MEMBRANE TRANSPORT

INTRODUCTIONCells must exchange materials with their environment. Without nutrients and oxygen and without disposal of metabolic wastes, cell death is inevitable. In addition, many specialized cellular functions require the selective movement of materials across cell membranes (e.g., secretion, nerve and muscle activity, etc.). Each cell is separated from its extracellular environment by a plasma membrane, the function of which is to regulate the continual exchange of materials between the fluid inside the cell (intracellular fluid) and the fluid outside the cell (extracellular fluid). The plasma membrane is selectively permeable, passing some substances freely and acting as a partial or a complete barrier to other substances.

All passive transport processes involve movement down a concentration, pressure or electrical gradient. Passive transport does not require an input of metabolic energy, so these processes can be studied using nonliving model systems. In the following exercises, you will examine a few of the physiological principles that govern movement through membranes.

I. OSMOSIS

Osmosis is a special case of simple diffusion and refers to the movement of water down its concentration gradient. It can be a little odd to think of water in terms of concentration since the term concentration is usually used to talk about solutes (the particles dissolved in the water). Pure water, which has no solutes in it, is as concentrated as water can get. As more and more solutes are added to water, they take up space, leaving less room for the water. Thus, as the concentration of the solutes increases, the concentration of the water goes down. So although it is absolutely correct to say that osmosis is the movement of water from an area of high water concentration to an area of low water concentration, it is usually easier (and just as correct) to say that osmosis is the movement of water from an area of low solute concentration to an area of high solute concentration.

Osmosis can only occur across a membrane that is semipermeable, meaning that some substance can cross the membrane (permeable substances) and others cannot (impermeable substances). Obviously the membrane must be permeable to water for osmosis to occur, but is also important that some solutes are impermeable. If water is across the membrane from solutes that cannot cross through the membrane, then the water will move towards those solutes (down the concentration gradient for water). Thus, any solute dissolved in the solution that cannot cross the membrane (is impermeable) will contribute to osmosis, and is said to be osmotically active. However, any solute that is permeable to the membrane will not contribute to osmosis and is not osmotically active. This is because a permeable solute will simply cross the membrane to even out its concentrations on either side of the membrane, and is therefore not contributing in any way to water’s concentration gradient.

II. OSMOLARITYThe osmolarity of a solution is a measure of the total number of moles of dissociated particles dissolved in that solution, and has the units of osmoles (Osm). Osmolarity is similar to “concentration”. However, while concentration is the number of moles of a single compound per liter of solution, osmolarity is the number of moles of particles from every compound dissolved in a liter of solution, including compounds that dissociate into several particles when dissolved in water (like ions). For instance, a solution containing glucose at a concentration of 50 mM and NaCl at a concentration of 100mM would have an osmolarity of 250 mOsm (50 mM glucose + 100 mM Na+ + 100 mM Cl-).

The osmolarity of our blood is about 300 mOsm. If the osmolarity of a solution is compared to the osmolarity of our blood, the solution is said to be iso-osmotic if it has the same osomlarity as our blood (iso means “same”). If the osmolarity of a solution is greater than our blood, the solution is said to be hyperosmotic (hyper means “above”), and if the osmolarity of a solution is less than our blood, the solution is said to be hypo-osmotic (hypo means “below”).

III. TONICITYRecall that osmolarity refers to the total number of particles in a solution. The tonicity of a solution refers to the effect the solution has on the shape or “tone” of the cell due to osmosis. An isotonic solution has the same concentration of osmotically active particles as normal body cells. When a red blood cell is bathed in an isotonic solution, there is no net movement of water into or out of the cell due to osmosis, so cell volume remains constant. If a red blood cell is placed in a hypotonic solution, there are fewer

H2O

Side AHigher solute conc.

(Lower water conc.)

Side BLower solute conc.

(Higher water conc.)

Semipermeablemembrane

Impermeable solute – osmotically activePermeable solute

Figure 1.

Water moves via osmosis from Side B to Side A because Side A has a higher concentration of impermeable solutes (and thus, a lower concentration of water). Only the impermeable particles are contributing to the osmosis.

osmotically active particles in the solution compared to inside the cell, causing water to enter the cell by osmosis and swell, perhaps to the point of rupturing (“hemolysis”). If a red blood cell is placed in a hypertonic solution, there are more osmotically active particles outside the cells, causing the cell to lose water by osmosis and shrink (“crenate”). Remember that these tonicity terms only tell you about the effect that a solution has on the shape of a cell.

