of the same molecules face outwards into the water, interacting with it through hydrogen
bonding. In this way phospholipids can form bilayers. A phospholipid bilayer is an energetically
stable structure in an aqueous (watery) environment - and it forms the basis of the cell membrane.
High power electron micrographs of cells can actually visualise a 3 layered structure of cell
membranes (2 layers of phospholipids with a tiny “gap” between). Just for interest’s sake, there is a
second stable structure that phospholipids form in watery solution – and that is a “micelle” which is
a tiny sphere with phospholipid tails facing inwards and polar groups outwards. Micelles have
nothing to do with neurophysiology.

Membrane permeability
The basic structure of the membrane, based on the bilayer of phospholipids, is a semi-fluid at
normal body temperature. That is, the membrane has no stiffness and can be readily deformed, but
it does not tear easily. Because of the inner layer of non-polar fatty acid chains, the membrane is
very impermeable to all charged or polar molecules and to all charged ions. Some non-polar
molecules (CO2, O2) can cross the membrane very easily. Most water movement across membranes
is probably through ion channels because water is quite polar. Other compounds which cross
membranes easily are “lipophilic” (ie: fat-loving) substances such as steroid hormones and some
drugs. These typically have low solubility in water, but are very soluble in organic solvents
(including oils). Unlike ions, some molecules (such as glucose or amino acids) may not have an
overall charge, but they are still polar – they have a slightly positive end and a slightly negative end.
Polar molecules like these also do not cross a phospholipid membrane easily. Generally, these
water soluble) molecules (eg: glucose, adenosine triphosphate, proteins, neurotransmitters, amino
acids etc) cannot cross the membrane directly – they need to go through via special pores,
transporters or channels etc.

The long chain fatty acids in the phospholipids can either be saturated or unsaturated (unsaturated
means containing one or more double bonds between the carbon atoms in the chain). Typically,
unsaturated long chain fatty acids do not form straight chains - they have a kink in them at the
double bond. This means that they do not pack as closely as straighter, saturated fats and this
makes the membrane rather more fluid than it would otherwise be. In many animal cells,
particularly nerve cells, at least one of the fatty acids on each phospholipid is unsaturated, so that
they don’t fit perfectly together and this makes nerve cell membranes especially fluid.

Other fatty components of cell membranes

Phospholipds are not the only constituent of cell membranes. There are also considerable (and
variable) quantities of cholesterol. This molecule consists of a 4-ring structure with an attached
short carbon chain. It is very non-polar and does not mix with water (it is “hydrophobic”), however
it mixes well with the non-polar fatty acid chains inside of the phospholipid bilayer. Cholesterol is
a constituent of most membranes and tends to further increase the fluidity of the membrane and
reduces its permeability. In some parts of the membrane, cholesterol may be concentrated. These
"lipid rafts" often contain high concentrations of membrane proteins including receptors, channels
etc.

The other major lipid ingredients of membranes are “glycolipids”. These are found only in the
outer leaflet of the membrane and consist of two long hydrophobic tails (based on long chain fatty
acids) with a polar sugar group making the third component (and NO phosphate group). The
hydrophobic tails insert into the inner parts of the bilayer and the charged sugar groups protrude out
into the extracellular space where they interact with water. Glycolipids are important in cell
recognition functions during growth and development of contacts between neighbouring cells.

Protein components of cell membranes

Cell membranes perform an essential function in all cells, separating potentially toxic and
disruptive chemicals in the external environment from the rather delicate machinery inside the cell.
However, this comes at a price. The cell needs to have supplies of raw materials (building blocks
such as amino acids, and energy sources such as glucose) entering the cell and waste products being
removed. For water-soluble molecules that cannot cross the membrane by themselves, this is
achieved via specific membrane channels, pumps and exchangers as well as processes such as
endocytosis, phagocytosis, pinocytosis and exocytosis. Thus the fourth major component of cell
membranes are the hundreds of different types of proteins embedded in them – many of which are
involved in moving substances across the cell membrane.

