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Membrane Potentials and Action Potentials

Membrane Potentials and Action Potentials. Chapter 5 And chapter 45. http://www.blackwellpublishing.com/matthews/default.html. Lecture outline. I. Review A. Permeability B. Concentration gradients C. Sidedness of the membrane II. Electrical gradients A. Potential

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Membrane Potentials and Action Potentials

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  1. Membrane Potentials and Action Potentials Chapter 5 And chapter 45 http://www.blackwellpublishing.com/matthews/default.html

  2. Lecture outline I. Review A. Permeability B. Concentration gradients C. Sidedness of the membrane II. Electrical gradients A. Potential B. Electrolytes C. Conductance (permeability) III. Resting membrane potential A. Caused by i. Proteins ii. Na+/K+ ATPase iii. K+ “leak” channels (pores) B. Nernst potential C. Why is RMP so close to K+ nernst potential IV. Excitable cells A. Gene expression of unique integral proteins B. Neurophysiology terms C. Action potentials i. Caused by a. passive diffusion of ions when channels open Ligand gated Voltage gated

  3. You don’t need to know the Nernst equation. I will tell you how to think about it. • You see body fluid compartments, separated by a membrane. • Will the solute diffuse down the concentration gradient? Yes, if it can get through. • Well, now I am adding that the solute also needs to be able to go through an electrical gradient.

  4. Cell Membranes • What two conditions must be met for diffusion of a species across a semipermeable membrane? • Is the membrane permeable to it? • Does it have a concentration gradient? • If the answer is yes to both questions, then the species will diffuse (Which way? Down it’s gradient)

  5. inside (in mM) 14 140 0.5 10-4 (pH 7.2) 10 5-15 2 75 40 outside (in mM) 142 4 1-2 1-2 (pH 7.4) 28 110 1 4 5 Na+ K+ Mg2+ Ca2+ H+ HCO3- Cl- SO42- PO3- protein “Sidedness” of the membrane and some reasons why Different permeability Pumps Protein channels Remember to ask: Is there a gradient? Can it diffuse? If allowed to diffuse, which way would it go?

  6. Not just separation of solutes, but charges, too! + + + + • Inside of the cell is negative due to : • Abundance of negatively charged proteins • Na+/K+ ATPase (net loss of positive charges~ 4mV) • Membrane is 100x more permeable (“leaky”) to K+ _ _ _ _ _ _ _ _ _ _ _ _ + + + + + + + + + + + + Let’s explore this concept more........

  7. Sidedness • “Sidedness” of the membrane • Sidedness means that the electrical charges on one side of the membrane (positive or negative) are different than on the other side. • Why does sidedness exist? • Different permeability • Pumps • Protein channels • How does a membrane become sided? • Primary and secondary active transport, or pores that allow only one particular solute to move. These things make a higher concentration on one side. Therefore, sidedness is caused by proteins.

  8. Take an electrode, pierce the membrane, attach to voltometer, compares the charges inside and outside of the cell. Inside of cell is more negative for three reasons: • Proteins are abundant inside cell,and are negatively charged at your normal pH. • Sodium-potassium-ATPase mechanism contributes toward the electronegativity inside the cell. Two K are pulled in while 3 Na go out. They are all +1, so net loss of one positive charge, so the inside is more negative than the outside. • Most important reason: potassium leak channels. If men are K and women are Na, ladies are most abundant outside of cell, more guys inside room. After class, guys want to leave, they can use the door. Ladies want to get into the room, but to get in, they have to come under the crack of the door. Not likely to get in. Sodium does not have leak channels, which are created by integral proteins. Potassium leaves by leak channels, contributing to negativity.

  9. How negative is the inside of the cell? • Minus 70 mV (milivolts), depends on the cell. Heart cells are minus 90, some are minus 50-60. • We have chemical and electrical sidedness on a cell membrane. The membrane has membrane potential (separation of charges). You can calculate voltage. If the charges on a battery reach equilibrium on both sides, the battery will be dead. That can happen to our cells, too. We need to use the electrical energy in our cells to do kinetic work. You have to turn on a machine to engage its electrodes. The cells don’t all allow the diffusion of electrical current to do work with.

