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MED 1200

MED 1200. Membrane Excitability Tutorial. Dr. Stephen Gee. You may only access and use this PowerPoint presentation for educational purposes. You may not post this presentation online or distribute it without the permission of the author. Objectives. Resting Membrane Potential

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MED 1200

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  1. MED 1200 Membrane Excitability Tutorial Dr. Stephen Gee You may only access and use this PowerPoint presentation for educational purposes. You may not post this presentation online or distribute it without the permission of the author.

  2. Objectives Resting Membrane Potential • Predict the effect of changing Na+, K+ or Cl- concentration gradients or permeabilities on resting membrane potential. • Predict the effect of inhibiting Na+/K+ ATPase on resting membrane potential. Action Potential • Predict the effect of changing Na+ or K+ concentration gradients on the amplitude and threshold of the action potential. • Predict the effect of blocking the voltage-gated and background (leaky) Na+ or K+ channels on the amplitude, threshold and duration of the action potential. Synaptic Membrane Potentials • Predict the effect of opening the following ligand-gated channels on postsynaptic membrane potential: Na+ channels, K+ channels, Na+/K+ channels, Cl- channels. Membrane Excitability Tutorial – Dr. Stephen Gee

  3. Question 1: • How would the resting membrane potential (Er) change by: • Doubling [K+]out ? • Doubling [Na+]out ? • Doubling [Cl-]out ? • How would Er change by: • Increasing PK? • Increasing PNa? • Increasing PCl?

  4. Membrane Potential All cells under resting conditions have a potential difference with the inside being more negative – the resting membrane potential. ECF assigned a voltage of zero If the potential across membrane is 70 mV and ICF has excess –ve charge, then the membrane potential is -70 mV. Fig. 6-8 Membrane Excitability Tutorial – Dr. Stephen Gee

  5. Charge Separation Across the Plasma Membrane There is a tiny excess of –ve ions inside the cell and a tiny excess of +ve ions outside. Excess charges collect at plasma membrane. The bulk of the ECF and ICF remains neutral. The actual number of charges that are separated is infinitesimally small compared to total number of charges in the cell. Only a very thin shell of charge difference is needed to establish a membrane potential. Figure 6-9 Membrane Excitability Tutorial – Dr. Stephen Gee

  6. Distribution of Ions in a Nerve Cell There are many other ions, including Mg2+, Ca2+, H+, HCO3-, HPO42-, SO42-, amino acids, and protein in both fluid compartments so that the bulk of the ICF and ECF are electrically neutral. Na+, K+ and Cl- are present in the highest concentration and membrane permeability to each is independently determined. Na+ and K+ play the most important role in generating the resting membrane potential but Cl- is a factor in some neurons. The resting membrane potential established and maintained mainly by Na+/K+-ATPase activity. Membrane Excitability Tutorial – Dr. Stephen Gee

  7. Membrane Potential The magnitude of the membrane potential depends mainly on two factors: [Ion] differences (outside versus inside) Membrane permeability to the different ions (number channels open for each ion) Consider a membrane that is permeable only to K+ (a) no potential difference because +ve ions are equal and channels are closed. (b) If K+ channels open, K+ will diffuse from 2  1 (c,d) Compartment 1 will have an excess of +ve charges and compartment 2, an excess of –ve charges. (e) The buildup of +ve charges in Compartment 1 (and –ve charges in Compartment 2) produces an electrical potential that exactly offsets the [K+] gradient. The membrane potential at which the flux due to concentration difference becomes equal in magnitude but opposite in direction is called the equilibrium potential for that type of ion. Fig. 6-10 Membrane Excitability Tutorial – Dr. Stephen Gee

  8. Membrane Potential…(cont’d) Consider a membrane that is permeable only to Na+: (a) no potential difference because +ve ions are equal and channels are closed. (b) if Na+ channels are opened, Na+ will diffuse from 1  2 (c,d) Compartment 2 will have an excess of positive charges and compartment 1, an excess of negative charges. (e) At the Na+ equilibrium potential: buildup of positive charge in Compartment 2 produces an electrical potential that exactly offsets the Na+ chemical concentration gradient. Membrane Excitability Tutorial – Dr. Stephen Gee