Movement of ions across membranes

The movement of ions is really what we are interested in for understanding the electrical activity of
nerve cells. That is: sodium ions moving through sodium channels, potassium ions moving through
potassium channels….you get the picture. Whenever ions move across a membrane, they carry
charge with them. They actually don't pass through the phospholipid bilayer- they move through
the pores in proteinaceous ion channels. However, they don't move freely through those ion
channels -they have to "squeeze" through. Thus, in electrical terms, the cell membrane acts as a
resistor - Rm - to the flow of ions/charge. The actual value of R is measured in Ohms - and is
determined by the number of open ion channels. The more open ion channels; the lower the
resistance. This resistance is crucial in allowing potential differences to be maintained between the
inside and the outside of the cell. If you punch a hole in the membrane (ie: zero resistance) the ions
instantly move to have identical concentrations inside and outside the cell. However, because
membranes are so fluid, they tend to seal over fairly quickly – so small amounts of temporary cell
membrane damage are usually not lethal.

You may also come across the term “conductance”. This refers to how easily charge moves across
the membrane. In some ways, this term makes a bit more sense than resistance. Resistance
DECREASES, when the number of open ion channels INCREASE. That can be a bit awkward to
think about. However, conductance is the opposite (actually the inverse) of resistance. Each open
ion channel has a certain conductance – that is, it lets ions move through it. Conductances add
together. So, the more open ion channels; the higher the conductance. The higher the conductance,
the easier it is for ions to move across the membrane. That makes sense. Unfortunately, many
electrophysiologists and their text books still use resistances. So you sometimes have to perform
mental gymnastics to picture what is going on.

The other electrical feature of the membrane is that it also acts as a capacitor – Cm (means
Membrane capacitance)- , since it consists of a very thin resistive layer (the phospholipid bilayer)
with a large surface area, separating two conductive layers (watery solutions of ions ie: the
intracellular fluid and the extracellular fluid). This configuration (conductor/resistance/conductor,
with a large surface area) causes the property of capacitance. Capacitance is an important concept,
but quite difficult to visualise. It allows the membrane to temporarily store charge... We’ll come
back to this in the lecture on “cable properties” of nerve cells. For now, you just need to know that
resistance and capacitance of the membrane, acting in parallel, have profound effects on the
electrical activity of nerve cells. The effect of this resistance and capacitance together is that the
membrane gets a “time constant” – meaning that it does not respond instantaneously to ionic
currents. This time constant is called “tau” with the symbol τ A simple equation relates these 3
factors: τ = Rm.Cm. That is, the time constant of the membrane (in seconds = tau) equals
membrane resistance in Ohms (W) multiplied by membrane capacitance in Farads (F). This is one
of a few equations that you need to memorise in this topic.

Membrane proteins
The proteins that are associated with the cell membrane can be divided up into 4 major functional
families. There are;
receptors, which have neurotransmitter binding sites on the outside face of the cell,
ion channels which make pores through the membrane and allow particular charged ions to
pass through, - there are sodium channels, potassium channels, chloride channels, calcium channels
and some that are permeable to several sorts of ions (eg: “cation” channels which are permeable to
all cations = positively charged ions)
transporter proteins which help polar molecules to pass in or out of the cell…. and
structural proteins which act as anchor points for either intracellular or extracellular
cytoskeletal elements (eg: actin microfilaments), giving the cells a shape, despite the fluidity of the
membrane

Protein structure
The conformation of proteins in the membrane is largely determined by their amino acid sequences.
Some amino acids are highly polar, others are non-polar. When several non-polar amino acids are
located side-by-side in a protein sequence, the protein can span the non-polar part of the membrane.
In this way, some proteins cross backwards and forwards across the membrane, anchoring
themselves in position with defined intracellular and extracellular components. The whole protein
actually floats and can move laterally around the cell via the fluid membrane unless it is tethered by
structural proteins). The transmembrane regions of proteins are determined by the amino-acid
sequence and the gross 3-dimensional shape of proteins generally remains quite constant. However,
the fine, 3Dimensional (3D) structure of the protein can change on a tiny scale - this is caused by a
number of factors:
the electrical charge across the membrane and/or,
the pH of the extracellulr or intracellular fluid and/or
charged molecules interacting with binding sites on the protein (eg: neurotransmitters)
if strongly charged groups (eg: phosphate groups PO43-) are bonded to specific sites on them
by intracellular enzymes. "Phosphorylation" is one of the main ways that ion channels and
receptors are controlled.
These fine, "conformational changes" can be very significant, for example allowing an ion
channel to open, at a particular membrane potential. Millions of years of selection have led to the
evolution of hundreds of remarkably complex and finely tuned ion channels, each with distinctive
properties which depend on their amino acid sequences. A mutation in a single amino acid in an
ion channel protein can radically alter the behaviour of that channel, with major effects (often
disastrous) on behaviour.

