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Sept., 2008
Introductory Medical Physiology
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i. Abstract
ii. Content
1. Excitable tissues
1.1 The meaning and significance of excitable tissues
1.2 Characteristics and roles of excitable tissues
1.3 Membrane potentials and ion channels
2. The intracellular and extracellular environments, and the semi-permeable
membrane
3. Concentrations, gradients, and permeability
3.1 Concentration gradients
3.2 Electrical gradients
4. Membrane potential
4.1 The meaning and significance of membrane potential
4.2 Simulation of membrane potential using a Membrane Model
4.3 Establishing resting membrane potentials
4.4 Maintaining gradients and potentials across the cell membrane
4.5 Graphical representation of membrane potentials
4.6. Calculation of equilibrium potential of ions using the Nernst equation
4.7 Calculation of membrane potential using the Goldman equation
Membrane potential exists across the plasma membrane of every cell in the body. At
rest (when not stimulated) the membrane potential reads -40 to -70 mV, reflectingunequal distribution of charges brought about by unequal ion distribution across the
plasma membrane. This phenomenon is basically due to the special characteristics of
the plasma membrane of every cell. Nevertheless, only excitable cells (neurons andmuscle cells) are able to respond to stimuli by transforming the resting membrane
potential into action potential and utilising it to carry information to the next cell. This
phenomenon is also due to a special characteristic of the excitable cells namely thepresence of gated ion channels in the plasma membrane of these cells. These ion
channels could be voltage-gated, ligand-gated, or mechanically-gated channels which
respond to voltage, chemical substances, or mechanical stimulation respectively toproduce information-carrying action potentials. The membrane potential could be
determined based on the distribution of ions across the membrane.
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iii. Checklist of topics and activities in this module
Content Page Comments on
mastery
i Abstract 4
ii Learning resources 4
iii Background knowledge 5
iv Terms to know 5
v Learning objectives 6
vi Learning activities 8
1 Excitable tissues 8
1.1 The meaning and significance of excitable tissues 8
ACTIVITY 5.1. Reception and processing of
information by cells
8
ACTIVITY 5.2: Excitable tissues 8
1.2 Characteristics and roles of excitable tissues 9ACTIVITY 5.3: Special property of excitable cells 9
ACTIVITY 5.4: Sensory receptors 9
ACTIVITY 5.5: Flow of electrical information inthe body
11
1.3 Membrane potentials and ion channels 11
ACTIVITY 5.6: Significance of membranechannels
11
2 The intracellular and extracellular
environments, and the semi-permeable
membrane
12
ACTIVITY 5.7. Movement of substances acrosscell membranes
13
ACTIVITY 5.8. Semi-permeable membrane 13
ACTIVITY 5.9: Phospholipid bilayer 14
3 Concentrations, gradients, and permeability 15
3.1 Concentration gradients 15
ACTIVITY 5.10: Diffusion of solute molecules 15
ACTIVITY 5.11: Concentration gradient and
diffusion
16
3.2 Electrical gradients 16ACTIVITY 5.12: Electrical and concentration
gradients
16
ACTIVITY 5.13: Permeability of plasmamembrane
17
4 Membrane potential 17
4.1 The meaning and significance of membrane
potential
17
ACTIVITY 5.14: Membrane potential 18
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ACTIVITY 5.15: Calculation of membrane
potential
18
4.2 Understanding membrane potential by working
with the Membrane Model
19
ACTIVITY 5.16: Simulation of membrane
potentials
19
ACTIVITY 5.17: Measuring membrane potential 21
4.3 Establishing resting membrane potentials 22
ACTIVITY 5.18: Membrane conductance 23
ACTIVITY 5.19: Equilibrium potential 24
4.4 Maintaining gradients and potentials across the
cell membrane
24
ACTIVITY 5.20: Maintaining equilibrium
potential
24
ACTIVITY 5.21: Na+ /K
+ -ATPase 25
ACTIVITY 5.22: Simulation of Na+ /K
+ -ATPase 27
4.5 Graphical representation of membrane potentials 28ACTIVITY 5.23: Graphical representation of
membrane potential
28
4.6. Calculation of equilibrium potential of ions using
the Nernst equation
30
4.7 Calculation of membrane potential using the
Goldman equation
31
vii Summary 32
ACTIVITY 2.24: Summary 32
viii Conclusion 33
ix Assessment 34
x Appendix A: Membrane template 43Appendix B: Membrane template elements 44
Appendix C: Model of voltmeter 45
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iv. Learning Resources
Please refer to the following sources for further information:
• Boron and Boulpaep, Ch. 2 & 3
• Guyton & Hall, pp 10-12 & Ch. 4
• Ganong, Ch. 1
• Marieb, Ch. 3. + Study partner
• Tortora & Grabowski, Ch. 3
• Vander, Sherman & Luciano, Ch. 3
• Supplementary materials provided
Weblinks:
http://www.lifesci.ucsb.edu/~mcdougal/neurobehavior/modules_homework/lect2.dcr
resting membrane potential
http://sky.bsd.uchicago.edu/lcy_ref/synap/resting.html Tutorial in basic neurobiology
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v. Background knowledge
Your Matriculation or STPM biology and the introductory lectures on General Anatomythat you have just had should be sufficient to get yourselves ready to enjoy going through
this module. It would also be helpful for you to review the following:
• the general structure and functions of cell membranes (Module 1).• the mechanisms of solute transport across cell membrane (Module 2).
• the definition of chemical and electrical gradients.