PROCEDURESA. Membrane Transport in Red Blood Cells

1. Label 10 clean test tubes “1” through “10” and place in a test tube rack.

2. Fill in the “Osmolarity” and “Predicted tonicity” columns of Table 1.

3. Using a 3-ml syringe, deliver 2 cc (2 ml) of the following solutions into the test tubes as indicated below.

Test tube 1: 2 ml of distilled waterTest tube 2: 2 ml of 33 mM NaClTest tube 3: 2 ml of 100 mM NaClTest tube 4: 2 ml of 150 mM NaClTest tube 5: 2 ml of 333 mM NaClTest tube 6: 2 ml of 833 mM NaClTest tube 7: 2 ml of 300 mM glucose (use as control)Test tube 8: 2 ml of 300 mM glycerinTest tube 9: 2 ml of 300 mM ureaTest tube 10: 2 ml of soap solution

4. Before adding any blood to the test tubes, gently rock the vial of non-human blood to re-suspend the blood cells. Using the eye dropper, dispense 2 drops (0.1 ml) of blood to each test tube. Mix gently by swirling the test tubes a few times.

H2O

H2O

Cell shrinksHypertonic

No changeIsotonic

Cell swellsHypotonic

Figure 2.

The tonicity of a solution is determined by what effect the solution would have on the shape of a cell if a cell were to be placed in the solution.

5. Every few minutes gently swirl the contents of each tube to re-suspend the cells. Hold the tube in front of a printed page to check for evidence of hemolysis. You can tell if lysis has occurred if you can see the printed words from a page held behind the test tube. When the blood cells are intact the solution will appear opaque; if and when the cells lyse, the solution will appear transparent. Changes in RBC shape occur immediately for all solutions except #8, which can take up to 40 minutes.

Table 1.Tube # Solution Osmolarity Predicted tonicity Observed tonicity

1 Distilled water

2 33 mM NaCl

3 100 mM NaCl

4 150 mM NaCl

5 333 mM NaCl

6 833 mM NaCl

7 300 mM glucose iso-osmotic

8 300 mM glycerin

9 300 mM urea

10 soap solution iso-osmotic

6. Prepare a wet mount slide from tube 7 first. Place one drop of the solution on a slide and cover it with a coverslip. Remember to re-suspend the cells before taking a drop of solution. Immediately observe the slide under the microscope before the solution dries on the slide. Note the appearance of the RBCs and use it as your “control”. This solution is isotonic (the cells should not shrink nor swell).

7. Make wet mounts only of the solutions in which the cells have not lysed. Remember to re-suspend the cells before taking a drop of each solution. Immediately observe the slide under the microscope before the solution dries on the slide. Compare the appearance of RBCs in each of these slides to the “control”. Determine whether the cells have swelled, crenated, or not changed shape. Rank the solutions by the degree of crenation, if possible. If the results from any test tube are not what you expected, prepare a second slide. Record the observed tonicity in Table 1.

Questions:

1. Discuss your observations of the red blood cells in the following solutions: 300 mM glucose, 300 mM glycerin and 300 mM urea. Why did you get different results when the molarity of the solutions was similar?

2. What happened to the red blood cells in soap? Explain a possible mechanism for your observation.

B. Membrane Potentials (The Bean Lab)*

Definitions:Before coming to lab, you should understand and be able to define the following:

Ion

Cation

Anion

Simple diffusion

Concentration gradient

Depolarization

Hyperpolarization

What ions are the predominant ions of the extracellular fluid? of the intracellular fluid?

* Used with permission, courtesy of Dr. Dee Silverthorn, University of Texas, Austin

Introduction:

This exercise is designed to help you understand the basis of resting membrane potentials, graded potentials, and action potentials in living cells. You will use different kinds of beans to represent different ions. A piece of paper divided into two sections represents the intracellular (ICF) and extracellular (ECF) compartments. The two compartments are the same size. The dividing line between the compartments represents the cell membrane, and colored rectangles of paper are used to indicate ion channels. When the long axis of a channel is perpendicular to the membrane, the channel is open and ions can flow through it.

