Neuromuscular Junction
Lecture Notes
These notes supplement the lectures and cover background material that will help place
the experiments in context.
© University of Minnesota
Version: July, 2011
Table of Contents
1
Muscle Contraction: cardiac, smooth and skeletal_________________________ 3
2
The Neuromuscular Junction ________________________________________ 13
3
Membrane Resting Potential _________________________________________ 21
4
NMJ References ___________________________________________________ 29
5
Readings _________________________________________________________ 31
NMJ Lecture Notes
1
1 Muscle Contraction: cardiac,
smooth and skeletal
1. Types On the basis of structure, contractile properties and control mechanisms, three
types of muscle can be identified: 1) skeletal muscle, 2) smooth muscle and 3) cardiac
muscle. Although there are significant differences between these muscle types, the forcegenerating mechanisms are similar.
Skeletal Muscle: most skeletal
muscle is attached to bone and its
contraction is responsible for
supporting and moving the skeleton.
The contraction of these muscles is
initiated by action potential
propagating down motoneurons to the
muscle and can be under voluntary
control.
Smooth Muscle: sheets of smooth
muscle surround various hollow
organs and tubes (e.g., stomach,
intestines, urinary bladder, uterus,
blood vessels and airways).
Contraction of these cells may propel
the luminal contents through the
organ or regulate internal flows by
changing tube diameters. Single and
groups of smooth muscle cells are
also found distributed throughout
organs and perform various other
functions: e.g., iris of the eye and
attachment of hair. Smooth muscle
contraction can be spontaneous or
controlled by: the autonomic nervous
system, hormones and other chemical
signals.
Cardiac Muscle: The muscle of the
heart surrounds four pumping
NMJ Lecture Notes
3
chambers. Contraction of cardiac muscle provides the impetus for the movement of
blood through the pulmonary and systemic circulatory systems. Spontaneous cycling
of an intrinsic pacemaker triggers each heartbeat (or contraction). However the
autonomic nervous system and circulating hormones modulate the frequency of this
activation.
2. Structure and Function of Skeletal Muscle
If one sections through a skeletal muscle, one can observed that it is organized into
bundles of fibers call fascicles. The individual muscle fibers, multinucleated cells,
contain long slender structures called myofibrils. These are made of myofilaments,
which are organized into sarcomeres, the functional unit of contractions.
Both skeletal and cardiac muscle have a striated appearance under a light microscope,
due to the organization of the myofilaments.
Each myofibril is composed of thick and thin filaments arranged in a repeating pattern
along their length. thick filaments are composed primarily of the protein myosin and the
thin filaments are made up the three proteins, troponin, tropomyosin and actin. It is the
cyclic binding between myosin heads of the thick filament and actin of the thin filaments,
crossbridge formation, that allows of force production or muscle shortening. It should be
noted, that there exist other proteins within sarcomere which have recently been shown to
have a role in contractile function, e.g., the elastic protein titan (also known as
connectin).
3. The Motor Unit A single motor unit consists
of one motor neuron and all of the muscle fibers it
innervates. The cell bodies of motor neurons are
located within the brainstem or spinal cord. The
axons of these neurons are myelinated and large in
diameter, and thus are able to propagate action
potentials at high velocities. Once an alpha motor
neuron is activated to produce an action potential,
all of the fibers innervated by this neuron are
activated and contract simultaneously. Each
motor unit is made up of one type of muscle
fibers: i.e., slow twitch, fast-twitch fatigable or
fast-twitch fatigue resistant.
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4. Excitation-Contraction Coupling This refers to the sequence of events by which an
action potential in the plasma membrane of the muscle fiber leads to force production via
an increase in intracellular calcium and crossbridge formation and turn-over. Excitation
begins at the neuromuscular junction and then the action potential spreads over the
surface membrane and inward into the fiber via the transverse tubule system
(invaginations of the sarcolemma). This inward excitation activates calcium release from
the sarcoplasmic reticulum. The calcium then binds to the thin filament and crossbridge
formation occurs.
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4. The Neuromuscular Junction
Each branch of a motoneuron
forms a single junction with a
muscle fiber. The myelin sheath
surrounding the motor axon ends
near the surface of the muscle
fiber and the axon divides into a
number of short processes that
lie embedded in grooves on the
muscle-fiber surface. This
region of the sarcolemma
(muscle membrane) is known as
the motor end plate.
Acetylcholine is the
neurotransmitter in these
synapses. End-plate potentials
(EPPs) can be recorded at the
motor end plate when the
presynaptic membrane is
activated to release vesicles containing the acetylcholine.
Steps in neuromuscular transmission:
1) nerve action potential.
2) calcium entry into the presynaptic terminus.
3) release of Ach quanta.
4) diffusion of Ach across cleft.
5) combination of Ach with post-synaptic receptors and Ach breakdown via
esterase.
6) opening of Na+/K+ channels (cation channels).
7) postsynaptic membrane depolarization (EPP).
8) muscle action potential.
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5. Molecular Mechanism of Contraction Excitation of the sarcolemma and transversetubule system causes activation of a population of voltage-gate calcium channels located
in the tubules themselves. The channels also known as the dihydropyridine receptors
signals, by yet some unknown mechanism, the adjacent calcium-release channels on the
sarcoplasmic reticulum (ryanodine receptors) to allow calcium to be released from this
storage site. Hence, the intracellular [Ca2+] increases (i.e., sarcoplasmic concentration)
which then diffuses and binds to troponin on the thin filaments which allows for
crossbridge formation between actin and myosin by removing the steric interaction
imposed by tropomyosin.