• the mechanism of signal transduction (Module 3).
vi. Terms to know
After studying the materials and doing the activities in this module, students should be
comfortable with the following terms:
action potentialactive transport
body fluid compartments
channelschemically activated gates
concentrationconcentration gradientsconductance
cytoplasm
depolarization
diffusiondynamic equilibrium
electrical gradients
electrodeelectrochemical gradient
electrogenic pump
fatty acid (tails)gated channels
hydrophilic
interstitial fluid (ISF)hydrophobic
ion binding sites
membrane permeabilitymembrane potentials
milliseconds (msec).Na
+ /K
+-ATPase
net charge
net movement
passive channels
phospholipid bilayerresting membrane potential
semi-permeable
solutesolvent
voltmeter
Please add other terms that you feel are relevant to your understanding of this module.
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vii. Learning objectives
Objectives from the American Physiological Society
NEU 1. Define, and identify on a diagram of a neuron, the following regions: dendrites,
axon, axon hillock, soma, and synaptic cleft.
NEU 2. Write the Nernst equation, and explain the effects of altering either the
intracellular or extracellular Na+, K
+, Cl
-, or Ca
2+ concentration on the equilibrium
potential for that ion.
NEU 3. Describe the normal distribution of Na+, K
+, Ca
2+, and Cl
- across the cell
membrane, and using the chord conductance equation, explain how the relativepermeabilities to these ions create a resting membrane potential.
NEU 4. Describe ionic basis of an action potential.
NEU 5. Contrast the generation and conduction of graded potentials with that of actionpotentials, identifying on the neuron the area in which each occurs.
NEU 6. Describe the basis for the calculation of the space constant and time constant ofneuron process.
After studying the materials in Module 5, the students should be able to:
1. Define “excitable tissue” by giving examples, by relating to its characteristics,and by stating its significance.2. Differentiate between intracellular and extracellular ionic constituents, and state
the significance of ionic imbalance across plasma membrane.3. Explain the establishment of resting membrane potential and describe how this
potential is maintained.
4. Differentiate between resting membrane potential and equilibrium potential.5. Calculate membrane potential and equilibrium potential given the appropriate
variables.
6. Relate membrane potentials to intracellular and extracellular ions and ionchannels in the membrane.
7. Represent membrane potentials in graphical forms.
Please set up more specific objectives after you have thoroughly studied the materialsin this module to help yourselves in your revision later on. Make notes that meet the
requirement of the new objectives.
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NEU 7. Define membrane capacitance and identify how membrane capacitance affects
the spread of current in myelinated and demyelinated neurons.
NEU 8. Compare conduction velocities in a compound nerve, identifying how thediameter and myelination lead to differences in conduction velocity, and the use of these
differences to classify neurons as group Ia, Ib, II, III, IV fibers or as A alpha, Abeta, Adelta, b,and c fibers.
NEU 9. Describe the ionic basis for inhibitory and excitatory post-synaptic potentials and
how these changes can alter synaptic transmission.
NEU 10. Distinguish the effects of hyperkalemia, hypercalcemia, and hypoxia on the
resting membrane and action potential.
NEU 11. Describe the effects of demyelination on action potential propagation and nerveconduction.
Neurochemistry NEU 12. Compare electrical and chemical synapses transmission based on velocity of
conduction, fidelity, and the possibility for neuromodulation (facilitation or inhibition).
NEU 13. Describe chemical neurotransmission, listing in correct temporal sequence
events beginning with the arrival of a wave of depolarization at the pre-synaptic
membrane and ending with a graded potential generated at the post-synaptic membrane.
NEU 14. Define the characteristics of a neurotransmitter.
NEU 15. Learn the synthetic pathways, inactivation mechanisms and neurochemicalanatomy and mechanisms of receptor transduction for the following neurotransmitters:
1. Catecholamines (DA, NE, E)2. Acetylcholine (ACh)
3. Serotonin (5-hydroxytryptamine; 5-HT)
4. Histamine5. GABA (gamma-aminobutyric acid)
6. Glutamate
7. Endorphins8. Enkephalins
9. Dynorphins
10. Substance P
NEU 16. Learn the major receptor classifications and representative receptor agonists and
antagonists for the above transmitters.
NEU 17. Describe the relationships between neurotransmitter dysfunction andneuropathology.
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viii. Learning Activities
1. Excitable Tissues
1.1. The meaning and significance of excitable tissues
From your previous knowledge you know that there are many types of cells in the body.
You also know that these cells are able to communicate among themselves (Module 4).You have studied the molecular mechanism of intercellular communication and we have
agreed that all cells can receive and process information. However, not all cells can
transform the information into an electrical signal to be transmitted to other cells.
Communication between adjacent cells could occur via direct contact (gap junctions, tight junctions and desmosomes) or via paracrine agents (Module 4). On the other hand,
communication between cells that are a distant apart could occur chemically via hormones
or electrically via neurons. You already have some idea now about the action of
hormones on target organs, and you’ll learn more about it in the Endocrinology course
(Semester 2). In this module, we’ll look at how some cells generate electrical signalsacross the plasma membrane and how they make use of the signals to communicate with
each other. These cells are called excitable cells.
Excitable tissues consist of cells with the ability to receive and respond to stimuli by
converting them into electrical signal. They are mainly nerve and muscle cells.
• What kinds of information do cells receive? Give examples.
• Why do cells need to be able to receive and process information?• How do cells receive and process information?
• What happens to the information eventually?
ACTIVITY 5.1: Reception and processing of information by cells
• What is the difference between excitable tissues and non-excitable tissues? Giveexamples of excitable and non-excitable tissues.
• Please explain why skeletal muscle, smooth muscle, and cardiac muscle are considered asexcitable tissues. What is the significance of muscle cells being excitable?