  9. The Nernst Equation: Equilibrium Potential The Nernst equation describes the equilibrium potential for any ion species – that is, the electrical potential necessary to balance a given ionic concentration gradient across a membrane so that the net flux on that ion is zero. Eion = 61/Z log (Co/Ci) Where, Eion = equilibrium potential in mV Ci = intracellular ion concentration Co = extracellular ion concentration Z = valence of the ion 61 is a constant that takes into account the universal gas constant, the temperature, and the Faraday electrical constant. ENa = 61/1 log (145/15) = +60 mV EK = 61/1 log (5/150) = -90 mV What does this mean? At typical concentrations (see Table 6.2), Na+ flux through open channels will drive the membrane potential towards +60 mV while K+ flux will bring it towards -90 mV. In an actual nerve at rest, there are many more open K+ channels than Na+ channels; chloride permeability generally falls in between. PK = 1, PNa = 0.04, PCl = 0.45. Membrane Excitability Tutorial – Dr. Stephen Gee

  10. Forces Influencing the Resting Membrane Potential (a) At a resting membrane potential of -70 mV both the concentration gradient and electrical potential favor inward movement of Na+, while the K+ concentration gradient opposes the electrical potential. (b) The greater permeability and movement of K+ maintains the resting membrane potential at a value near Ek. In general, the greater the permeability for an ion, the higher the influence of this ion on Er. In order to predict the influence of a given ion species on membrane potential, one needs to know the equilibrium potential for this ion, which in turn depends on its concentration gradient, and is calculated by the Nernst equation. An ion will always have a tendency to bring the membrane potential towards its own equilibrium potential. Voltage (V) = current (I) x resistance (R) Membrane Excitability Tutorial – Dr. Stephen Gee

  11. The Goldman-Hodgkin-Katz Equation • How do we calculate the membrane potential if more than one type of channel is open? • We need to consider the permeability (P) for each ion species (how many channels are open) and the concentration difference. • The GHK equation is essentially a modified form of the Nernst equation that takes into account the individual ion permeabilities. • Vm = 61 log PK[Ko] + PNa[Nao] + PCl[Cli] PK[Ki] + PNa[Nai] + PCl[Clo] *Note that for chloride, [Cli] is in the numerator instead of the denominator. This is because it is a negatively charged ion Membrane Excitability Tutorial – Dr. Stephen Gee

  12. Resting membrane potential (Er) - Question 1A: How would Er change by: Doubling [K+]out ? Membrane depolarization. Since [K+] gradient is decreased, K+ ions would have less tendency to leave the cell. In other words, doubling [K+]out would cause EK to become less negative**, and the membrane potential would also become less negative. ** You can verify this with the Nernst equation. Thus, using [K+]in = 150 mM, Ekwould go from -90 to -72 mV if [K+]out increases from 5 to 10 mM. Doubling [Na+]out ? Membrane depolarization. ENa would become even more positive such that Na+ ions would have a greater tendency to rush in. However, changes would be much less pronounced with Na+ than with K+ because PNa << PK Doubling [Cl-]out ? Membrane hyperpolarization. The membrane should hyperpolarize since ECl would become more negative. Final effect would depend on PCl which varies with cell type. Cl- Cl-

  13. Resting membrane potential (Er) - Question 1B: How would Er change by: Increasing PK? Hyperpolarization since EK = -90 mV (i.e. more K+ ions would leave the cell if PK is increased). Remember that a ion will always have a tendency to bring the membrane potential closer to its equilibrium potential. Increasing PNa? Depolarization since ENa = + 60 mV (more Na+ ions would enter the cell if PNa is increased) Increasing PCl? Varies among cells, depending on ECl. • If ECl = Er (e.g.: in most skeletal muscle cells)  No effect of changing PCl. • If EClis more negative than Er (e.g.: in most neurons) hyperpolarization; Cl- would rush in to make it more negative inside. Cl- Cl-