As mentioned before, ion channel proteins are responsible for allowing most of the
movements of charged ions across the membrane and thereby set its overall resistance. In fact,
membrane resistance is inversely proportional to the total number of open ion channels at that
moment. The more open channels, the lower the resistance. The fewer ion channels open, the
higher the resistance. The phospholipid bilayer (basically, its total surface area) sets the
capacitance of the cell - the other important electrical property

The Na+/K+ ATPase

Before we get onto ion channels, we need to think about a transporter protein (an ion pump). One
particularly important molecule plays a key role in nerve cell membranes (and in all other cells too).
The “sodium-potassium ATPase” is a transporter protein embedded in the cell membrane. It is
classified as an ion pump – a specialised type of transporter protein. On the intracellular face of the
membrane, the molecule has a site that can cleave ATP to produce ADP+free phosphate – this
releases lots of stored energy. Thus, the molecule can also be considered as an enzyme (hence the
name: ATPase). The energy from ATP is used to rotate the rest of the molecule which acts as a
pump for sodium and potassium ions. For each ATP used up, the pump forces three sodium ions
out of the cell, in exchange for 2 potassium ions entering. In this way, in a live cell, there is an
accumulation of potassium ions to quite high concentrations inside the cell and a depletion of
sodium ions inside to quite low concentrations. Pumping ions against their concentration gradients
requires work to be done – ATP provides the energy for this process. As long as most of the ion
channels in the cell membrane are closed, the sodium potassium ATPase will set up quite strong
concentration gradients for Na+ and K+. This is a crucially important characteristic of all
mammalian cells, which also make possible the sophisticated electrical activity of nerve cells. ONE
IMPORTANT POINT TO NOTE: the Na+/K+ ATPase controls the intracellular concentrations of
Na+ and K+ - NOT the extracellular concentrations. Remember, the extracellular fluid is in
continuity with the blood stream, which is regulated by homeostatic mechanisms - mostly
controlled by the kidneys.
Terminology: When the membrane potential becomes less negative - or more positive (say, moving
from a resting potential of -60mV to -40mV) the cell is “depolarised”. When membrane potential
becomes more negative (eg: from -60mV to -80mV) the cell is “hyperpolarized”. Nerve cell
action potentials consist of a depolarisation, followed by a hyperpolarisation (where membrane
potential is more negative than resting potential). (You need to understand this terminology. It is
not acceptable to say “the membrane potential gets smaller” – does that mean it becomes more
positive, or more negative? I can’t tell, so you won’t get full marks if you use imprecise
terminology.)

Nerve cells, like all other cells in the body, are characterised by having a negative resting
membrane potential (inside the cell is negative compared to outside of the cell membrane ie:
extracellular fluid). This arises because of the differential concentration of ions inside and outside
the cell. In particular, resting membrane potential is due largely to the fact that the concentration of
K+ ions is much higher inside the cell than outside. The exact concentrations vary between different
cells, but typically the concentration of K+ inside is 15-40 times higher inside than outside. This
concentration gradient is due to the specific membrane pump mentioned above – the Na+/K+
ATPase. But that sounds very counter-intuitive! How does a high concentration of positive K+ ions
inside the cell lead to the inside being negatively charged????

Resting potential
Here's the quick answer. The concentration gradient ([K+] high inside, low outside) makes K+ ions
diffuse out of the cell. If any K+ channels are open (and there are some open, all the time) then
small numbers of K+ ions will leave the cell and enter the extracellular fluid. They do this because
substances always diffuse from areas of higher concentration into areas of lower concentration. As
positive K+ ions leave the cell, each one takes a positive charge with it. Therefore, the inside of the
cell starts to become more and more negative. As this negative charge increases, it makes it harder
for more K+ ions to leave (as they are now moving against an electrical potential gradient which
attracts positive K ions back into the negatively charged cell – this is energetically unfavourable).
Eventually, an equilibrium potential is set up where the tendency of K+ ions to move down their
concentration gradient is exactly counterbalanced by their tendency to move (in the opposite
direction) down their newly-formed electrical gradient. Two things are important about this. First,
only K+ ions can go through K+-channels, so other ions can't counteract the effect of K+
movement. Second, only tiny numbers of actual K+ ions actually leave the cell before this
equilibrium potential is reached. Because very few K+ ions actually move across the membrane
that there is a negligible effect on their overall concentration either side of the membrane. But these
few ions moving are enough to set up a membrane potential close to -90mV (inside compared to
outside of the cell). This value of -90mV is specific for the concentrations of K+ ions that exist on
either side of a normal nerve cell membrane - about 150 millimolar inside the cell and 5millimolar
outside.