• What is the significance of nerve cells being excitable? Give examples to demonstrate thesignificance of nervous tissues in the body.
ACTIVITY 5.2: Excitable tissues
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1.2. Characteristics and roles of excitable tissues
All cells, excitable or non-excitable, have resting membrane potential (Section 4).
Resting membrane potential is actually the voltage difference between the inside and the
outside of the cells at rest (not stimulated). When stimulated, only excitable cells can
respond to the stimulus by generating an electrical signal called action potential. In otherwords, when stimulated, the resting potential is changed to action potential, and this can
only be carried out by excitable tissues. You will explore this phenomenon in Section 4.
What are action potentials or electrical signal impulses used for? Examples (Fig. 5.1):
• Impulses from the sensory receptors are transmitted to the central nervous systemfor perception or processing.
• Impulses are passed on from one neuron to another in the central nervous system.
• Impulses from the central nervous system are passed on to the effectors (especiallymuscles and glands) for action in response to the stimuli. Impulses that reach the
muscles and are distributed along the membranes cause the muscles to contract.
From the knowledge you gained previously in Module 3, please explain why only excitable cellscan respond to stimuli and convert them to electrical signals. Hint: presence of gated ionchannels.
The electrical signal is produced in the membrane of the excitable cells due to movement ofspecific ions across the membrane via the gated ion channel. In terms of the presence of ionchannels, what is the difference between excitable cells and non-excitable cells?
ACTIVITY 5.3: Special property of excitable cells
Some specialized excitable cells (e.g. sensory receptors) can be stimulated by specific stimulifrom the external or internal environment to produce electrical signals. Please give examplesof the sensory receptors and their external stimuli:Receptors Stimuli
Please give examples of the visceral receptors and their internal stimuli:Receptors Stimuli
In addition, a few types of cells can produce electrical signals spontaneously (no stimulusrequired). Please give an example.
ACTIVITY 5.4: Sensory receptors
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Fig. 5.1. Flow of information from the receptor to the control centre and finally to the effector
In summary, a very important characteristic that distinguishes excitable tissues from non-excitable tissues is that excitable tissues possess specific gated ion channels that would
open when stimulated, thereby allowing movements of certain ions across the membrane
to produce action potentials. What can stimulate these channels to open? Once action
potential is produced, it can be transmitted along the axon to carry a message to the targetcell. Normally, electrical signals complete a circuit between a receptor that detects
changes in internal or external environment and transmits the message to the appropriate
control centre and eventually to the effector to stabilize the change in the parameterdetected.
Receptor
Control centre
Effector
With reference to Fig. 5.1 please write a short essay on the flow of information via the neuronsand the skeletal muscle when you pick up a glass of water. Where and how is the informationinitiated? How is it passed on from one neuron to another? Where is the instruction to theeffector made? How is the instruction sent to the effector? How does the effector respond?
You can conclude that electrical signals can be transmitted from receptors to control centresand eventually to effectors for the purpose of responding to changes in external and internal
environmental parameters.
ACTIVITY 5.5: Flow of electrical information in the body
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1.3. Membrane potential and ion channels
• Membrane potential is the voltage difference between the inside and the outside of the
cells. At rest (not stimulated), it is called resting membrane potential; it does not
carry any information (why?). However, when excitable cells are stimulated by
appropriate stimuli, the resting membrane potential is converted to action potentials or impulses that are propagated along the membrane of excitable tissues (Section 4).
• Membrane potential occurs due to differential distribution of ions across the cellmembrane caused by the movement of ions in and out of the cell through specific ion
channels.
• Types of ion channels (review Module 2):i. Leakage channels (number of K+ channels >Na+ channels; this is the
reason why membrane is more permeable to K+ compared to Na+).
ii. Voltage-gated channels: participate in the generation and conduction ofaction potentials (Module 5) when stimulated by electrical stimuli.
iii. Ligand-gated or chemical-gated channels: open and close in response to
neurotransmitters, hormones and particular ions, thus establishing electricalsignals.iv. Mechanically-gated channels: open or close in response to mechanical
stimulation eg: vibration, pressure or tissue stretching, thus establishing
electrical signals.
• In which cells are each of the ion channels listed above located?
• Draw a section of plasma membrane with the four types of ion channels embedded. Usingexamples, describe the significance of these channels.
• What is “membrane potential”? Is there any movement of ions across the membrane whenthe cell is at rest (not stimulated?). Why? Is there any change in concentrations of the ionsacross the membrane at rest? Why?
• How does movement of ions across the membrane produce electrical signals?
ACTIVITY 5.6: Significance of membrane channels
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2. The intracellular and extracellular environments, and thesemi-permeable membrane
Let us assume for the moment that membrane potentials in excitable cells are produced by
the movement of ions (cations and anions) across the cell membrane (we shall verify this
in Section 4). Thus, it is important to first look at the ionic composition of theextracellular and intracellular fluids.
The chemical makeup of cytoplasm inside a cell is quite different from that of the
interstitial fluid (ISF) that bathes it (Table 5.1). It is with the ISF that cells directly
exchange molecules and ions on a continuous basis. What are some other molecules that
are exchanged?
Table 5.1. Chemical makeup of the body fluids
Substance Interstitial Fluid
Concentration (mM)
Intracellular Fluid
Concentration (mM)Na
+ 145 15
K+ 4.5 120
Ca+ 1.0 0.0001
Mg+ 1.2 58.0
Cl- 116 20
HCO3- 24 15
Phosphate ion 28 10
Glucose 4-6 0-3Amino acids 4 75Proteins 20k 160k
pH 7.4 7.0
Figure 5.2 is a simple diagram of three fluid compartments in the body (review Module
1).