  14. Question 2: How would inhibition of the Na+/K+ pump affect: • [K+]in? • [Na+]in? • Er?

  15. Establishing the Resting Membrane Potential The Na+/K+ ATPase establishes concentration gradient and generates a small negative potential; pump uses up to 40% of the ATP produced by the cell. Greater net movement of K+ than Na+ makes the membrane potential more –ve on the inside of the membrane. (c) At a steady state negative resting membrane potential, ion fluxes through the channels and activity of the pump balance each other. Fig. 6-13 Membrane Excitability Tutorial – Dr. Stephen Gee

  16. Inhibition of the Na+/K+ ATPase Pump – Question 2: Whatis the effect on the following? [K+]in This inhibitionwould result in a loss of K+ ions ([K+]in would decrease) [Na+]in A gain of Na+ ions ([Na+]in would increase). Er Depolarization because of the consequent dissipation of [K+] gradient. After blocking the pump, the cell would start to lose its K+ ions, such that EK would progressively become less and less negative (until it gets close to 0 mV as [K+]in approaches [K+]out ). The resting membrane potential should therefore progressively depolarize as it follows EK. (Remember that K+ permeability is higher in the resting membrane so changes in [K+] have more of an effect on Er) Membrane Excitability Tutorial – Dr. Stephen Gee

  17. Objectives Resting Membrane Potential • Predict the effect of changing Na+, K+ or Cl- concentration gradients or permeabilities on resting membrane potential. • Predict the effect of inhibiting Na+/K+ ATPase on resting membrane potential. Action Potential • Predict the effect of changing Na+ or K+ concentration gradients on the amplitude and threshold of the action potential. • Predict the effect of blocking the voltage-gated and background (leaky) Na+ or K+ channels on the amplitude, threshold and duration of the action potential. Synaptic Membrane Potentials • Predict the effect of opening the following ligand-gated channels on postsynaptic membrane potential: Na+ channels, K+ channels, Na+/K+ channels, Cl- channels. Membrane Excitability Tutorial – Dr. Stephen Gee

  18. Changes in Membrane Potential The resting membrane potential is ~ -70 mV. The membrane is polarized. The membrane is depolarized if the potential is less –ve (0) and hyperpolarized if it is more –ve (towards EK). The membrane polarity can be reversed if the potential becomes positive (inside of cell +ve) – overshoot. Figure 6-14 Membrane Excitability Tutorial – Dr. Stephen Gee

  19. Action Potentials An action potential (AP) is an “all-or-none” sequence of changes in membrane potential resulting from changes in ion permeability due to the operation of voltage-gated Na+ and K+ channels. APs are generally very rapid (can be a few milliseconds) and can have frequencies of several hundred/sec Voltage-gated channels give the membrane the ability to generate and propagate APs. Fig. 6-18 Membrane Excitability Tutorial – Dr. Stephen Gee

  20. Action Potential Mechanism Absolute refractory period Relative refractory period PNa PK Membrane Excitability Tutorial – Dr. Stephen Gee

  21. Threshold Potential Depolarization of excitable membranes triggers an AP only when the membrane potential exceeds a threshold potential (~-55 mV) . A threshold potential is defined as a potential that will generate an AP. It depends on: 1. physical properties of the Na+ channel, 2. the number of functional channels and 3. the Na+ gradient. Regardless of the size of the initial stimulus, if the membrane reaches threshold, the APs generated are all the same size. Because the amplitude of a single AP does not vary in proportion to the amplitude of the stimulus, a AP cannot convey information about the magnitude of the stimulus that initiated it. Fig. 6-21 Four action potentials, each the result of a stimulus strong enough to cause depolarization, are shown in the right half of the figure. Membrane Excitability Tutorial – Dr. Stephen Gee