Electricity Review

Atoms, the smallest units of matter, are electrically neutral entities. They are composed of positively charged protons, negatively charged electrons, and uncharged neutrons, but in balanced proportions so that an atom is neither positive nor negative. The removal or addition of electrons to an atom creates a charged particle known as an ion. Positive ions (cations) that are important in the human body include Na+, K+, and H+. For each of these positive ions, somewhere there is a matching electron, usually found as part of a negative ion (anion). For example, when Na+ in the body enters in the form of NaCl, the "missing" electron from Na+ can be found on the Cl-.

The following principles are important to remember when dealing with electricity in physiological systems:

(1) The Law of Conservation of Electric Charge states that the net amount of electric charge produced in any process is zero. This means that for every positive charge on an ion, there is an electron on another ion. Overall, the human body is electrically neutral.

ECF ICF

Cell membrane

Open channel

Closed channel

(2) Opposite charges (+/-) are attracted to each other, but two charges of the same type (+/+ or -/-) repel. With ions, the ion species does not matter: only the net charge on the ion is important. Thus a Cl- has one negative charge that is equal and opposite of one Na+ or one K+.

(3) To separate positive and negative charges, it is necessary to use energy. For example, energy is needed to separate the protons and electrons of an atom.

(4) If separated positive and negative charges can move freely toward each other, the material through which they are moving is called a conductor. Water is a good conductor of electricity. But if separated charges are unable to move through the material that separates them, the material is known as an insulator. The phospholipid bilayer of the cell membrane is a good insulator, as is the plastic coating on electrical wires.

In each of the following exercises, you will distribute ions (represented by beans) between the two compartments, set the permeability of the dividing membrane, then predict the movement of the ions between the compartments based on their electrochemical gradients. Write in the answers to the questions as you work. If you don’t understand something, talk with neighboring groups or your TA.

Exercise 1: What happens if a membrane is freely permeable to all ions?

Place 10 Na+ and 10 Cl- ions in the ECF compartment. Place 10 K+ and 10 protein anions (A- ) in the ICF compartment. Because the compartments are the same size, equal numbers of ions in the two compartments are the same as equal concentrations.

(Q1) Is the ECF electrically neutral? _______ Is the ICF electrically neutral? ________

(Q2) Is there an electrical gradient that would move ions from one compartment to the other? ________

(Q3) Assume that the membrane is freely permeable to all four types of ions. Is there a concentration gradient for Na+ ? __________ for K+ ? __________

for Cl- ? __________ for A- ? __________

Rearrange the ions so that any concentration gradients are abolished.Without a selectively permeable membrane around cells, the body would be unable to create or maintain the ion gradients needed for electrical signaling.

Exercise 2: What happens when a membrane is selectively permeable, allowing certain ions to cross while preventing other ions from crossing?

Molecules that cannot diffuse freely through the phospholipid bilayer of the cell membrane require protein transporters. Channel proteins have water-filled passageways that link the intracellular and extracellular compartments, allowing ions and water to move rapidly between the ECF and ICF. Channel proteins may restrict ions based on size and electrical charge. For example, there are separate Na+ and K+ channels. Ion channels are either leak channels or gated channels. Leak channels are open all of the time, allowing ions to move back and forth across the membrane without regulation. Gated channels spend most of their time in the closed state, with no movement through them. The opening and closing of gated channels is either controlled by 1) binding to the channel by a messenger molecule or ligand (chemically gated channels), 2) by the electrical state of the cell (voltage-gated channels), or 3) by a physical change such as a stretching of the membrane that pops the channel open (mechanically gated channels).

From now on, assume that the cell membrane of your model is impermeable to all ions unless a transport protein for an ion is placed on the membrane.

Rearrange the beans so that you again have 10 Na+ and 10 Cl- ions in the ECF compartment, and 10 K+ and 10 protein anions (A- ) in the ICF compartment.

(Q4) Is the ECF electrically neutral? _______ Is the ICF electrically neutral? ________ (Q5) Is there an electrical gradient that would move ions from one compartment to the other? ________

(Q6) Can any ions cross the membrane due to their concentration gradients? If so, which ones?

_______________________________________________________________

Now place the rectangle representing the K+ leak channel on the membrane in the open position.

(Q7) Can K+ now cross the membrane? ______ Will it move to the ICF or ECF? _______

(Q8) What force is moving the K+ ?___________________________________________

Move two K+ beans from the ICF to the ECF.

(Q9) Now what is the net electrical charge on the ECF? __________

(Q10) What is the net electrical charge on the ICF? __________

(Q11) Like charges repel and opposite charges attract. Why don’t the A- ions of the ICF follow the K+ to the ECF, or some of the Na+ of the ECF move into the ICF?