• A capillary (A) is drawn to represent the blood or plasma compartment, whichexchanges water and solutes directly with the interstitial fluid compartment (C).
• The intracellular "compartment" (B) is actually the millions of individualcytoplasmic spaces inside each cell of the body. Label the compartments in Fig.
5.2.
CB
Figure 5.2. Fluid compartments of the body
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Regulation of molecular exchange between the ISF and the intracellular compartments isaccomplished by the cell membrane (Module 2). You have learnt that the cell membrane is
semi-permeable.
Fig. 5.3 represents a piece of the cell membrane with the interstitial fluid above and the
cytoplasm below. Label the compartments.
Figure 5.3. Cell membrane, solutes, and gradients
o
oo
o
o
o
o
o
o
o
o
o o
o o
o o
o
oo o
o
Intracellular
extracellular
The walls of most capillaries are quite leaky (due to gaps between endothelial cells, discussedin Module 2), and the gaps allow free exchange of water, small molecules and ions betweenthe plasma and interstitial fluid.
• In Figure 5.2, draw a small circle to represent an oxygen molecule (O2) in the blood, thenuse an arrow to indicate the direction of movement between the blood and the cell. Do thesame for CO2. Explain the forces that cause the movement of these molecules.
• Some large particles like protein molecules in the blood cannot normally cross the capillarywall, nor can most of the blood cells. Thus they are kept afloat in the vessels. What is thesignificance of this phenomenon?
ACTIVITY 5.7. Movement of substances across endothelial membranes
What is the meaning of semi-permeable membrane? Relate your answer to the structure of themembrane and the molecules that move across it.
If the smallest circleswere Na
+, what would be
their tendency in termsof movement across themembrane? Why thendoesn’t theconcentration change
with time at rest? Doyou expect other ions tobehave the same withrespect to movementacross the membrane?
ACTIVITY 5.9.Movement of ionsacross semi-permiablemembrane
ACTIVITY 5.8. Semi-permeable membrane
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Missing in Fig. 5.3 are cholesterol molecules and the large proteins that are usually shown
embedded in the plasma membranes. We will study more about these important elementslater.
3. Concentrations, gradients, and permeability
3.1. Concentration gradients
Activity 5.10 helps you to figure out the meaning and significance of concentration
gradients across plasma membrane.
Concentration gradients exist in situations where the number of molecules per unit
volume (concentration) at one location differs from that at another. In the above example,
you discovered that a concentration gradient exists across the cell membrane. The "high"end of the gradient is the location where the concentration is greatest. The other is the
"low" end. In diffusion, the net movement of molecules by random motion is down theconcentration gradient, from the high to the low end. Do molecules in a gradient ever
diffuse up the gradient?
The movement of molecules is driven by the concentration gradient that exists betweenthe two sides. A good definition of concentration gradient is "the difference in
concentration of a solute molecule over a distance between two points." One point is at the
high end of the gradient, the other point is at the low end. In this module, we will bedealing primarily with very short distances i.e. from one side of the cell membrane to the
other.
You may remember that in all fluid compartments of the body, solute molecules or ions (forexample sodium ions or glucose) are dissolved in the solvent (water). At physiologicaltemperatures, the solute particles move rapidly (Brownian motion), colliding with one another
and with the membranes of the cell. How do you relate this phenomenon to diffusion? Hint:Effect of concentration and temperature on diffusion.
Look at Figure 5.3 again. Assume for our work that the volume of solution both inside andoutside the membrane is 0.1 ml. What would be the concentration of small molecules outside inthe interstitial fluid? Use the measure "particles/ml" as the unit of measure. What are theconcentrations of medium-sized molecules both in- and outside the cell?
ACTIVITY 5.10: Diffusion of solute molecules
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3.2. Electrical gradients
Electrical gradient is the difference in total charges between the inside of the cell and theoutside of the cell. You remember that these charges are carried by the ions, and you also
remember that there is unequal distribution of ions across the membrane. Therefore, there
is an electrical gradient across the membrane.
Electrical gradients are especially important in moving charged particles into or out of a
cell. You may remember from basic principles of magnetism that like charges repel one
another, while opposite charges attract. In an electrical gradient, like that across the cellmembrane, there are more negative charges inside and positive charges outside. If the
membrane is permeable, positively charged ions (Na+, K
+ , and Ca
2+ ) will be attracted
inward, attracted to the negative charges in the cytoplasm.
Diffusion of solutes across the membrane depends on concentration and electrical
gradients, and also on membrane permeability to the solutes. Two factors that influencethe permeability of a membrane to a particular kind of solute particle are:
The direction and rate of net movement due to diffusion depends largely on the difference inconcentration for a molecule on the two sides of the membrane. The larger the difference inconcentration, the faster the rate of movement can be across the membrane. Why?
In Figure 5.3, how many concentration gradients can you identify?
If a cell membrane is permeable to solute particles, some will move across to the adjacentcompartment. In the Figure 5.3, draw some tiny solute molecules between the largerphospholipid molecules of the membrane, possibly denoting their ways from one side to theother. Add arrows to a few of the small and medium sized molecules to indicate in whatdirection you think they are heading. Explain your prediction.
Activity 5.11: Concentration gradient and diffusion
Consider the electrical and concentration gradients together (Table 5.1). Do the two gradientsfor Na
+ work in the same or opposite directions? What about K
+? Predict how you think Na
+
and K+ will move across the cell membrane. Which will have the strongest flow? Why?