  10. The ones that can increase resistance have something special that other cells don’t have. Cells that can increase resistance have proteins that create channels. Examples of this type of cell are neurons and muscle cells; they allow charges to move across the membrane, because they express genes that make the integral proteins that create these channels. Charged ions, K+, Na+, Ca++ are ions, so they are called electrolytes. When they move, they carry an electrical charge. What do electricians use to reduce resistance of wires? They use Insulation, in the form of plastic coating on wires. We need resistance (block flow of charges). A cell creates this resistance by blocking the channel.

  11. Myelinated neurons carry current faster (the current skips over the Nodes of Ranvier and just has to travel down the bare portions of the axon). Another thing that affects speed of electrical transmission is the size of neuron: bigger neurons carry current faster (expand the freeway to add extra lanes, you will get home faster). • Conductivity means the permeability. If conductivity increases, it means that permeability increased. • Ions diffuse at a faster rate when there is less resistance. The more resistance there is, the less conductivity, and less resistance will cause more conductivity. Myelin reduces resistance because it makes electrical charges move faster.

  12. - - - - - - - + + + + + - + Electricity • Current: the flow of charge • Voltage: separation of opposite charges (mV) • Voltage • Voltage difference • Potential difference • Potential • Resistance: opposition to charge movement (friction) • Conductance: allowing a charge to move (permeability) What are the charged things that run through our body fluids? Electrolytes! ions: Na+ K+ Cl- Ca++

  13. - - - - - - - + + + + + - + = Na+ When dealing with things that are charged …. You must ask an additional question! • Is the membrane permeable to it? • Is there a chemical gradient for it? • Things tend to move from high to low concentration • Is there an electrical gradient for it? • Things tend to move to regions of opposite charge Only then, can you predict! • How do you measure each of these gradients or forces? Sometimes, the chemical gradient is favors one ion to go in one direction, and the electrical gradient favors it to go in the other direction. The stronger pull will win.

  14. Every cell has a separation of charge; the #1 reason is the leakiness of K. It leaks out all the time, and the Na pushes K back in. We use this electricity to do work. Blood pressure, peristalsis of intestines, muscles, etc, use this electricity for work.

  15. Because of this separation of chemicals and electrical charges, every cell has a Resting Membrane “Potential” • A difference in electrical charge across the membrane (a potential difference) • More negative inside; more positive outside • Our cells are like batteries and some cells can tap into this “potential energy” to do work (“kinetic energy”) • What generates it? • Mainly, ion concentration gradients and differences in membrane permeability (leaky to K+ but not to protein) • -70-90mV

  16. Why is the resting membrane potential negative? Because K has leakiness, so it escapes with its positive charge, leaving the inside of the cell more negative. • Which force is “winning” at rest? Potassium • How can simple diffusion cause this potential? There is not much Na inside the cell, so sodium wants to diffuse in with its positive charge.

  17. The inside of the cell is negative because there are K leak channels, that means there is greater permeability for K, so it will diffuse out of the cell down its concentration gradient. • The membrane potential (how negative or positive is) is a number that is a reflection of the ion with the greatest permeability. If our cells are minus 70 mV, it’s because they are most permeable to K.Therefore, K will diffuse out its concentration gradient, taking its positive charges with it, leaving the inside of the cell more negative. What if the cell was more permeable to Na? Sodium would diffuse down its concentration gradient to the inside of the cell, taking its positive charges with it, making the inside of the cell more positive.

  18. Na+ Cl- Na+ Na+ K+ Na+ Cl- Cl- Na+ Cl- Na+ K+ K+ K+ Na+ K+ K+ - Cl- Proteins Organic phosphates - - - - - - - Na+ - - Cl- Na+ Cl- K+ Membranes are leaky! Solutes diffuse down their EC gradients. Most leaky to K+ Not permeable to proteins (too big!) What happens? “Diffusion potential” Let’s explore this concept more........

  19. So, we have a battle: diffusion of a chemical gradient and the diffusion of the charges (Electrical potential ) + - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + - - When will the negatively charged molecules stop entering the cell? The Nernst potential (equilibrium potential) is the theoretical intracellular electrical potential that would be equal in magnitude but opposite in direction to the concentration force. In other words: when does the attraction between opposite charges oppose the diffusion of a chemical gradient?