_____________________________________________________________________

On the number line below, mark and label the net charge for the ECF and the net charge for the ICF.

-------|--------|--------|--------|--------|--------|--------|--------|--------|--------|--------|------ -5 -4 -3 -2 -1 0 +1 +2 +3 +4 +5

When there is uneven distribution of ions between the intracellular and extracellular compartments, such as you just created in your model, the electrical gradient between the two compartments is known as the membrane potential difference, or membrane potential for short. Although the name sounds intimidating, let's break it apart and see what it means.

(1) The "potential" part of the name comes from the fact that an electrical gradient is a source of stored or potential energy. When oppositely charged molecules move together, they release energy that can be used to do work; in the same way, molecules moving down their concentration gradient can perform work. The work done by electrical energy includes opening voltage-gated membrane channels and sending electrical signals through nerve cells.

(2) The "membrane ... difference" part of the name is to remind you that this number represents a difference in the electrical charge inside and outside the cell (i.e. across the membrane. The word “difference” often gets dropped.

In your model, you created a membrane potential difference in which the ECF has a net charge of +2 and the ICF has a net charge of -2. But in living systems, we cannot accurately measure the absolute number of electrical charges on two sides of a

membrane. So instead, we measure electrical gradients on a relative scale with a voltmeter, which measures the difference in electrical charge between two points. The voltmeter artificially sets the charge on the extracellular fluid at zero millivolts (this is known as the ground) and measures charge on the intracellular fluid relative to the ECF.

(Q12) Go back to the number line above. If you shift your mark for the ECF to zero, and the difference in charge between the ECF and ICF remains the same, what is the new value for the ICF charge?

_______________________________________________

In biological systems, the actual value for the membrane potential of living cells is usually in the range of -70 to -90 millivolts (mV), meaning the ICF is negative relative to an ECF value of zero mV.

Exercise 3: Which of an ion’s gradients (concentration or electrical) affects the ion more?

An ion’s concentration gradient produces a concentration force that pushes that ion from an area of high concentration to an area of low concentration. The larger an ion’s concentration gradient, the larger the concentration force pushing on the ion. Similarly, when an ion is within an electrical gradient, the electrical gradient produces an electrical force that pushes the ion toward the side of the gradient with the opposite charge as the ion. The larger an ion’s electrical gradient, the larger the electrical force pushing on the ion.

Look at your model, which now has 2 K+ in the ECF and 8 K+ in the ICF. The K+ moved out of the cell in response to a concentration gradient. But K+ is an ion, so it must also be affected by its electrical gradient.

(Q13) If the net charge inside the cell is now -2, in whichDirection would K+ move along its electrical gradient? __________________

(Q14) With K+ being acted by both a concentration and an electrical force, in which direction do you think K+ will move, toward the ECF or ICF?____________

That is a tough question because the ion is faced with being moved in two directions: out of the cell by its concentration force, but back into the cell by its electrical force. Which force will “win”? The simple answer is that the ion will be moved by whichever of the two forces is stronger. But how do we know which is stronger? Before determining which force is stronger is helpful to understand when the forces would be the same.A substance is said to be at equilibrium when all of the forces acting on that substance sum to zero (i.e. cancel each other out). This means that for an ion, equilibrium will be achieved when the ion’s concentration force and electrical force have exactly the same magnitude but are going in opposite directions. Unfortunately, we cannot directly compare the concentration and electrical gradients because they have different units (mM and mV), which would be like comparing apples and oranges.

Fortunately, there is a mathematical equation known as the Nernst equation that calculates the electrical gradient needed to exactly oppose an ion’s particular concentration gradient. Since we always set the outside of the cell to 0 mV, in reality the Nernst equation calculates the charge inside the cell that would be needed to create an electrical force that exactly cancels out the ion’s concentration force. The membrane potential of the cell when an ion is at equilibrium is known as the equilibrium potential of that ion.

For example, if a cell had a K+ concentration in the ICF of 150 mM and a K+

concentration in the ECF of 5 mM, the concentration force would tend to drive K+ out of the cell and into the ECF (see Figure 3). We could use the Nernst equation (which will not be explained in this lab), to determine that the equilibrium potential of K+ (given these K+ concentrations) would be -90 mV. Therefore, we could say for certain that when the ICF has a membrane potential of -90 mV (relative to the ECF), the electrical force pulling K+ back into the cell would be equal in magnitude but opposite in direction to the concentration force pushing K+ out of the cell.

