the neuromuscular junctionwkdurfee/projects/itasca/lecturenotes.pdf · nmj lecture notes 7 5....

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

NMJ Lecture Notes 1

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 3

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 force-

generating 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 4

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.

NMJ Lecture Notes 5

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.

NMJ Lecture Notes 6

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.

NMJ Lecture Notes 7

5. Molecular Mechanism of Contraction Excitation of the sarcolemma and transverse-

tubule 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 8

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 9

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 10

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.


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 z-

lines.

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.


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

http://www.me.umn.edu/labs/hmd/lab/docs/widmaier_samplech9.pdf

http://www.rad.washington.edu/atlas2/

http://www.nlm.nih.gov/medlineplus/muscledisorders.html


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.


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


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

Reaction 2[A] + [R] < -- > [AR] + [A] < -- > A2R < -- > A2R*

Channel closed closed closed open

States:

AR A2R

Desensitized 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) ACHRs can be irreversibly blocked by the snake poison

vii) Curare block binding of ACH to its receptors. Curare is a non-activating

or non-depolarizing block.

viii) Nicotine acts at the NMJ and binds to the ACHR.

ix) 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


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.

Acetyl Transferase

AcetylCoA + Choline ------------------------ > 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 voltage-

gated channel.

a) 4 Ca2+ act cooperatively to release one quantum.

b) Reducing extracellular Ca2+ reduces ACH release.


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 voltage-

gated 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 .


e) The typical EPP amplitude is –30 mV, which represents current through

approximately 100,000 open ACH endplate channels.


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.


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.


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 ion-

selective, 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.


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 -COO-

groups 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:

Inside Outside Ratio of

outside/inside

Sodium 12 mM 120 mM 10

Potassium 110 mM 4mM 0.0363

Chloride 9 mM 124 mM 13.78

"A” ~(113 mM) -- --

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

2 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.


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.

i

o

i

o

C

C

zF

RT

C

C

zF

RTE log3.2ln

where Co = outside concentration; Ci = inside concentration; z = the charge(valence) of

the ion; and 2.3 RT/F = 61.5 mV, at 37C.

Substituting in the concentration values for potassium, we get:

mVxK

KE

i

oK 9044.15.61)04.0log(5.61

110

4log5.61log5.61

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

mVCl

Cl

Cl

ClE

o

i

i

oCl 70log5.61log5.61

This would be the value of the membrane potential if the membrane were permeable only

to chloride ions. For sodium, the equilibrium potential is

mVNa

NaE

i

oNa 5.61)10log(5.61

12

120log5.61log5.61

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

+.


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 sodium-

potassium '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.


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

oCliNaik

iCloNaok

ClPNaPKP

ClPNaPKP

F

RTE ln

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).


o

K

Cl

i

K

Na

i

i

K

Clo

K

Nao

ClP

PNa

P

PK

ClP

PNa

P

PK

F

RTE ln

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:

ii

oo

ii

oo

NaK

NaK

NaK

NaK

F

RTE

b

b

b

blog5.61ln

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


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 three-

dimensional 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!


-100

-90

-80

-70

-60

-50

-40

-30

-20

-10

0

10

20

30

40

50

60

70 61.5 mV

EK

ENa

Resting (PNa/PK) = 0.02

Hyperpolarization < 0.02

Depolarization > 0.02

Potential (mV)

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.


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. McGraw-

Hill.

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.


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.


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

http://www.me.umn.edu/~wkdurfee/publications/wiley-chap-2006.pdf


**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)

http://www.me.umn.edu/~wkdurfee/publications/wiley-chap-2006.pdf

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