  20. In the previous picture, just look at the number of blue-dot particles (ignore the charges). There are more blue dots outside of the cell, so there is a chemical gradient for the blue dots to move inside of the cells. Insert a voltage rod to inject positive charges into the cell. The negative charge of the blue dots will want to enter the cell because they are attracted to the positive charges there. Thus, there are two reasons why these blue dots will quickly enter the cell. But when will the blue dots stop going into the cell? They will be attracted to the positive charge, but if they are all inside, the chemical concentration gradient makes them want to diffuse out of the cell. When the electrical and chemical gradient is equally powerful (in opposite directions), that is the Nernst potential: No net gain or loss.Cells with resting membrane potential are at minus 70mV. They are not at their resting K potential. If you open more K channels than just the leak channels, there will be more movement of K out of the cell, and the potential will get closer to minus 94 mV (at which time, the cell will reach equilibrium, and the cell will die; but the body does not let it get that far).

  21. The Nernst potential (equilibrium potential) is the theoretical intracellular electrical potential that would be equal in magnitude but opposite in direction to the concentration force. • In other words: when does the attraction between opposite charges oppose the diffusion of a chemical gradient? • In this case we’re changing the electrical potential across the membrane to see what happens to the concentration gradient of the ion. In reality, the concentration gradient is changed during various cellular processes which determine the Nernst potential.

  22. Membrane Potential (Vm): - charge difference across the membrane - outside inside …how can passive diffusion of potassium lead to development of negative membrane potential? K+ K+ Na+ Na+ Answer: Potassium leaks out of the cell, taking its positive charge with it, leaving the inside of the cell more negative.

  23. Simplest Case Scenario: inside outside If a membrane were permeable to only K+ then… K+ K+ K+ would diffuse down its concentration gradient until the electrical potential across the membrane countered diffusion.

  24. As K leaves the cell, it takes a positive charge outside with it, so the inside is more negative. However, as the inside of the cell is becoming more negative, the outside of the cell is becoming more positive, and the positive charges will want to flow back inside of the cell since they are attracted to the negative charges. This is electrical potential that counters the net diffusion of K. • The electrical potential that counters net diffusion of K+ is called the K+ equilibrium potential (EK). • The equilibrium potential of K is minus 94 mV • So, if the membrane were permeable only to K+, Vm would be -94 mV (cell death from equilibrium)

  25. Simplest Case Scenario: inside outside If a membrane were permeable to only K+ then… K+ K+ The electrical potential that counters net diffusion of K+ is called the K+ equilibrium potential (EK). So, if the membrane were permeable only to K+, Vm would be -94 mV

  26. Simplest Case Scenario: inside outside If a membrane were permeable to only Na+ then… Na+ Na+ would diffuse down its concentration gradient until potential across the membrane countered diffusion. Na+ The electrical potential that counters net diffusion of Na+ is called the Na+ equilibrium potential (ENa). So, if the membrane were permeable only to Na+, Vm would be +61 mV

  27. But the body prevents the cell from reaching equilibrium. Since the resting membrane potential is minus 70 mV, (a negative number), it tells us that potassium has the greatest membrane potential at rest. Don’t confuse this with thinking that potassium has a negative charge. It has a +1 charge, the same as sodium. But when potassium leaves the inside of the cell, it takes its positive charge with it, leaving the inside of the cell more negative. The inside of the cell is negative because proteins have a negative charge, and the cell contains so many proteins.

  28. At resting membrane potential, cell voltage is at minus 70 mV. • Since potassium’s chemical equilibrium is minus 94, potassium’s chemical equilibrium is not met yet. • That means that it will WANT to flow out of the cell. • But the difference between the voltage of where it is (minus 70) and where it wants to be (minus 94), is only 24 mV. • This is not a very strong difference. • Since sodium’s chemical equilibrium is plus 61 mV, sodium’s chemical equilibrium is not met yet. • That means that it will WANT to flow into of the cell. • The difference between the voltage of where it is (minus 70) and where it wants to be (plus 61), is 131 mV. • This is a much stronger difference, compared to potassium. Therefore, the ion with the strongest driving force is sodium because its equilibrium potential is much different from the resting membrane potential. • If you want to harness the electrical current to use it to do work, use the sodium driving force instead of potassium.