Shown to the right is the association between changes
in intracellular [Ca2+] and force. The change in Ca
concentration is detected using a fluorescent calcium
indicator dye so that changes in relative light is related
to changes in calcium concentration. Note that a rise in
calcium precedes force production and intracellular
[Ca2+] decrease well before force.
NMJ Lecture Notes
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Functional Overview
A neuromuscular junction
sends excitatory signals from
the CNS via the
neurotransmitter,
acetylcholine which binds to
nicotinic receptors on the
post-synaptic membrane.
The binding causes a local
change in the voltage of the
sarcolemma affecting
neighboring channels (Na+ to
enter and eventually K+ to
flow out). This ion
movement produces the
action potential which
propagates along the
sarcolemma and inward via
the transverse tubule system.
This rapid voltage change
initiates the gating of
dihydropyridine receptors
which in turn causes the
release of calcium from the
sarcoplasmic reticulum via
the ryanodine receptors. The
released calcium binds to
troponin inducing a
conformation change in
tropomyosin, also a
component of the contractile
apparatus, which in turn
allows crossbridge formation between actin and myosin (an energy dependent process). The crossbridge
formation leads to muscle fiber shortening and the generation of force. Crossbridge cycling will proceed
until calcium dissociates from troponin and the inhibitory influence of tropomyosin is reestablished. The
dissociation occurs because calcium release stops and its active uptake (requiring ATP) into the
sarcoplasmic reticulum causes a reduction.
7. Metabolic pathways producing the ATP utilized during muscle contraction
There are three
primary ways a
muscle fiber can
form ATP during
contractile activity:
1) phosphorylation
of ADP by creatine
phosphate; 2)
oxidative
phosphorylation of
ADP in
NMJ Lecture Notes
8
mitochondria (need myoglobin for oxygen transfer); or 3) substrate phosphorylation of
ADP, primarily by the glycolytic pathway in the cytosol (forming lactic acid).
The phosphorylation of ADP by creatine
phosphate provides a very rapid means of
forming ATP at the onset of contractile
activity. In a resting muscle fiber, the
concentration of ATP is always greater
than ADP leading to the reformation of
creatine phosphate. During rest muscle
fibers build up a concentration of creatine
phosphate to a level approximately five
times that of ATP.
6. Force production: the frequency of stimulation and the length-tension
relationship The amount of tension developed by a muscle fiber and thus its
strength can be altered not only by the frequency of stimulation, but also by changing the
length of the fiber prior to or during contraction.
If the frequency of stimulation increase such that relaxation in not complete force will
begin to superimpose. Eventually the frequency of stimulation becomes high enough that
NMJ Lecture Notes
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the force becomes fused. Further increase in the frequency will cause more force to be
produced until eventually a maximum is reached.
If one stretches skeletal or cardiac muscle the magnitude of subsequent contractions will
be altered. If the muscle is unloaded, i.e., the sarcomere spacing compressed, there is
little force or shortening that can occur. Skeletal muscle has an optimal length (l0) at
which force is maximal due to the greatest possible numbers of crossbridges can be
formed. Most muscles in the human body are attached so to have near their optimal
length at rest.
Because skeletal muscle can shorten allowances need to be made for the sarcolemma to
also conform to this changes without being damaged. Structural proteins are present
which link the myofilaments to the surface membrane and extracellular matrix. One of
these proteins is dystrophin which is lacking is patients with Muscular Dystrophy.
7. Contraction in Smooth Muscle
This type of muscle lacks cross-striated banding patterns and the nerves which can
innervate it arise from the autonomic nervous system. Nevertheless, smooth muscle also
uses cross-bridge movements between actin and myosin molecules to produce force.
NMJ Lecture Notes
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Each smooth-muscle fiber is a spindle-shaped cell with a diameter ranging between 2 and
10 µm. Smooth muscle cells have only a single nucleus and can continue to divide. Two
types of filaments are present in the cytoplasm: thick filaments containing myosin and
thin composed of actin. The actin filaments are anchored either to the plasma membrane
or to cytoplasmic structures known as dense bodies, the smooth muscle equivalent to zlines.
Tension produced
by smooth
muscle also
varies with
length, but the
range of length
and amount of
shortening that
smooth muscle
can achieve is
greater than
skeletal muscle.
The pathways
leading to an
increase in
cytoplasmic
[Ca2+] and to
force generation
differs
significantly
between smooth
and skeletal
muscle.
Crossbridges in
smooth muscle
can form once
myosin is phosphorylated by a calcium dependent process (enzyme). There are two
sources of calcium which leads to an increase in cytoplasmic concentration prior to
contractions: 1) the sarcoplasmic reticulum , and 2) extracellular calcium.
NMJ Lecture Notes
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Some smooth muscle will generate action potentials, but they differ from skeletal muscle
in that Ca2+ is the primary ion responsible for the membrane depolarization. Some cells
spontaneously produce action potential and have pacemaker like properties. Other types
of smooth muscle are innervated by nerves. The released neurotransmitter can be
released onto varicosities along the fibers or in spaces between fibers which then requires
greater diffusion of the neurotransmitter to the receptor sited. In addition in certain
multiunit smooth muscle arrangements, excitation may be initiated on some cells via
innervation and then transmitted to additional cells via gap junctions between cells.
8. Cardiac Muscle The cardiac-muscle cells of the myocardium are arranged in layers
that are tightly bound together and completely encircle the blood-filled chambers.
Cardiac muscle combines the properties of both skeletal muscle and smooth muscle. The
cells are striated as the result of an arrangement of thick myosin and thin actin filaments.