  22. Action Potentials: Amplitude and Excitability AP Amplitude: Amplitude= Peak - Er ≈ +30 – (-70) = 100 mV The peak value (+30 mV) depends mainly on [Na+] gradient. Er (-70 mV) depends mainly on [K+] gradient. Membrane Excitability: Depends on the difference between threshold and Er. The greater the difference, the less excitable the membrane is. Peak Amplitude Threshold Er An AP is like a fire that starts at a spot and spreads. You need a certain amount of heat (analogous to Na+ influx or current) to ignite the fuel (Na+ channels) initially. The temperature at which the fuel ignites is the threshold. The burning is all or none. Once burned, it cannot burn again until it has regenerated. If there is insufficient heat generated by the burning then it will not ignite the fuel adjacent to it and the fire will not propagate. One needs sufficient fuel (channels) and heat (Na+ ions) to start the fire and keep it burning.

  23. Question 3: Effect of Ion Gradients on Action Potentials How would the action potential amplitude and membrane excitability be affected by the following events? • a decrease in [Na+]out • a decrease in [K+]out • an increase in [K+]out • inhibition of the Na+/K+ ATPase

  24. Question 3A: Effect of decreased [Na+]out Amplitude Threshold Er • AP amplitude would decrease due to  peak value (because the driving force for Na+ entry is decreased), with little effect on Er. • Membrane excitability would decrease due to an increased threshold. • Why does the threshold increase with low [Na+]?

  25. Question 3A: Effect of decreased [Na+]out cont’d… • The voltage threshold for the action potential varies because a minimum size current is needed to trigger an AP. • With a decrease in [Na+]out, the current going through an open channel is smaller. So you need to open a greater percentage of the population of voltage-gated channels to generate a current large enough to trigger an AP. • Furthermore, these channels must be activated synchronously because there's a very brief window for summation to occur because they rapidly inactivate. • Threshold voltage depends on the properties of the Na+ voltage-gated channels, but also depends on the density of the channels. For example, the axon hillock has a high concentration of Na+ channels and has a lower threshold potential (~-60 mV). • Low [Na+] (and Na+ channel blockers) decrease excitability because they make the threshold voltage more positive. If you decrease [Na+]out, or block Na+ channels, you need to depolarize further in order to compensate for the diminished inflow of Na+ ions. • The more depolarized the membrane is, the higher the probability that a voltage-gated Na+ channel will open (This also applies to voltage-gated K+ channels).

  26. Voltage-dependent activation and inactivation of the cardiac sodium channel. • Using the patch-clamp technique, the membrane potential dependence of activation is studied by applying 50 ms depolarizing voltage steps from a holding potential of −120 mV (inset). The activation curve is obtained by dividing the amplitude of the resulting sodium current at each voltage step by the maximum peak sodium current amplitude, and plotting versus the corresponding voltage. • The membrane potential dependence of inactivation is studied by applying 500 ms depolarizing voltage steps from a holding potential of −120 mV to inactivate the channels (pre-pulse). Next, the fraction of channels that is not inactivated by the pre-pulse is measured by applying a voltage step to -20 mV (test pulse). The inactivation curve is obtained by dividing the amplitude of sodium current at each test pulse by the maximum peak sodium current amplitude, and plotting versus the corresponding pre-pulse voltage. More channels active here… …than here Fraction of channels NOT inactivated At -55 mV ~95% of Na+ channels are inactive! Only a small fraction are needed to generate an AP Amin et al., 2010, PflügersArchiv - European Journal of Physiology 460: 2

  27. Question 3B: Effect of decreasing [K+]out (hypokalemia) Amplitude Er • AP amplitude would increase due to Er, with little effect on peak value. • Membrane excitability would decrease due to increased difference between threshold and Er.

  28. Question 3C: Effect of increasing [K+]out (hyperkalemia) Amplitude Er Er • A moderate increase in [K+]out would cause a moderate depolarization that would bring Er closer the threshold  Increased excitability (and decreased amplitude) • A large increase in [K+]out would cause a large depolarization which would eventually lead to a sustained closure of the voltage-gated Na+ channels (i.e. their inactivation) and a loss of excitability of the membrane.