Based on what you have just learned, see if you can answer the following questions. Remember that the cell contains protein anions (A-) that cannot leave the cell and that the ECF fluid contains anions such as Cl- that cannot enter the cell.

(Q15) Looking at the cell in Figure 3, if the membrane potential of the cell was -75 mV (instead of -90 mV) and the equilibrium potential of K+ was -90 mV, which force would be larger, the concentration force or the electrical force of K+?

Q16) Would K+ move into the cell, out of the cell, or have no net movement? _____________

Q17) As K+ moved across the membrane, would the membrane potential get closer to or farther away from -90mV? ____________________

By comparing the membrane potential to the ion’s equilibrium potential we can determine if the electrical force of an ion is larger, smaller, or the same size as the concentration force of the ion.

K+

[K+] = 150 mM

K+

-90 mV

Conc. force

[K+] = 5 mM

0 mV

Concentration gradient

Electrical gradient

Elec. force

Figure 3.

Example showing the electrical gradient needed to create an electrical force that would exactly cancel out the concentration force created by the concentration gradient. Since the concentration and electrical forces cancel each other out, K+ is at equilibrium.

Now, based on what you learned about equilibrium potentials, see if you can use your model to answer the following question. Rearrange the beans so that you have 12 Na+ and 12 Cl- ions in the ECF compartment, and 12 K+ and 12 protein anions (A-) in the ICF compartment. Remove the K+ leak channel and replace it with a Na+ leak channel.

(Q18) Will Na+ move across the membrane, and if so, in which direction?_________

_____________________________________________________________________

(Q19) If Na+ moves, what happens to the electrical neutrality of the model?___________

_____________________________________________________________________

(Q20) Based on the above model, predict the sign (e.g., positive or negative) of the equilibrium potential for Na+. (Remember, the equilibrium potential is the electrical charge inside the cell that would exactly oppose the ion’s concentration force.)

_____________________________________________________________________Exercise 4: What creates and maintains the ion concentration gradients in living cells?

In the examples above, we started with systems in which the ECF contained NaCl and the ICF contained K+ and proteins anions. But how did the system first get that way? In order to create concentration gradients between the ECF and ICF, cells must use energy. By the process known as active transport, cells use protein carriers and energy from ATP to move substances against their concentration gradient. We will use the carrier known as the Na+/K+-ATPase to show how cells create their concentration gradients for K+ and Na+. The Na+/K+-ATPase, also called the sodium-potassium pump, is considered an electrogenic pump because as it works, it creates a charge difference across the membrane.

This occurs because it does not exchange ions in a 1:1 manner. The usual ratio is 3 Na+ pumped into the cell in exchange for 2 K+ pumped out of the cell. Let us set up the model to show how this pump creates both concentration and electrical gradients.

On your model, remove any membrane transporters. Place 40 Cl- in a dish at one corner of the ECF compartment and 40 A- in a dish at one corner of the ICF compartment. Within each compartment (both the ICF and ECF) place 20 Na+ and 20 K+.

(Q21) Are the ECF and ICF electrically neutral at this point? ______________________

(Q22) Is there a concentration gradient for K+ ? ____________ for Na+ ? _____________

In order to disrupt a system at equilibrium, energy must be put into the system. This energy will come from potential energy stored in chemical bonds and transferred to the transport protein of the Na+/K+-ATPase by ATP. Place the paper representing the ATPase on the membrane so that the protein is pumping Na+ out of the cell and K+ into the cell.

Roll the die to determine how many turns of the pump you will make. For each turn of the pump, move 3 Na+ from ICF to ECF, and move 2 K+ from ECF to ICF.

(Q23) Now count the Na+ and K+ in each compartment:

ECF Na+ __________ ICF Na+ __________

ECF K+ ___________ ICF K+ ___________

(Q24) Are the ECF and ICF electrically neutral at this point? _____________________ (Q25) If not, what is the net charge on the ECF? _________ on the ICF? _____________

(Q26) What is the membrane potential if the ECF is considered to have a relative charge of zero? _____________________________________________________________________

From this exercise, you can see how the Na+/K+-ATPase plays an essential role in establishing and maintaining the concentration gradients for Na+ and K+.

Exercise 5: Which ion has the largest effect on the cell’s membrane potential?