Now note that I’ve only talked about K+ ions – their gradient and their ion channels. For the time
being, we can forget about Na+ ions and Cl- ions and Ca++ ions (I’ll tell you why later).

A German chemist, Walther Nernst developed an equation that can predict the balance point (called
the Equilibrium Potential) for ions, with known concentrations, moving down their specific
concentration gradient. This is purely physical chemistry – it is not specific for biological
membranes – however, it is very accurate for describing what happens in nerve cells.

The equation is horrible: here is its full form:

EK = RT/zF ln [K+]out/[K+]in
Where EK = the Equilibrium potential for K+ ions (ie: the point where the tendency of K+ ions to
move down their concentration gradient is exactly counterbalanced by their tendency to move down
the electrical gradient.
R= the gas constant;
ln = the natural logarithm
z = the charge or valence on the ion (ie: 1+, 2+)
T = the temperature in ˚K
F = the faraday constant (converts between moles and coulombs)
[K+]out/[K+]in = potassium ion concentration outside the cell divided by K+ concentration inside the
cell

Fortunately, for any particular ion and temperature, this simplifies to something that can be learned:

EK = 61.45mV * log10 [K+]out/[K+]in

So in a cell, if [K+]out = 10mM (ten millimoles per litre) and [K+]in = 100mM

Then [K+]out/[K+]in = 10/100 which equals 10-1. Log10 of 10-1 = -1. So the Nernst equation then
simplifies to:

EK = 61.45mV * -1 = -61.45mV.

Thus if [K+]out=10mM and [K+]in =100mM, then the equilibrium potential for K+ will be -61.45mV
(inside negative).

Obviously this equation only operates if K+ ions can actually move across the membrane – that is,
there must be some open K+ channels that allow K+ ions to pass through them. In normal cells,
there are always some K+ channels open and the Nernst equation predicts the resting membrane
potential moderately well. In fact, in most cells the ratio of [K+]out : [K+]in is more than 1:10, so
typically both EK and membrane potential are usually between -60 and -90mV.

____________________

Recording from nerve cells

Cell resistance and capacitance, as well as electrical activity (action potentials and synaptic
potentials), can be recorded using “intracellular microelectrodes” (also called “micropipettes”).
These “sharp” electrodes have tiny tips – usually less than 0.1µm in diameter (10-4 mm). They are
made by pulling glass capillary tubing, after first heating it to nearly white heat. This produces a
finely tapered tube, which is hollow right down to its tip. The microelectrode can be filled with a
highly conductive solution such as 3 Molar KCl (ie: 3 moles per litre of solution). By putting a
wire in the blunt end of the electrode, so that it contacts the conductive KCl solution, it is possible
to record the potential between the fine tip of the electrode and the bath. If the electrode is mounted
in a fine holder (“micromanipulator) it can be poked into a nerve cell body. The fluid membrane
then reseals around the electrode, and recordings can be made of the cell’s membrane potential
(between the inside and outside of the cell). In this way, it is possible to record resting membrane
potential, synaptic potentials and action potentials.

Really advanced difficult stuff for people who like that sort of thing: It is also possible (with the
right amplifier) to inject current into a cell through the microelectrode. Ohms law (V=IR) states
that voltage = current (“I”, measured in Amps) multiplied by Resistance (measured in Ohms). If
we inject a known amount of current, this will cause a change in the cell’s potential (ie: Volts).
From this, we can calculate the cell’s membrane resistance and get an idea of whether ion channels
are opening or closing if the resistance changes. If we calculate resistance and measure the time
constant of the cell’s responses (tau), then we can calculate the cell’s capacitance (tau = R x C.)
Capacitance is usually proportional to the surface area of the cell. The resistance value give a
good picture of whether ion channels are open at any moment (as they determine resistance) and
the voltage recording shows synaptic or action potentials as they occur.

Simon Brookes