Recognizing that an ion may be a part of two gradients at the same time, we can talk about itscombined electrochemical gradient. In the case of Na
+, both the chemical and electrical
gradients favour the movement of Na+ _______ the cell. However, in the case of K
+, the
chemical concentration gradient tends to move potassium ions ______________ the cell, butthe electrical gradient moves them ___________.
ACTIVITY 5.12: Electrical and concentration gradients
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a. The size of the particle. Smaller particles pass across more easily than larger
ones. Very large ones may not pass at all. The membrane is impermeable tothem.
b. The electrical charge of the particle and/or it solubility in lipids. Electrically
charged particles, called ions (example, Na+), do not dissolve well in lipids, and
the phospholipid bilayer is rather impermeable to them. The lipid connection topermeability is due to the fact that the center of the cell membrane is made of
lipids (fatty acids). Many solutes must dissolve in the lipids in order to passacross.
The movement of particles in Fig 5.3 will result in a stable distribution on either side of
the membrane for some of the particles. In this dynamic equilibrium, exchange acrossthe membrane still occurs. However, the number of particles crossing in one direction is,
over time, equal to the number crossing in the other direction, so the net movement is
zero.
In the case of large particles like carbohydrates that don't cross the membrane in Fig 5.3,
there is no dynamic equilibrium and a concentration gradient is maintained by theimpermeability of the membrane.
No electrically charged particles are shown in Fig 5.3. Based on size alone predict thepermeability of the membrane to the three types of solute particles.
If we allow diffusion to occur, starting from the arrangement of solutes shown in Fig. 5.3, predictwhat changes will occur in the distribution of particles in and outside the cell membrane.Explain your predictions.
ACTIVITY 5.13: Permeability of plasma membrane
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4. Membrane Potential
4.1. The meaning and significance of membrane potential
All cells in our bodies have membrane potentials because there is a difference in net
charge across the plasma membrane. The remainder of this module is devoted tounderstanding how membrane potential is established and maintained when the cell is at
rest. Later in Module 5, we’ll learn how membrane potential changes over time whenthere is an appropriate stimulus.
The concepts introduced here build upon your foundation of knowledge about cellmembranes and gradients. Therefore, it is essential that you have a strong grasp of the
material in the prior sections. Review if you lack confidence in understanding the earlier
material.
It is important to distinguish between charge and membrane potential. An ion carries a
positive or negative electrical charge that contributes to the net charge in one locationsuch as in the cytoplasm of a cell. The net charge is the algebraic sum of all positive and
negative charges in that area. For example if there were 5 +ve charges and 3 -ve charges,
the net charge is +2. When the net charges in two separated areas are compared there maybe a difference (gradient). The difference in charge on the two sides of the membrane isthe membrane potential.
• Based upon what you learned before, define “membrane potential” or describe what youthink a membrane potential is.
• Does a membrane potential involve a gradient? Explain.
• How does the gradient involved here differ from the concentration gradients you studiedearlier?
ACTIVITY 5.14: Membrane potential
Draw a large circle to represent a cell. Then draw:• 20 small circles with the symbol Na
+ outside the membrane and 2 inside the cell.
• 20 small circles with the symbol K+ inside the cell and 1 outside
• 16 small circles with the symbol Cl- outside the membrane and 2 inside• 5 larger circles with symbol P
- to represent proteins in the cell.
Assume the ions do not move across the membrane.
Calculate:• the gradient of each of the ion across the membrane • the net charge outside the cell • the net charge inside the cell• the difference in the net charge between the outside and the inside. This is the electrical
gradient, and it is called the membrane potential.
ACTIVITY 5.15: Calculation of membrane potential
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4.2. Simulation of membrane potential using a Membrane Model
Membrane potentials are vital to the life of every cell. They are used to produce and
conduct signals (especially in neurons and muscle cells), transport molecules across the
cell membrane (for example, in moving glucose across the wall of the gut), and many
other functions. Cells spend great amounts of energy maintaining membrane potentials by"pumping" ions in and out across the cell membrane. Understanding the cellular basis of
many physiological processes ultimately involves knowledge of membrane potentials.
Find the cell membrane template at the end of this module (Appendix A). Make a photocopyand place it on a table with the various elements that are used with it ( Appendix B).
You will notice that the membrane on the template has two gaps. In these locations you can
place a number of different elements to represent the structure of the membrane. For example,membrane "patches" simply complete the phospholipid bilayer, while channels of various typesadd functional proteins that are involved in solute transport. Finally, the molecular pumpsprovide active transport of substances across the membrane using metabolic energy (ATP).Also the template has large, negatively charged proteins shown in the cytoplasm.
In the diagram (Appendix A), what is the total negative charge of protein molecules?
Paper circles in Appendix B with plus (+) or minus (-) signs on them represent ions. For ourpurposes, we will define each ion as contributing one unit of positive or negative charge to theinside or outside of the cell. When we measure the difference in net charge on the two sides ofthe membrane, we will express the resulting membrane potential in millivolts (mV). For thissimulation each difference of one unit of charge will be recorded as a millivolt of potential. So ifthe outside has a net charge of +2 and the inside -2, the membrane potential will be -4 mV. Inactual life, each ion carries a very tiny amount of charge. Differences in the millions of ions areneeded to produce each millivolt of membrane potential.
To prepare for the simulations, cut out the elements as needed and punch out the ions using asingle-hole punch. One set will be sufficient for your group. Please do not use glue to stick theelements on the membrane model.
ACTIVITY 5.16: Simulation of membrane potentials
Fig. 5.4. Measurement of membrane potential
using a sensitive voltmeter
An "instrument" for measuring potentialdifference between the inside and outsideof the cell is the voltmeter (Appendix C).