However, cardiac cells are considerably shorter than skeletal muscle fibers and have
several branching processes. Adjacent cells are joined end to end at structures called
intercalated disks, within which are desmosomes that hold the cells together and to which
the myofibrils are attached.
Approximately 1 percent of cardiac-muscle tissue have specialized features that form the
conducting system of the heart. They send information to the contractile cells via gap
junctions. The conducting system initiates the heartbeat and helps spread the impulse
rapidly through the heart.
The heart receives a rich supply of sympathetic (norepinephrine) and parasympathetic
(acetylcholine) innervation contained in the vagus nerve.
On-Line Muscle References
The excellent chapter on muscle from Vander, Sherman & Luciano's Human Physiology
text, one of the most widely used physiology texts in the world. The chapter from the
2004 edition is available online at
www.me.umn.edu/labs/hmd/lab/docs/widmaier_samplech9.pdf
The Dept of Radiology at the University of Washington has an excellent upper and lower
extremity Muscle Atlas with images suitable for presentations. The site has a simple and
free copyright form to use the images for academic purposes.
www.rad.washington.edu/atlas2/
Medline Plus has an excellent section on muscle disorders.
www.nlm.nih.gov/medlineplus/muscledisorders.html
NMJ Lecture Notes
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2 The Neuromuscular Junction
Each branch of a motoneuron forms a single junction with a muscle fiber. The myelin
sheath surrounding the motor axon ends near the surface of the muscle fiber and the axon
divides into a number of short processes that lie embedded in grooves on the muscle-fiber
surface. This region of the sarcolemma (muscle cell membrane) is known as the motor
end plate. Acetylcholine is the neurotransmitter in these synapses. End-plate potentials
(EPPs) can be recorded at the motor end plate when the presynaptic membrane is
activated to release vesicles containing the acetylcholine.
1) Nerve and muscle have separate, intact, plasmalemmas, which are separated by a 50
nm gap (500Å) known as the synaptic cleft.
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2) An unmyelinated motoneuron terminal (i.e. presynaptic end of the axon) sits in a
specialized groove of the skeletal muscle fiber to form the neuromuscular end plate.
a) There is only one presynaptic nerve per muscle fiber.
b) Each motoneuron has several ending; each innervates only one muscle fiber.
c) All of the muscle fibers in a given motor unit contract in unison when their
motoneuron fires an action potential.
d) All muscle fibers in a motor unit are of the same fiber type (either all-slow or fast
twitch).
3) The junction or end plate region of the skeletal muscle fiber is specialized and
different from the rest of the plasmalemma.
a) Synaptic infoldings of the plasmalemma in the cleft greatly increase the
membrane surface area.
b) Receptors (protein molecules) for acetylcholine (ACHR) are located near the cleft
edge of the infoldings. In denervated muscle fibers, the ACHRs spread of the
entire muscle
plasmalemma
(sarcolemma).
i)
The skeletal
neuromuscular
junction ACHR is
nicotinic sensitive
receptor.
ii)
The nicotinic
ACHR structure is
well characterized
(i.e., cloned and
sequenced)
iii)
In its protein
moiety, the ACHR
contains:
(1) Binding sites for
acetylcholine
(ACH) and like
molecules (agonists and antagonists).
(2) Ligand-gated cation channel
(3) Several types of modulator sites
NMJ Lecture Notes
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c) The are 5 subunits of the ACHR: 2 alpha, beta, gamma and epsilon.
d) The channel and the ACH binding sites are on the alpha subunits.
e) The ACHR is a non-specific cation channel which opens and closes in response to
ACH binding and unbinding (insensitive to TTX or TEA)
i)
In the presence of an elevated [ACH] in the cleft ACH binds to the
extracellular side of the receptor and the channel opens.
Fast
2[A] + [R] < -- > [AR] + [A] < -- > A2R < -- > A2R*
closed
closed
closed
open
Reaction
Channel
States:

AR
Desensitized


A2 R
desensitized
ii)
The binding of the 2 ACH molecules to a single receptor elicits positive
cooperativity.
iii)
When the ACHR channel opens at a normal muscle fiber resting
membrane potential, Em =90 mV, the net current through the channel is
inward and depolarizing.
iv)
ACH unbinds from the ACHR after the channel closes when the [ACH] in
the endplate decreases due to diffusion from the cleft and is broken down by
an acetylcholinesterase (ACHE). Prolonged ACH (> 100 msec) stimulation
leads to inactivation of the channels through a change to the desensitized state.
v)
Then the end plate channel opens, net current (I) from all cations in
inward: this positive charge (q+) movement through the myoplasm to the
surrounding sarcolemma cause a capacitate change (depolarization) which
then affects the gating of the voltage-sensitive Na+ and K+ channels.
vi)
vii)
ACHRs can be irreversibly blocked by the snake poison
Curare block binding of ACH to its receptors. Curare is a non-activating
or non-depolarizing block.
viii)
ix)
Nicotine acts at the NMJ and binds to the ACHR.
There are substances, which mimic ACH, but are not readily broken down
by ACHE, thus that cause and initial opening of the channel and then
NMJ Lecture Notes
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inactivation through desensitization. Succinylcholine is one of these
depolarizing muscle relaxants.
4) The nerve terminal has vesicles (50 nm in diameter) containing ACH which fuse with
the plasmalemma and release ACH into the cleft after the nerve AP depolarizes the
membrane and Ca2+ enters through channels in the nerve terminal.
a) Formation of ACH in nerve terminal.