In a living cell, different types of ions can cross through the membrane at the same time. If Na+, K+, Cl-, and other ions are all moving into or out of the cell in order to bring about their own equilibrium, which ion “wins” and has the largest influence on the cell’s

membrane potential (the charge in the ICF when the ECF is set to 0 mV)? To explore that question, you will use your model again.

On your model, remove any membrane transporters. Place 40 Cl- in a dish at one corner of the ECF compartment and 40 A- in a dish at one corner of the ICF compartment. Within the ICF compartment place 40 K+. Within the ECF compartment place 40 Na+. In the membrane, place a Na+ channel in the closed position and a K+ channel in the closed position.

There is now a concentration gradient for Na+ and a concentration gradient for K+. Because each compartment is electrically neutral, there is no electrical gradient for any ion. This means that initially, Na+ and K+ only have concentration forces acting on them.

Q27) Is either Na+ or K+ ions at equilibrium? _________________________

Q28) If the Na+ channel were to open to allow Na+ to cross themembrane, would the ICF become more positive or more negative? _________________

Q29) If the K+ channel were to open to allow K+ to cross themembrane, would the ICF become more positive or more negative? _________________

Switch both the Na+ channel and the K+ channel to their open positions. Both ions are now equally permeable to the membrane, meaning that one can cross through the membrane just as easily as the other. Let’s assume that each open ion channel can allow 4 ions to cross the membrane in a given amount of time. Move 4 Na+ to the ICF and 4 K+

to the ECF.

Q30) With both ions being equally permeable to the membrane, what effect did this have on the charge in the ICF?

__________________________________________________

Reset the model by moving the 4 Na+ back into the ECF and the 4 K+ back into the ICF. Add another K+ channel to the membrane and put it in the open position. K+ ions are now twice as permeable as Na+ ions. Again, assume that each open ion channel can allow 4 ions to cross the membrane in a given amount of time. Move 4 Na+ to the ICF and 8 K+ to the ECF.

Q31) With K+ having a greater permeability the membrane, what effect did this have on the charge in the ICF?

____________________________________________________

The greater the permeability of an ion, the greater influence that ion will have on the membrane potential. In a living cell, the permeability of K+ is typically far greater

(50 –75 times) than that of Na+ or any other ion, because there are far more leak channels for K+ compared to other ions. Thus, the membrane potential of a cell is typically influenced almost exclusively by K+ ions.

Exercise 6: Changes in K+ concentration in the ECF changes the membrane potential.

As K+ ions, and to a lesser degree Na+, Cl- and other ions, move across the membrane due to the concentration and electrical forces acting on them, the cell’s membrane potential will become relatively stable. The potential at which membrane stabilizes is known as the cell’s resting membrane potential. Since it is primarily K+ that influences the resting membrane potential (because it has the largest permeability), any change in the forces acting on K+ will cause a significant change in the resting membrane potential.

(Q32) Refer back to Figure 3, which showed a cell with 150 mM of K+ in the ICF and 5 mM of K+ in the ECF. We know already that with this concentration gradient, the equilibrium potential of K+ is -90 mV. Since the membrane potential of the cell is at -90 mV, we know that K+ is at equilibrium. However, suppose you add some KCl (electrically neutral) to the ECF so that the concentration of K+ in the ECF goes from 5 mM to 10 mM. [Note: you are adding KCl from another source to the ECF, not simply moving it from inside the cell to outside the cell] What would happen to the concentration force driving K+ out of the cell?

(Q33) Assume that K+ is so much more permeable than all the other ions that K+ is the only ion that influences the resting membrane potential. When the concentration force acting on K+ changes, as it did in the previous question, does the resting membrane potential of the cell become more positive or more negative? Does the resting membrane potential get closer to zero (depolarize) or move farther from zero (hyperpolarize)?

_____________________________________________________________________

From this model cell, you can begin to understand how changing the K+ concentration of the ECF (which includes the blood) alters the resting membrane potential of a cell. If the body’s homeostatic mechanisms for maintaining ECF K+ within a narrow range fail, the

resting membrane potential of cells will change. This can have serious consequences for excitable cells, such as nerves and heart muscle.

Exercise 7: Electrical signals in cells

A cell’s membrane potential is usually stable at the cell’s resting membrane potential. Remember that this stable resting membrane potential is primarily determined by K+ ions since K+ is normally far more permeable than any other ion. However, some cells can temporarily change their membrane potentials away from the resting state by increasing the permeability of different ions (like Na+ or Cl-).