Fig. 5.4 shows the diagram of a voltmeter
used to measure potential difference in anaxon.
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Membrane potential problem.
Cytoplasm Interstitial Fluid
Large protein (number/ total charge)
________
Large protein (number/ total charge)
________
+ve cations (number/ total charge)
________
+ve cations (number/ total charge)
___________
-ve anions (number/ total charge)
___________
-ve anions (number/ total charge)
___________
Net charge inside the cell ___________ Net charge outside the cell ___________
Membrane Potential __________________
From the above activity, you can conclude that membrane potential is determined bythe difference in net charges between the cytoplasm and the interstitial fluid.
In the model you have, the scale on the face of the meter measures between -25mV and +25mV, with zero at the midpoint. Assemble the voltmeter as directed by your instructor. Align it sothat the reference electrode is on the outside of the membrane and the recording electrode is inside. Electrodes are made of materials that conduct tiny currents to the voltmeter. With thisarrangement, the meter should display the difference between the inside charge and theoutside charge, which is the membrane potential. For example if the inside net charge was -7units and the outside net charge was +5 units, what would the membrane potential be? _______. Adjust the indicator arrow on the meter to represent this value.
Set up the voltmeter by cutting out the 2 pieces and connecting them with a paper fastener atthe center of the meter. The arrow should be set to rotate over the range of values on themeter.
• For the first exercise place membrane patches into the gaps on the template.• Next, add paper ions to the template so that the total charge outside the membrane is +8
units.• Then add ions inside the cell so that the membrane potential is -11 mV.• Record your information in the data box below. Align the arrow on the voltmeter to show
the correct reading.
ACTIVITY 5.17: Measuring membrane potential
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4.3. Establishing Resting Membrane Potentials
Although the phospholipid bilayer of the cell membrane is largely impermeable to ions
like sodium (Na+ ), potassium (K
+) and chloride (Cl
-), these ions may pass through the
membrane from time to time through passages in large protein molecules called channels
(review Module 2). With your model template you have three channels, two for Na
+
andone for K+. Channels have several important properties you should know in order to
understand transport of ions across the cell membrane:a. Channels are selective in what ions they allow to pass through them. For example,
the structure and internal electrical charges in a K+ channel enable rapid facilitated
diffusion of K+ ions to occur down the concentration gradient, but permit little or
no flow of Na+ and Cl- ions.
b. Channels may be passive or gated. Some channels, like the ones represented in this
simulation are passive; they are open all the time, allowing ions to trickle constantly
through them at some rate (for example 100 ions/sec). In Module 5 you will beconstructing gated channels that can open or close in response to changes in the
cellular environment.c. Channels can vary the conductance of the cell membrane for different ions.
Membrane conductance for a particular ion is a measure of the ease with which it can flowacross the membrane through all its channels. To understand this, set your template up withmembrane patches in both gaps. In this situation what is the Na
+conductance?
If now you substitute one Na+ channel for a membrane patch, what happens to the
conductance?
What happens if you add 2 Na+ channels?
What can you infer (in terms of channel numbers) if we say that the membrane conductance forK
+ is higher than for Na
+?
Set your template up as follows: fill the gaps with one K+ channel and one membrane patch.
Remove all ions from the ISF (outside the membrane). Add 10 K+ ions to the cytoplasm.
Record the net charge inside and outside the membrane, and the membrane potential. Setyour voltmeter to the correct reading.
Now move one K+ ion through the membrane to the outside. What happens to the net charges
inside and outside the cell? What happens to the membrane potential? What is causing the K+
ions to move outward?
Move two more K+
ions out. What happens?
You probably discovered that each time a K + ion left the cell, the inside of the cell got more
negative and the outside (ISF) got more positive . Also, the membrane potential got larger . Theresult is that a larger and larger electrical gradient is being produced with a positive end thattends to repel cations like K
+. Does the electrical gradient work with or against the outward
diffusion of K+ ions across the membrane? Will the electrical gradient inhibit the outflow of K
+?
ACTIVITY 5.18: Membrane conductance
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In the above situation where the concentration and electrical gradients oppose one another,
eventually a dynamic equilibrium would be reached. Then the number of K+ ions moving
out of the cell down the concentration gradient would exactly equal the number moving
down the electrical gradient (moving from the outside which is more positive to the inside
which is more negative). For the model, let us assume that dynamic equilibrium is reached
when 3 K
+
s have left the cell. Determine the membrane potential at that point using themodel. ________mV. This is called the equilibrium potential for K+.
In actual cells, the resting membrane potential is determined largely by the dynamic
equilibrium potential of the K+ as it has the highest conductance across the cell membrane.
Both Na+ and Cl
- have very low conductances across the membrane and influence the
membrane potential in a smaller way.
What is equilibrium potential?
If Na+ and Cl- ions had high conductance, in what direction would they move across the cellmembrane? What would be the consequence on membrane potential for each one? Pleaseexplain.
ACTIVITY 5.19: Equilibrium potential
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4.4. Maintaining gradients and potentials across the cell membrane
Your prediction is most probably right! However, we know that at rest the membrane
potential stays constant at ~-70mV, [K+] inside the cell is higher compared to outside, and
[Na
+
] outside the cell is higher compared to inside. This means that there’s a mechanismto maintain such stability of ionic composition on both sides of the membrane. Can you
suggest what this mechanism is?
Ions and molecules do regularly move against their concentration gradients across cellmembranes, but at a cost of energy to the cell. This is the process of active transport
(review Module 2).