  29. Question 3D: Effect of Na+/K+ ATPase Inhibition Depolarizationof Er(similar to hyperkalemia): After blocking the pump, the cell would start to lose its K+ ions, such that EK would become less negative (until it gets close to 0 mV as [K+]in approaches [K+]out ). The resting membrane potential should therefore progressively depolarize as it follows EK AP amplitude decreases because of i) depolarized Er and ii) loss of Na+ gradient. Excitabilityincreases at first since Er becomes closer to the threshold level. Eventually, loss of excitability when depolarized Er becomes less negative than the threshold voltage, which causes inactivation of Na+ channels. Examples of Na+/K+ ATPase inhibition: Lack of ATP (following hypoxia); cardiac glycosides (ouabain, digoxin) Amplitude Er Er

  30. Question 4: Effect of Ion Channel Inhibition on Action Potentials How would Er, excitability and action potential amplitude (and duration, in the case of voltage-gated K+ channels) be affected by the following events? • inhibition of K+ leak channels • inhibition of the voltage-gated Na+ channels • inhibition of the voltage-gated K+ channels

  31. Question 4A: Effect inhibiting K+ leak channels Effects similar to hyperkalemia. Block of those channels would cause membrane depolarization. • Partial block could lead to a moderate depolarization that would enhanceexcitability. • Complete block would render the neurons unexcitable because of the strong depolarization that would lead to inactivation of voltage-gated Na+ channels. Amplitude Er Er

  32. Question 4B: Effect of inhibiting voltage-gated Na+ channels Effects similar to decreasing [Na+]out • Er : No change (voltage-gated Na+ channels are closed at resting membrane potential). • AP amplitude would decrease because of reduced inflow of Na+ ions. The action potential would be completely inhibited with a complete block of all the Na+ voltage-gated channels. • Excitability would decrease; i.e. more difficult to trigger an action potential because less voltage-gated Na+ channels are available. Total loss of excitability (i.e. no AP) if all voltage-gated Na+ channels are blocked. Examples of Na+ channel inhibitors: local anaesthetics, TTX (tetrodotoxin), other toxins Amplitude Threshold Er

  33. Question 4C: Effect of inhibiting voltage-gated K+ channels Er: little change (voltage-gated K+ channels are mostly all closed at resting membrane potential). Threshold : no effect (voltage-gated K+ channels do not normally contribute to the threshold because they open only AFTER the entry of Na+ ions, i.e. only after the triggering of the action potential). AP amplitude: no effect. However, AP duration increases due to a slower repolarization. The function of the voltage-gated K+ channels is to accelerate repolarization by allowing a greater outflow of K+ ions (during the AP, K+ ions can cross the membrane by two different types of channels: the “leak” channels (always open) and the voltage-gated channels. If the latter are blocked, K+ ions can leave only through the leak channels, thereby slowing repolarization. Examples of K+ channel inhibitors: some ions (Cs+), some toxins. Amplitude Duration Er No after-hyperpolarizing phase

  34. Objectives Resting Membrane Potential • Predict the effect of changing Na+, K+ or Cl- concentration gradients or permeabilities on resting membrane potential. • Predict the effect of inhibiting Na+/K+ ATPase on resting membrane potential. Action Potential • Predict the effect of changing Na+ or K+ concentration gradients on the amplitude and threshold of the action potential. • Predict the effect of blocking the voltage-gated and background (leaky) Na+ or K+ channels on the amplitude, threshold and duration of the action potential. Synaptic Membrane Potentials • Predict the effect of opening the following ligand-gated channels on postsynaptic membrane potential: Na+ channels, K+ channels, Na+/K+ channels, Cl- channels. Membrane Excitability Tutorial – Dr. Stephen Gee

  35. Question 5: Synapses • Predict the effect of opening postsynaptic ligand-gated channels that are selectively permeable to: • K+ ions • Na+ ions • Cl- ions • Suppose that the postsynaptic ligand-gated channels are equally permeable to Na+ and K+ ions (i.e. they are not selective for specific cations), what would their opening lead to? • EPSP, IPSP, or nothing?