  29. Anatomy of a neuron

  30. Receptors on the dendrites act to bind proteins, can direct things into and out of the cells. • Neurotransmitters from one synaptic knob are released onto another dendrite; a channel opens within the receptor on the dendrite. If the channel was for sodium only, sodium will move inside of the cell. The sodium travels down to the axon hillock. If enough sodium is allowed to enter the soma, the sodium that reaches the axon hillock can trigger the opening of the first voltage-regulated gated channel on the axon. Channels distal to the hillock are locked down (bound), and will open only if electrical charges have been reduced on inside of cell (they are voltage regulated). As the sodium gets to the axon hillock, the axon will get less negative, so the inside of the cell is less negative, will it will open K voltage channels. The first opened gated channel will open the next gated channel, and so on, down the axon. The axon hillock is the trigger zone where you start to see voltage gated channels.

  31. When you are firing a gun, even if you squeeze the trigger slowly, there is a trigger point where the bullet will fly out of the gun. Pulling the trigger slow or fast does not change velocity of the bullet. The voltage gated channels are same; if one opens, they will all open one by one, like a wave. If enough sodium diffused in when a ligand channel opened, the first voltage gated channel will open. A ligand is an integral protein in a cell membrane that binds to a chemical, and then transports it into the cell. Thus, it serves as both a receptor and the transporter.

  32. If you want more electrical current, open the sodium channels first (instead of the potassium channels). When you increase sodium conductance (permeability), sodium can move down its electrical gradient as well as its chemical gradient. Sodium’s equilibrium potential (+61), will make inside of the cell very positive, which is the opposite of the resting membrane potential (minus 70). The reversal of the membrane potential is called the ACTION POTENTIAL.

  33. But you cannot let sodium continue on into the cell until it reaches equilibrium, or the cell will not be able to metabolize, and it will die. To prevent too much sodium from entering the cell, you have to open the K channel to allow K to diffuse out by its chemical and electrical gradients. This is called the dance of the gates. During an action potential, the sodium gate opens first, the potassium gate opens second. Are the ions where they should be? If not, something needs to push K back into the cell and Na out: Na-K ATPase (the mother protein, or housekeeping protein). Mom organizes the house and the kids mess it up again. She directs the kids to take their toys and put that here, and put that there. During action or resting potential, Na-K ATPase is active all the time, constantly trying to reestablish the gradients. She never stops working. When is the Na-K ATPase active? ALWAYS!

  34. Normal conditions Vm -74 ENa+61 EK -94 0 mV 20 mV 135 mV What is the net driving force on K+ ions? What is the net driving force on Na+ ions? Which way do the ions diffuse? What effect does increasing Na+ or K+ permeability (or extracellular concn) have on Vm? Why is Vm so close to EK? Ans. The membrane is far more permeable to K+ than Na+. The resting membrane potential is closest to the equilibrium potential for the ion with the highest permeability!

  35. What keeps the ion gradients from running down? The sodium/potassium ATPase (“the housekeeper”) Do we want our cells to be like a “dead battery?” outside inside K+ Na+ Na+ ATP K+ 3 Na+ 2 K+ Remember: sodium is pumped out of the cell, potassium is pumped in... ADP • Integral membrane protein found in all cells which “pumps” (against their gradients across the membrane) Na and K. • Fueled by ATP • ATP ADP + Pi + energy This pump is electrogenic, it contributes slightly to RMP

  36. The “Resting” Cell Cl- Na+ Cl- Na+ K+ Na+ Cl- Na+ Na+ Cl- Na+ Diffusion, leak K+ K+ Na+ K+ + Na+ K+ K+ + + - Cl- Proteins Organic phosphates - - Diffusion, leak - - - - - The Housekeeper - - Na+ K+ - Na+ K+ - - Cl- Na+ Cl- K+ Cl-

  37. Integral membrane proteins found in all cells will “pump” (against their gradients across the membrane) Na and K. This is fueled by ATP. • ATP  ADP + Pi + energy

  38. http://bcs.whfreeman.com/thelifewire/content/chp44/4402001.htmlhttp://bcs.whfreeman.com/thelifewire/content/chp44/4402001.html http://www.sumanasinc.com/webcontent/animations/biology.html

  39. Na+ Cl- Cl- Na+ + + Na+ Cl- + + Cl- Na+ - - - - Na+ Cl- Na+ Cl- Na+ Cl- -70 mV What if….. • What if a membrane suddenly became MORE PERMEABLE to Na+????? • Even for just a moment in time….. • What would Na+ do? (Ask yourself the 3 questions) Which way is the electrochemical gradient for Na+? Electrical: inward Chemical: inward Answer: Most definitely INWARD Sodium WANTS IN! What would happen to the membrane potential of the cell when this event occurs?