Frame 1 of Fig 5.5 shows a diagram of a Na+ /K
+ ATPase protein embedded in the lipid
bilayer. You will notice the protein has several parts. Part B anchors it in the bilayer
because it is hydrophobic. The other sections (A, C, & D) are hydrophilic, facing into the
ISF or the cytoplasm. Segments C & D have multiple binding sites for ions. The sites withlighter outlines bind Na+, while those with heavier outlines are specific for K+. The
binding sites change shape during the transport process indicating their availability to bind
with the ions. In Frame 1, for example, the Na+
sites are open (available), while the K+
sites are closed (unavailable) to binding.
Thus far, we have discussed mostly the movement of ions down their concentration and/orelectrical gradients. Unless there were movements in the other direction (against the gradients)what do you predict would eventually happen?
ACTIVITY 5.20: Maintaining equilibrium potential
Turning to your template again, place a "pump" protein in each of the gaps in the membrane.One of the best known of these pumps is the Na
+ /K
+ -ATPase molecule (Fig 5.5). Embedded
in the cell membrane, this protein uses the energy of ATP to pump Na+ out of the cell and K
+
in, both against their gradients.
ACTIVITY 5.21: Na+ /K
+ -ATPase
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ISF
Cytoplasm
ISF
Cytoplasm
ISF
Cytoplasm
+
+++
A
B CD
EISF
Cytoplasm
+
+
+
+
++
Protein changes shape sobinding sites open for K
+
ions as Na+ ions are released
Two K+
ions attach
to open
binding sites
on the
protein
Protein changes shape so
binding sites face the ISF
again. K+ ions diffuse into
the cytoplasm. Cycle
com lete.
Na+ ions in
the
cytoplasmattach to
open
binding sites
on the
protein
pump
1 2
34
+ +
+
+
+
+
+
+
K+ ion Na+ ion
A: a hydrophilic segment of the protein pumpB: a hydrophobic segment of the protein pump
C: binding sites for Na+ ions on a hydrophilic segment of the protein pump
D: binding sites for K+ ions on a hydrophilic segment of the protein pumpE: phospholipid molecule in the membrane bilayer
Note: the energy molecule ATP and its by-products are not shown in the diagram.
Figure 5.5. Active transport across the cell membrane by the Na+/K+ ATPase protein pump
+ +
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Because of the effect the pump has on the membrane potential, it is called an electrogenic
pump. It not only restores the gradients, but also actually alters the membrane potential,
making the inside more negative compared to the outside. However, the Na+
does not
accumulate outside as it diffuses back into the cell via the leakage channel, and K+ does
not accumulate inside the cell as it diffuses out down its concentration gradient. The
balance of diffusion and active transport of K+ and Na
+ maintains the stability of the
resting membrane potential (Fig 5.6).
The ongoing work of Na+ /K
+-ATPase pumps in the cell membrane restores and maintains
the critical gradients needed to carry out other vital cell activities, especially transport andsignaling.
Fig. 5.6. The balance of diffusion and active transport of K+ and Na
+ maintains the stability of
the resting membrane potential
View the frames in Fig 5.5 to follow:a) the binding of ions (frame 2),b) the change in protein shape which exposes the binding sites to the opposite side of
the membrane (frame 3),
c) the release of Na+
ions and binding of K+
in the ISF (frames 3 & 4), andd) the completion of the cycle (back to frame 1).
To check the effects of this action on membrane potential, set up your model (Appendix A) withthe protein pumps in the gaps. Then add the following ions. Inside: 4 Na
+, 10 K
+ , 1 Cl
-; outside:
7 Na+, 3 K
+, and 3 Cl
-. Determine and record the membrane potential before the pump is
activated. _____________.
Now run a simulation, going through the steps you studied in Fig 5.5 for one cycle, moving Na+
and K+ ions across the membrane. Try to do each step. Record the new membrane potential.
In comparing your result, answer the following:a. What did the pump cycle do to the size of the gradient for each ion?
b. What did the pump cycle do for the membrane potential?
ACTIVITY 5.22: Simulation of Na+ /K
+ -ATPase
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4.5. Graphical representation of membrane potentials
Although membrane potential is actually the difference in net charges brought about bymovement of ions on both sides of cell membrane, we normally present it in graphical
form. The graph represents a recording, over time, of the membrane potential from a
single cell. You may remember that the recording electrode is inside the cell (cytoplasm)while the reference electrode is outside in the ISF (Fig. 5.4). Thus, the negative or
positive values of a membrane potential reflect the net charge inside the cell as compared
to the outside.
M e m b r a n e
o t e n t i a l ( m V )
Time (msec)
1 2 3 4 5 6 7 8 9 10
-100
-80
-60
-40
-20
0
20
12 13 14 1511
Fig. 5.7. X and Y axis for
representing membrane
potentials graphically
Examine closely the axes on Fig. 5.7. The Y-axis represents the membrane potential with theconvention we used consistently in this module. Notice especially the location of zero on the Y-axis. At zero there is no potential difference between inside and outside the cell, meaning thenet charge inside the cell is the same as the charge outside.
Going down the scale from zero, is the inside of the cell is more or less negative compared tothe outside? ___________. Is the potential difference across the membrane larger or smaller? ___________ .
Going up the scale from zero, is the inside of the cell is more or less positive compared to theoutside? ___________. Is the potential difference across the membrane larger or smaller? ___________ .
The X-axis represents time in milliseconds (msec). How many marks on the axis per msec? ________ .
Refer to Activity 5.19 and 5.22. Draw on the graph paper the membrane potential of each ofthe exercise (1-15 msec).