AcetylCoA + Choline
Acetyl Transferase
------------------------ > ACH
+ CoA
b) ACH is stored in vesicles in the nerve terminal.
c) Quantum: Smallest amount of ACH released. Probably the amount of ACH in a
“standard” presynaptic vesicle is: Quantum = 2,000 to 10,000 ACH molecules.
5) The NMJ cleft is filled with extracellular fluid and ground substance, which also
contains the enzyme acetylcholinesterase (ACHE).
ACHE
ACH -------------- > Acetate + Choline
6) ACHE acts only on unbound ACH. Acetate and choline are transported back into the
nerve terminal. Acetate is converted to acetylCoA (in mitochondria) and then
combines with choline to reform ACH.
a) Organophosphates inhibit ACHE and thus prolong ACH lifetime. Inhibitors of
ACHE are: physostigmine and neostigmine, which are used clinically to reverse
neuromuscular blockage.
Characteristics of ACH release:
1) As a result of AP depolarization, Ca2+ enters the nerve terminal through a voltagegated channel.
a) 4 Ca2+ act cooperatively to release one quantum.
b) Reducing extracellular Ca2+ reduces ACH release.
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c) Mg2+ competes with Ca2+ and does not active ACH release; increasing
extracellular Mg2+ decreases ACH release.
2) One nerve AP causes the release of approximately 300 quanta (vesicles)
1 AP ------ > 300 quanta released ----------- > 1,500,000 ACh molecules
(assuming 5,000 ACH/quantum) Some of this ACH diffuses out from the cleft and
some is broken down by ACHE: approximately 200,000 molecules bind to ACHR to
open channels in the endplates.
3) Factors which alter or block nerve APs will alter ACH release:
a) Local anesthetics (e.g., procaine) inhibit voltage-gated Na+ channels and interfere
with AP transmission in the nerve. Some local anesthetics also act on the ACHR
by promoting desensitization and/or by blocking the channel.
b) An increase [K]o causes prolonged depolarization of the nerve and thus partial
inactivation of the voltage-gated Na+ channels, thus alters AP transmission.
Hemicholinium inhibits uptake of choline into the nerve terminus and thus decreases
ACH production and storage. The result is decreases ACH/quantum.
Botulinum toxins block the release of ACH from the nerve terminals (i.e., paralysis from
bad tuns).
Characteristics of the End Plate Potential (EPP):
1) Opening of the end plate channels and the subsequent net inward current sets up a
transient depolarization of the sarcolemma adjacent to the end plate.
a) The end plate potential (EPP) spreads electrotonically and thus decrements in
amplitude with distance from the end plate region. The EPP itself in not
propagated but serves as the stimulus to drive the Em to threshold for an AP to be
initiated. The EPP can be seen in isolation of an AP by treating the muscle fibers
with tetrodotoxin (TTX) which blocks the voltage-gated Na+ channels.
b) The EPP brings the adjacent sarcolemma to and beyond threshold: the voltagegated channels in the non-endplate region of the membrane are then responsible
for the propagation of the AP throughout the length of the muscle fiber.
c) EPPs last approximately 5-10 msec.
d) Normally: 1 nerve AP -- > 1 EPP -- > 1 muscle AP -- > a single twitch .
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e) The typical EPP amplitude is –30 mV, which represents current through
approximately 100,000 open ACH endplate channels.
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2) Mini-EPPS (MEPPs) occur spontaneously
independent of the nerve AP, although
membrane depolarization increases and
hyperpolarization decreases their frequency of
occurrence.
a) Are due to the release of quantum =
approximately 5,000 ACH molecules.
b) Amplitudes are approximately 0.5 to 1
mV.
c) Ca2+ and Mg2+ do not alter the magnitude
or time course of MEPPs, but due alters
the number released.
NMJ Lecture Notes
19
Review: Steps in neuromuscular transmission:
1) nerve action potential.
2) calcium entry into the presynaptic terminus.
3) release of Ach quanta.
4) diffusion of Ach across cleft.
5) combination of Ach with post-synaptic receptors and Ach breakdown via
esterase.
6) opening of Na+/K+ channels (cation channels).
7) postsynaptic membrane depolarization (EPP).
8) muscle action potential.
NMJ Lecture Notes
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3 Membrane Resting Potential
The nervous system uses electrical signals to communicate over relatively enormous
'biological distances'. It does so with speed and accuracy, and deals in a vast traffic of
signals distributed to millions of cells. Present-day emphasis on the nerve membrane
and the resting potential is far more than a political dogma to bedevil the student. For in
understanding these processes, we glimpse the basic strategy used by neurons to carry-on
their mission of information processing. This strategy--sometimes loosely called the
ionic hypothesis - appears to be common to all nerve cells, and to be a specialization of
the general phenomenon of irritability present in all cells from the dawn of life.
Our knowledge of the, subject is by no means complete, but sufficient elements have
been assembled to allow the conclusion that this strategy itself is a remarkably simple
one, but also elegant in that there is a capacity for many individual variations on a
common theme. The necessary elements appear to be: 1) the presence of an ionselective, semi-permeable membrane;
2) the ability of the cell to concentrate different
amounts of sodium, potassium and chloride across the membrane, 3) passive physical
forces of diffusion and electrical gradients; and 4) an active process (principally operating
on sodium and potassium ions), that aids in maintaining the various ionic concentration
profiles.