A change in membrane potential can serve as a signal that directs the cell to initiate a particular response. For example, if a change in membrane potential exceeds a certain level known as the threshold, an additional electrical signal known as an action potential is generated in nerve cells and muscle cells.

One exciting new discovery in physiology is the fact that nerve and muscle cells, traditionally considered the excitable tissues, are not the only tissues that use electrical signals. Research into the mechanism by which endocrine cells of the pancreas sense blood glucose concentrations has revealed that these cells also use electrical signals to trigger the release of the hormone insulin. You will now use the model to demonstrate different ways that cells can alter their resting membrane potential.

On your model of a cell, remove all the channels from the membrane. Place 10 Na+, 2 K+, and 10 Cl- in the ECF compartment. Place 8 K+ and 10 A- in the ICF compartment. Place an open K+ leak channel on the membrane. Put the Na+/K+-ATPase on the membrane so that it pumps Na+ out of the cell and K+ into the cell. Put a gated Na+ channel on the membrane in the CLOSED position. You have now created the cell at rest.

(Q34) What is the resting membrane potential of this cell (chargein the ICF relative to the ECF): positive, negative, or zero? ____________________

(Q35) Change the Na+ channel to the open position. Which way will Na+ move, and what forces are acting on it?

___________________________________________________

Now move the ions in the appropriate direction.

(Q36) What happens to the membrane potential of the cellas Na+ moves? Does it become more or less negative? ___________________________

On the graph above, draw in what happens to the membrane potential as Na+ moves.

(Q37) If the membrane potential moves closer to zero mV, the cell is said to have depolarized. If the membrane potential moves farther from zero mV, the cell is said to have hyperpolarized. Did this cell hyperpolarize or depolarize?

_____________________

Gated Na+ channels can be opened when a chemical binds to them (chemically-gated channels) or when the cell membrane is deformed due to pressure on the membrane (mechanically-gated channels). The movement of ions that changes the membrane potential serves as a signal to the cell. (Q38) Return the ions to their starting positions. How do living cells do this? [Hint: Think about how ions can be moved “upstream”]

_____________________________________________________________________

Figure 4.

Continue the trace on the graph to show what happens to the membrane potential after Na+ channels are opened. Does it get closer to zero (depolarizes) or farther from zero (hyperpolarizes)?

0

-70

Mem

bran

e Po

tent

ial (

mV

)

Time

Na+ channel opens

Replace the gated Na+ channel with a gated Cl- channel in the CLOSED position. Assume that the equilibrium potential for Cl- is - 72 mV and that the membrane potential of this cell is -70. Now open the gated Cl- channel.

(Q39) Will Cl- move? If so, in which direction?_________________________________

(Q40) What forces favor and oppose Cl- movement? _____________________________________________________________________

On the graph above, draw in what happens to the membrane potential of the cell when the Cl- channel opens. (Q41) Did the cell depolarize or hyperpolarize?__________________________________

The depolarization created when Na+ enters a cell, as well as the hyperpolarization caused when Cl- enters the cell are both known as graded potentials (“graded” means “variable”). This is because the amount the membrane potential deviates from the resting state can be a small amount or a large amount, depending on how many channels for Na+

or Cl- open.

Exercise 8: The action potential (To be done outside of the lab before the exam)

Now see if you can use the principles you have learned to explain the action potential to your lab partner. The person who thinks he/she understands this the LEAST should be the

Figure 5.

Continue the trace on the graph to show what happens to the membrane potential after Cl- channels are opened.

0

-70

Mem

bran

e Po

tent

ial (

mV

)

Time

Cl- channel opens

person to begin. If you like, you can use the beans to demonstrate. Be sure to include all the terms listed below. Some hints and leading questions are included to help you.

axon axon hillockconduction of action potential graded potentiallocal current flow thresholdvoltage-gated K+ channels voltage-gated Na+ channels

What is needed for an action potential to begin?What force opens the voltage-gated Na+ channels ?What force opens the voltage-gated K+ channels?Why don’t the two types of voltage-gated channels open at the same time?Which way does Na+ move when the voltage-gated Na+ channels open? What happens to

the membrane potential?Which way does K+ move when the voltage-gated K+ channels open? What happens to

the membrane potential?

On the graph, draw in the voltage changes that occur with an action potential and explain what channels are open and which ions are moving at each part of the action potential.