ACTIVITY 5.23: Graphical representation of membrane potential
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4.6. Calculation of equilibrium potential of ions using the Nernst equation
Make sure that you are very comfortable with the term “equilibrium potential”. You know
that equilibrium potential of an ion depends on :
a) intracellular concentration of the ion,
b) extracellular concentration of the ion,c) the valence (charge) of the ion.
In addition, equilibrium potential is also influenced by the temperature and the gas
constant. The relationship between the equilibrium potential of an ion to the variables is
given by the Nernst equation. Eg: for Cl-
ECl = RT ln [Clo-]
FZCl [Cli-]
Where
ECl = equilibrium potential for Cl-
R = gas constant
T = absolute temperature
F = the faraday (number of coulombs per mole of charge)ZCl = valence of Cl
-
ln = natural log
[Clo-] = Cl
- concentration outside the cell
[Clo+] = Cl
- concentration inside the cell
Converting from the natural log to the base 10 log and replacing some of the constants
with numerical values, the equation becomes:
ECl = 61.5 log [Cli-] at 37
oC
[Clo-]
Note that in converting to the simplified expression the concentration ratio is reversed
because the –1 valence of Cl- has been removed from the expression.
Similarly, for K+
EK = RT ln [Ko+] = 61.5 log [Ko
+] at 37
oC
FZK [Ki+] [Ki
+]
From the equation, notice the difference in the ratio of intracellular and extracellularcomponents between anion and the cation.
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Table 5.2 Concentration of some ions inside and outside mammalian spinal motor neurons
Ion Concentration (mmol/L of H2O) Equilibrium potential(mV)Intracell Extracell
Na+ 15.0 150.0
K+ 150.0 5.5
Cl- 9.0 125.0
Based on the values in the above table, please calculate the equilibrium potential for Cl-,
K+, and Na
+.
Suppose the membrane potential measured is –70 mV. What does it mean with regard to
the equilibrium (movement across the membrane) of the ions?
4.7. Calculation of membrane potential using the Goldman equation
The equilibrium potential for an ion is the membrane potential at which the ion on both
sides of the membrane is in equilibrium i.e. there is no net movement of the ion into or outof the cell. However, since there are more than one ion species in the ISF and in the
intacellular fluid, how is the membrane potential determined?
The magnitude of the membrane potential at any given time depends upon the
distribution of Na+, K
+, and Cl
- and the permeability of the membrane to each of these
ions. According to the Goldman equation:
EM = RT ln PK+[Ko
+] + PNa
+[Nao
+] + PCL
-[Cli
-]
F PK+[Ki
+] + PNa
+[Nai
+] + PCL
-[Clo
-]
Where:
VM = membrane potential
R = gas constant
T = absolute temperatureF = the faraday (number of coulombs per mole of charge)
PK+, PNa+, PCl- = permeabilities of the membrane to K+, Na+, and Cl-
[ ] = concentration
i = inside of the cello = outside of the cell
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vi. Summary
When we measure the membrane potential of a neuron at rest with a voltmeter by puttingone electrode inside the cell and another outside the cell, the reading says that the inside is
~70 mV more negative than the outside (by convention it is written as –70 mV). This
resting membrane potential does not carry any message to other cells, but is important as abasis for generation of graded potentials and action potentials, which are the message-carrying potentials (Modules 5 & 6). The question is: how is this resting membrane
potential established and how is it maintained?
Membrane potential results from unequal distribution of charges of the ions (mainly Na+,
K+, Cl
- and protein
-). The concentration of K
+ and protein
- is higher in the cytosol; the
concentration of Na+ and Cl
- is higher in the ISF. Since the membrane is more permeable
to K+ (there are more K+ passive channels on the membrane) than Na+, more K+ is leaving
the cell than Na+ coming in. This makes the inside of the cell more negative compared to
the outside. The electrochemical gradient is maintained by the Na+,K
+-pump.
Draw a creative and comprehensive concept map that encompasses the main ideas in thismodule.
Describe what you have learnt from this module, including non-academic outcomes.
Comment on the activities, and suggest innovations for improvement of the module.
ACTIVITY 5.24: Summary
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vii. Conclusions
Please make sure that you achieve all the objectives set up at the beginning of this module:
Objectives Comments
1. Define “excitable tissue” by givingexamples, by relating to itscharacteristics, and by stating its
significance.
2. Differentiate between intracellularand extracellular ionic constituents,
and state the significance of ionicimbalance across plasma membrane.
3. Explain the establishment of restingmembrane potential and describe
how this potential is maintained.
4. Differentiate between restingmembrane potential and equilibriumpotential.
5. Explain how resting membranepotential is maintained.
6. Calculate membrane potential andequilibrium potential given the
appropriate variables.
7. Relate membrane potentials tointracellular and extracellular ions
and ion channels in the membrane.
Don’t forget the objectives that you have constructed yourselves!
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Appendix A: Membrane template
ISF (outside cell)
Cytoplasm (inside cell)
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Appendix B : Membrane template elements
+ +
+
+
+
+
+
+
+
+
+
+
+
+ ++ +
+
+
+
+
+
+
+
++
+
+
++
+
+
+
+
+
+
++
- - - - - -
- - - - - -
Bilayer patches
Na+ Channel Proteins
Na+ /K
+ ATPase pumps
Channel gates with
receptor sitesChemical
signal
Na+ K+
Cl-
Na+ Na
+ Na
+ Na
+ K
+ K
+
K+ Channel Proteins
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Model of a voltmeter
0 +5
+10
+15
+20
+25-25
-20
-15
-10
-5
Millivolts (mV)
Carefully push the paper
fastener through the white
circle of the indicator
arrow and the black circle
of the face of the
voltmeter
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