1) The Nerve Membrane. The nerve membrane has been extolled to you in
biochemistry, biology and physiology, so we will not dwell in detail on it here. Suffice it
to say that there exists a membrane composed basically of a bi-layer of lipid leaflets
embedded in which at various distances are proteins. Many of these proteins have water
permeable pores or channels in them which allow one or more ion species through. Some
of these channel-bearing proteins are "gated" (can open and close as the result of
something that forces a change in their structure), some are theorized to be passive and
always open. These latter channels are what are called “resting channels", and we will
concentrate on these, as it is thought they are responsible for the resting permeability of
the membrane and therefore for the resting potential. The "gated" channels operate to
produce changes in the membrane potential and therefore mediate information
exchanges.1
In the absence of any active -information processing, the nerve membrane maintains a
potential difference between the inside of the cell and outside of some 70 to 90 millivolts,
with the inside negative (1 mV = 10-3 volts), and this voltage difference is called the
1
No one has ever "seen" a resting channel, and there are various alternative explanations that could account
for the passive, resting permeability of the membrane. For instance, gated channels that flicker open and
closed on a probabilistic basis could account for much of the resting permeability However, for present
didactic reasons, it is easier to just theorize that resting channels exist.
NMJ Lecture Notes
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resting potential. 70 millivolts may seem like a very small electrical force compared to
the 120 volts of city power you may have painfully sampled on occasion, but this 70 mV
is acting across a correspondingly small distance. To obtain a "feel" for the actual force
one has to look at the ratio of the resting potential to the thickness of the membrane. 70
mV across an approximately 100 membrane corresponds to a force of 70,000
volts/centimeter. This is more than adequate to make proteins with charged groups
imbedded in the membrane stand-up and salute!2 Indeed, such a powerful electrical field
can and does regulate the three- dimensional configuration of some of the gated
membrane protein channels ("voltage gated channels") causing them to open and/or close
depending on voltage changes that occur across the membrane.
2) Ionic Profiles. The typical mammalian extracellular solution contains about 120 mM
(millimoles) of sodium, 4 mM of potassium and 124 mM of chloride ions. Inside the
cell, the relative concentrations are almost reversed. We find about 12 mm of sodium,
110 mM of potassium and 9 mM of chloride ions. There are sufficient, large,
impermeable anions (bicarbonate, glutamate, aspartate and organo-phosphates and -COOgroups of cellular proteins) to bring the sum total of negative charges inside about equal
to the number of negative ions outside. Thus from a macroscopic view, the inside and
outside contain an almost equal number of both positive and negative ions. The table
below sums up the ion concentrations:
Sodium
Potassium
Chloride
"A”
Inside
Outside
12 mM
110 mM
9 mM
~(113 mM)
120 mM
4mM
124 mM
--
Ratio of
outside/inside
10
0.0363
13.78
--
From a macroscopic view, then, the inside and outside contain an almost equal number of
both positive and negative ions. There must exist, however, a small imbalance of charge
immediately across the membrane or there would be no resting potential. It should be
emphasized though, that the required amount of excess negative charge inside, separated
from the outside by less than 100 Angstroms, is almost immeasurably small. In fact, the
ability of small amounts of charge to produce significant voltage changes across the
nerve membrane is one of the great utilities of the system: small amounts of charge
migrating the small distance across the membrane introduce very rapid, precise
changes in voltage
3) Passive Physical Forces of Diffusion and Voltage: The Nernst Fquation.
Given the ionic profiles, we are faced with explaining how they lead to the resting
potential. As a first step, consider what the situation would be if the membrane were
permeable only to one ion species (had only one kind of protein channel that was
permeable to only one kind of ion). Taking potassium (K+) first, the relatively large
1 Ả = 10-8 cm., 100 Ả = 10-6 cm. To scale the membrane and voltage to recognizable dimensions,
multiply both the membrane thickness and the voltage by 106. This gives I cm. for the membrane now, and
70 x 10-3 xIO6 =7o x 103 volts = 70,000 volts for the scaled electric field.
2
NMJ Lecture Notes
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concentration of the K+ ions on the inside should cause K+ ions to diffuse outward
toward the more dilute solution of K+ ions outside. Every ion of K+ that diffuses across
the membrane will carry a positive charge with it, leaving behind an excess of negative
charge. It is just this separation of charges which will produce an electric field, or
potential. Moreover, increasing the number of charges which are separated, will tend to
impede subsequent, positively charged K+ ions from diffusing outward. Indeed, it can be
shown that an equilibrium will soon be reached such that the electrical field will exactly
oppose the force of diffusion! In this state, as many potassium ions are attracted across
the membrane from outside to inside by excess negative charges, as are shoved from
inside to outside by the force of diffusion. The quantitative expression of this balance is
the Nernst Equation for the single ion species.
E
RT  Co
ln 
zF  Ci

C
RT
  2.3
log o
zF

 Ci



where Co = outside concentration; Ci = inside concentration; z = the charge(valence) of
the ion; and 2.3 RT/F = 61.5 mV, at 37C.
Substituting in the concentration values for potassium, we get:
K 
 4 
E K  61.5 log o   61.5 log
  61.5 log( 0.04)  61.5 x 1.44  90mV
 110 
 Ki 
EK = -90 mV if the membrane were permeable only to potassium ions. In other words, a
voltage difference of -90 mV would exactly oppose the diffusion force for the listed ionic
concentrations of potassium across the nerve cell membrane. This is called the
equilibrium potential for potassium. Looking at chloride, we obtain3
 Cl 
 Cl 
ECl  61.5 log o   61.5 log i   70mV
 Cli 
 Clo 
This would be the value of the membrane potential if the membrane were permeable only
to chloride ions. For sodium, the equilibrium potential is
 Na 
 120 
E Na  61.5 log o   61.5 log
  61.5 log(10)  61.5mV
 12 
 Nai 
That is, the membrane potential would be 61.5 millivolts, inside positive, if the
membrane were permeable only to sodium ions.