  40. What would happen to the membrane potential of the cell when you open up a sodium channel? • If we instantly increase sodium permeability, sodium will enter the cell, creating a large electoral current, then K permeability will increase, and the membrane potential will return to negative.

  41. Excitable cells (neurons and muscles) are those that want this large electrical current to use for work. • They have proteins that are sodium channels. Not all cells have these proteins. All cells have the genes to make these proteins, but only the excitable cells EXPRESS these genes, and actually make the proteins that fuse with the cell membrane and form a sodium channel. Muscle cells use the electrical force to contract, and neurons use it to excite the neurons touching them.

  42. EXPRESSION ! Excitable Cells • Cells that can experience a momentary change in membrane voltage are “excitable” cells • That temporary change in voltage is due to a momentary change inpermeability • The membrane, for only a moment, becomes more permeable to Na+ than to K+ • When it’s called an Action Potential-it is a reversal of the membrane potential! • Cell becomes positive inside!!! • Question: What can allow membrane permeability to these ions? • Why are neuronal cells and muscle cells able to change their membrane potential? Integral proteins that can open and close depending on stimulus How did we discover these unique integral proteins?

  43. Hodgkin-Huxley Expts, 1952 Squid Giant Axon Few neurons, large diameter Large enough to insert microelectrodes Stimulating microelectrodes (inject current) to disturb cell with electrical stimuli Recording microelectrodes (see current changes in cell and record them) http://www.science.smith.edu/departments/NeuroSci/courses/bio330/squid.html

  44. Definitions: • There is a potential difference (pd) across the cell membrane • (minus 70 mV) is called the “Resting Membrane Potential” • Because a charge is present (it is not zero), we say the membrane is “polarized”

  45. If it becomes less negative, it is called depolarization (happens when sodium is entering the cell). • If it becomes more negative than minus 70, it is hyperpolarization. (happens when K leaves the cell) • In either case, when you go back towards minus 70, it is repolarization. • Thresholdis the point at which the first voltage-regulated sodium channel opens. Question • To depolarize a cell, what kind of charge must be put into the cell, positive or negative? Positive

  46. 0 mV excitability repolarization + threshold depolarization resting potential -90 mV +- hyperpolarization 70 mV Voltmeter - - - - - “Resting Membrane Potential”(70 mV) • excitability • Depolarization-a current entering the cell that decreases the polarity (voltage) across the membrane (that is, bring voltage closer to 0 mV). • To depol, what kind of charge must be put into the cell, positive or negative? • Hyperpolarization-a current that increases the voltage across the membrane (brings it farther from 0 mV.) • repolarization • towards resting potential

  47. When you take the mantle off the giant squid, the nerves are right there, and are so big, you can touch them. Hodgekin and Huxley put one of these neurons in an isosmotic solution and inserted four wires along the axon, distal to the hillock. The first wire was attached to an instrument that can inject a positive charge into the cell (increasing its membrane potential). The next three wires (R1, R2, R3) received the signal and measured the resulting charge. In this way, they could find out if the injected positive charge would continue down the axon or dissipate.

  48. With a small injection of a positive charge, they found the membrane potential was less as it got farther from electrode (the charge dissipated). Then, they put in a lot more positive charges, there was a greater potential in the other three receiving electrodes, but it still dissipated. Then they gave it a whopping positive charge, RMP rose to minus 160, and they found a reproducible membrane potential, which continued through length of axon, and they were able to maintain this reversal of the RMP. The voltage gated sodium channels allowed the Na to come in, allowed more Na gated channels to open, one after another. This is called an action potential. Sodium ions entering the cell is what created the action potential. You can have hundreds of thousands of action potentials in a second, but there is not a flood of sodium coming in; only a few ions need to go in to make an action potential.

  49. First try: a small depolarizing stimulus (-65 mV) +40 - +30 - +20 - +10 - 0 - -10 - Voltage (mV) -20 - -30 - -40 - -50 - -60 - -70 - time Stim Elec REC 1 REC 2 REC 3 ++++ ++ + +++

  50. Next try: a slightly larger depolarizing stimulus (-60 mV) +40 - +30 - +20 - +10 - 0 - -10 - Voltage (mV) -20 - -30 - -40 - -50 - -60 - -70 - time Stim Elec REC 1 REC 2 REC 3 ++++++ ++++++ +++ ++

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