3
Note that multiplying a log ratio by –1 simply reverses the position of the numerator and denominator.
This is an algebraic trick employed to keep minus signs out of textbook equations. Its end result here is
that the equilibrium expression for chloride has the inside and outside concentrations reversed when
compared with K+ and Na+.
NMJ Lecture Notes
23
Looking at the values obtained we can draw a number of conclusions about the actual
state of affairs. First, the actual resting potential of -70 mV agrees well with the chloride
equilibrium potential. This implies that chloride is distributed passively across the
membrane. If we artificially pass current into the cell to change the resting potential,
chloride concentrations will also change (over a period of time) such that they will
balance out the right hand side of the Nernst equation to equal the artificially imposed
resting potential. Nothing is acting on chloride other than the forces of diffusion and
voltage. Second, neither potassium nor sodium appear to be in equilibrium. We would
predict that with a resting potential of -70 mV and the given concentrations of Na and K,
a small amount of potassium should tend to diffuse out of the cell and a much larger
amount of Na should diffuse into the cell if both ions were equally permeable to the
membrane. In fact, potassium permeates the resting cell membrane about 50 times more
readily than sodium, and about an equal amount of potassium tends to diffuse out as
sodium goes in. Our third, conclusion is that given the figures on sodium and potassium,
some force other than the passive forces of diffusion and electrical field is necessary to
maintain the measured quantity of potassium so high (and that of sodium so low) inside
the cell. This is where the role of the sodium-potassium 'pump' (sometimes just called the
'sodium pump") figures in. Active transport in terms of the ATP-dependent sodiumpotassium 'pump' is constantly at work to maintain the K+ and Na+ concentrations
at their stated values. This is homeostasis in action.
The Na-K active transport may be considered as a background process in that the pump
cranks along, using cellular energy to maintain the K+ and Na+ gradients constant. If
something happens to transiently change the leakage rates of these two ions, it is known
that the pump will speed up a little bit or slow down a little bit to catch things back-up
within a few seconds or minutes, thereby maintaining the desired gradients. Indeed, the
ATP-ase activity of the sodium-potassium coupled transport is one of the principle
homeostatic mechanisms of most cell membranes studied. Present studies are moving
closer to physically characterizing the structure and exact function of this system.
One other thing to note: by exchanging one Na+ for one K+ ion, the pump is "electrically
neutral"; i.e., it does not change the charge concentration across the membrane, and
therefore it does not contribute directly to either the resting potential or to changes in the
resting potential. It works indirectly by maintaining the differential ion concentrations,
which in turn exert their passive forces as expressed in the Nernst equation above and in
the Goldman- Hodgkin-Katz equation explained below.4
4
There are actually a number of different, generic Na-K pumps and for most of them, the exchange
ratios are not exactly 1:1. In these cases, the pumps are considered "electrogenic" in that they do contribute
directly to the resting potential via unequal pumping ratios, but they still continue to contribute indirectly as
well by maintaining ionic gradients. The electrogenic effects of all such pumps studied are actually minor
ones, however, and they contribute only a few mV of potential. Much is made of them in many recent
texts, unfortunately, as they are a more recent and "hot" research discovery topic, and authors of textbooks
tend to overplay new things. Students should be as astute as we are, however, and realize that this aspect,
while exciting to study in the research laboratory, should not detract from the basic concept and utility of
the over-all homeostatic process, which is to maintain the constant ionic concentration gradients across the
membrane.
NMJ Lecture Notes
24
The Goldman-Hodgkin-Katz Equation. We are still left with the question, why 70
millivolts for the resting potential? A clue to the answer lies in realizing that the cell
membrane is not permeable to just one ion species but is instead permeable to all three
major ions, Na, K and Cl. We must quantitatively account for the relative permeabilities
of these ions in order to understand why -70 mV, and to do that we have to go beyond the
Nernst equation. If one were to stir a small quantity of oranges and a large quantity of
grapefruit on a platform with many holes the size of oranges and only a very few large
enough for grapefruit, the net result would be that more oranges than grapefruit would
fall through. The same principle holds for relative permeability of ions. The relative
permeability of the membrane to the various charged ions will determine which ion
species is most important in carrying charges across the membrane, and therefore which
of the ion species will have the greatest influence on 'setting' the resting membrane
potential.
The mathematics of the diffusion process gets complicated in so doing, but the three men
named above were able, over about 10 years time of independent and of collaborative
effort, to mold a relatively simple expression which considers both concentrations and
permeabilities. This is the GHK Equation, expressed as
E
RT  Pk K o  PNa Nao  PClCli 
ln 

F  Pk K i  PNa Nai  PClClo 
where:
PK = 1 x 10-6 (membrane permeability of potassium compared to free diffusion)
PCl = 1 x 10-6 (membrane permeability of chloride compared to free diffusion)
PNa = 2 x 10-8 (membrane permeability of sodium compared to free diffusion)
Note that the Goldman-Hodgkin-Katz equation gives a potential for the steady-state
condition where as many plus charges are flowing out across the membrane as are
flowing in. The same is true for minus charges. Only chloride is in thermodynamic
equilibrium, and the resting potential is a steady-state potential defined as no net flow of
charge. It is assumed, of course, that the concentration profiles remain constant due to
the active transport of Na and K by the sodium- potassium pump. In fact, membrane
permeabilities of both K and Na are so low that a nerve axon can be metabolically
poisoned to block active transport, and the concentration profiles will change measurably
only after many minutes or hours (depending on the diameter and volume of the fiber).
While not dwelling excessively on mathematics, it is instructive to play a few algebraic
games with the G-H-K equation to illustrate the relative importance of the terms within
the brackets. Divide all terms upstairs and down by PK. (This does not change the
equation, as PK/PK = 1, but it allows for rearrangement of terms).
NMJ Lecture Notes
25

 PNa 
P  
 Na o   Cl Cl i 
 K o  
RT 
 PK 
 PK  
E
ln

F 
P 
P 
 K i   Na  Nai   Cl Cl o 


 PK 
 PK 
Now, disregard the terms with chloride, since we know that chloride will passively
distribute its concentration to agree with whatever K and Na determine. (You might try
calculating E with and without deleting the chloride term to convince yourself of this).
Finally, if you get tired of writing PNa/PK, let this quantity be b, substitute it in, and we
obtain:
E
 K o  bNao 
RT  K o  bNao 
ln 
  61.5 log 

F  K i  bNai 
 K i  bNai 
Note that in the resting state, b is small because PNa is about 50 times smaller than PK
which divides it in the term. If you substitute in the concentrations, you will see that the
potassium concentration dominates (because Na terms are multiplied by about .02). We
expect, and we do find, that changing extracellular K concentration will greatly affect the
resting membrane potential while changing extracellular Na causes little change.
The Strategy.
One last feature remains to be considered, and herein lies the insight into the basic
strategy employed by the neuron in affecting voltage changes as its information signaling
mechanism. Since Na and K are, in fact, not in thermodynamic equilibrium, and because
the cell will maintain the ionic profiles cited above, a small change in the membrane's
permeability to either K or Na will cause the trans-membrane potential to change. The
resting state is, then, a true source of potential energy. Increase sodium permeability for
a fraction of a second, and the cell will depolarize (become less negative inside or less
polarized) towards the sodium equilibrium potential. Increase PK and the membrane will
hyperpolarize (become more negative or more polarized) towards the potassium
equilibrium potential. Few ions need to flow (and these will generally be handled over
the long haul by active transport). The concentrations remain effectively the same, but
the membrane potential will fluctuate rapidly towards the equilibrium potential of
whatever ion appears to dominate in the equation. Figure 1 at the end of this essay
summarizes the situation.
The change in permeability may be brought about by chemicals acting on chemically
gated channels in the membrane (as it is in synaptic transmission), or it may be that
forced changes in the electrical field across the membrane may trigger a voltage gated
channel (as is the case for the action potential). The permeabilities of both Na and K may
either increase (by opening gated channels) or decrease (by blocking resting channels),
they may do so simultaneously or in sequence, as there are a large number of different,
chemically gated and voltage gated channels. Other ions (chloride for instance, calcium
in cases such as in cardiac muscle) may also figure into the permeability changes, but
NMJ Lecture Notes
26
they do so in the same manner as sodium and potassium. In other words, the equation
can be extended within the brackets to incorporate other ion species, and while it makes
the equation more complicated to look at, the strategy by which membrane potential is
changed remains the same.
What isn't known is all the physical details of the different types of channels. We do
know that the major ion species all have membrane proteins which are pretty much
selective to each of them, and this makes common sense in considering how exquisite
and important is the control over permeabilities. Moreover, there are some channels that
are equally permeable to both Na and K (try putting b = 1 into the GHK equation and
predicting what direction the membrane potential will go when this type of gated channel
opens up). We also know that the permeability sensitivity of neuronal membranes to
various chemicals or to changes in electrical potential varies from cell to cell (i.e.,
different cells have different kinds of gated channels) and indeed, varies across the
different parts of each neuron. Thus different drugs may affect different neurons or may
block permeability changes on only a specific part of the neuron because of the drug's
effect on a specific channel.
Modern research now accepts the overall concepts given here, and the exciting new
research emphasis is exactly concentrating on understanding the proteins that make up
the gated channels. Considerable progress has been made on a few of these: the sodium
ion channel that is voltage gated and responsible for action potentials in nerve axons has
been isolated, cloned and sequenced; the acetylcholine receptor of the neuromuscular
junction, which contains a large channel that lets both Na and K through simultaneously,
has been isolated, purified, and sequenced; in addition numerous antibodies that bind to
specific parts of this protein have been made and used to help us understand its threedimensional structure. Other channels have been sufficiently isolated to allow for their
placement into "artificial" membranes in such a way as to make study of them easier.
Understanding these channels better should lead to a more complete understanding of
membrane. As pathologies of some channels lead to specific disease syndromes,
understanding their structure should eventually lead to better clinical control of the
diseases. As an example, auto immune attack on the above mentioned acetylcholine
receptor leads to the disease called myasthenia gravis, and you will hear more about this
later in the course. As the molecular biology of this receptor gets better understood,
means for curing this disease becomes increasingly probable. That may be as it 'is, and
one can get very excited about the research. What is just as exciting, however, is that we
now understand the basic functional concepts underlying the membrane potential and
how it is controlled. As explained above, it really isn't all that complicated, but it is an
elegant system in its purity, efficiency and in the number of combinations and
permutations that can lead from it. Indeed, understanding the membrane potential and
the natural strategy for controlling it through permeability changes really reduces much
the rest of basic neurophysiology to practical examples, and that should make any student
of this subject happy!
NMJ Lecture Notes
27
Potential (mV)
70
60
50
40
30
20
10
0
-10
-20
-30
-40
-50
-60
-70
-80
-90
-100
61.5 mV
ENa
Depolarization > 0.02
Resting (PNa/PK) = 0.02
Hyperpolarization < 0.02
EK
Figure 1. The membrane potential depends on its relative permeability to ions.
Permeability, and thus the membrane voltage, changes as voltage-dependent
or chemically-dependent gated channels open and close. At the resting
potential, PNa/PK = 0.02. When the ratio becomes larger than 0.02 (i.e.
increases or decreases), depolarization results. When the ratio becomes less
than 0.02 ( decreases or increases), hyperpolarization results. The two
equilibrium potentials, E = 61.5 mV and E = -90 mV, set the upper and
lower limits of the possible potential changes.
NMJ Lecture Notes
28
4 NMJ References
If you want to read more about neuromuscular physiology, try these references.
Vander A, Sherman J, Luciano D (2001). Human Physiology: The Mechanisms of Body
Function, 8th ed. McGraw-Hill.
An excellent overview of human physiology. Covers everything, but has nice sections on nerve
and muscle. Used in the core undergrad intro to physiology courses (PHYSL 3051, 6051) at the
University of Minnesota.
Kandel E, Schwartz J, Jessell T (2000). Principles of Neural Science, 4th ed. McGrawHill.
The bible of neurosciences. Excellent chapters on nerve and the NMJ. Every student of the
neurosciences should own this book.
Hodgkin A (1992). Chance and Design. Cambridge University Press.
Short autobiography of one author of the Hodgkin-Huxley equations. A wonderful book about his
work, starting as a student, which elucidated the ionic basis of neuronal and muscle excitability.
Koch, C (1999). Biophysics of Computation. Oxford University Press.
Reference for biophysics of neurons. Chapters 1-4, 6 and 8 of particular interest for this week.
Hille, B (2001) Ion Channels of Excitable Membranes. 3rd Ed. Sinauer, Sunderland ,
Mass.
Standard textbook and reference on ion channels.
Oakley B, Schafer R (1978) Experimental Neurobiology: A Laboratory Manual,
University of Michigan Press.
One of the only step-by-step guides on methods of basic neuroscience experiments. Out of print. If
you get lucky, you might find one in a used book stores.
Loeb G, Gans C (1986). Electromyography for Experimentalists. University of Chicago
Press.
Excellent coverage of experimental equipment for neurosciences, including how to build your
own. The book is a little old, but most of the information is still valid.
Adrian RH (1956) The effect of internal and external potassium concentration on the
membrane potential of frog muscle. J Physiol 133:631-658.
NMJ Lecture Notes
29
The classic paper on this topic. Included in the readings section of these lecture notes.
Magleby KI (1984) Neuromuscular transmission. In: The Anatomy, Physiology, and
Biochemistry of Muscle. Chapter 13, pp. 393-418.
Coverage of the NMJ, classic experiments on recording EPPS and MEPPs. Included in the
readings section of these lecture notes.
Matthews, G.G. (1998). Cellular Physiology of Nerve and Muscle. 3rd ed. Blackwell
Science.
Excellent overview of the principles at work in excitable cells. Textbook used for Univ of Minn
physiology courses.
Aidley, D. (1998). The Physiology of Excitable Cells. 4th ed. Cambridge Univ. Press
Excellent overview of the principles at work in nerve and muscle cells.
NMJ Lecture Notes
30
5 Readings
This section contains primary source material that should be read prior to the course.
Contents
Adrian RH (1956) The effect of internal and external potassium concentration on the
membrane potential of frog muscle. J Physiol 133:631-658.
A classic paper on how potassium concentration changes change the resting potential of a
membrane. You will be doing a similar experiment in this course.
Magleby KL (1984) Neuromuscular transmission. In: The Anatomy, Physiology, and
Biochemistry of Muscle. Chapter 13, pp. 393-418.
Coverage of the NMJ, classic experiments on recording EPPS and MEPPs. Relevant to all
microelectrode recording experiments you will do this week. Included in the readings section of
these lecture notes.
Engle AE (1994). Congenital myasthenic syndromes. In Neurologic Clinics of North
America, 12(2):401-437.
Overview of NMJ disorders. You only need to skim this one.
Durfee, W.K. and P.A. Iaizzo. Rehabilitation and muscle testing. In: Encyclopedia of
Medical Devices and Instrumentation, 2nd ed . J.G. Webster, ed., Vol 6, pp 62-71,
Hoboken, John Wiley & Sons, 2006.
Review of clinical human muscle force testing. (available on-line at
www.me.umn.edu/~wkdurfee/publications/wiley-chap-2006.pdf
NMJ Lecture Notes
31
**Note to handout assembler: Replace this page
with the following articles:
Adrian RH (1956) The effect of internal and external potassium concentration on
the membrane potential of frog muscle. J Physiol 133:631-658.
Magleby KL (1984) Neuromuscular transmission. In: The Anatomy, Physiology,
and Biochemistry of Muscle. Chapter 13, pp. 393-418.
Engle AE (1994). Congenital myasthenic syndromes. In Neurologic Clinics of
North America, 12(2):401-437.
Durfee, W.K. and P.A. Iaizzo. Rehabilitation and muscle testing. In: Encyclopedia
of Medical Devices and Instrumentation, 2nd ed . J.G. Webster, ed., Vol 6,
pp 62-71, Hoboken, John Wiley & Sons, 2006.
(available on-line at www.me.umn.edu/~wkdurfee/publications/wiley-chap-2006.pdf)
NMJ Lecture Notes
33