  36. Graded Potentials Graded potentials are changes in membrane potential whose magnitude can vary. They are confined to a relatively small (and usually specialized) region of the plasma membrane – synaptic potential, receptor potential, etc. When a graded potential occurs (due to channels opening), charge flows from the stimulus origin to adjacent regions of the plasma membrane (Fig. 6-15 [a]). Near the stimulus, the outside will be become less +ve and the inside less –ve so the potential difference will be less (closer to zero). The size of a graded potential (here, graded depolarizations) is proportionate to the intensity of the stimulus – how many channels open and how long they stay open. Fig. 6-15 Membrane Excitability Tutorial – Dr. Stephen Gee

  37. Graded Potentials…(cont’d) Graded potentials can be: Excitatory (depolarizing) – an action potential is more likely Inhibitory (hyperpolarizing – an action potential is less likely) Graded potentials decay as they move over distance due to loss of charge through open membrane channels (leak channels) – within a few mm from point of origin. Thus graded potentials can functions as signals only over short distances. Graded potentials can undergo temporal and/or spatial summation Fig. 6-16 Fig. 6-17 Local current is decremental Membrane Excitability Tutorial – Dr. Stephen Gee

  38. Excitatory Chemical Synapses An excitatory postsynaptic potential (EPSP) is a graded depolarizationthat moves the membrane potential closer to the threshold for firing an action potential. Activated receptors usually are non-selective cation channels. Postsynaptic neurons generally receive excitatory input through morphological specializations called dendritic spines that protrude from the main shaft of the dendrite. A typical mature spine has a single synapse located at its head. Glutamate is the major excitatory neurotransmitter in the brain. Fig. 6-28 Membrane Excitability Tutorial – Dr. Stephen Gee

  39. Inhibitory Chemical Synapses An inhibitory postsynaptic potential (IPSP) is a graded hyperpolarization that moves the membrane potential further from the threshold for firing an action potential (inhibition). The neurotransmitter GABA and glycine are most often inhibitory. Hyperpolarization can be induced by: Opening of chloride channels (Cl- moves inside to make cell more –ve) in cells where chloride is actively pumped out. Increased potassium permeability (resting membrane potential moves towards EK) Fig. 6-29 Membrane Excitability Tutorial – Dr. Stephen Gee

  40. Question 5A: Predict the effect of opening postsynaptic ligand-gated channels that are selectively permeable to: • K+ ions • Na+ ions • Cl- ions Cl- Cl-

  41. Answer to Question 5A: To predict the effect of opening ligand-gated channels, one only needs to know the equilibrium potential for the ions that can permeate these channels • Opening of ligand-gated K+ channels (EK more negative than Er)  IPSP (hyperpolarization) • Opening of ligand-gated Na+channels (ENamore positive than Er)  EPSP (depolarization) • Opening of ligand-gated Cl-channels. Since ECl is more negative than Er in most neurons, opening of Cl- channels should make Ermore negative and, hence, would cause a hyperpolarizationIPSP

  42. Question 5B: Suppose that a postsynaptic ligand-gated channel is equally permeable to Na+ and K+ ions (i.e. it a non-selective cation channel). What would be the effect of opening channels of this type? EPSP? IPSP? Nothing? Cl- Cl-

  43. Answer to Question 5B: An example of a non-selective cation channel is the nicotinic acetylcholine receptor (nAChR)at the neuromuscular junction. There are several ways to explain why the opening of a channel that is equally permeable to K+ and Na+ ions would induce a depolarization (see also SLM): i) By considering the driving forces for the two ions: • For K+ ions, their tendency to leave the cell down their concentration gradient is opposed by the negative membrane potential that tends to cause an entry of those positive ions . K+ outflow is small • For Na+ ions, the two forces act together: the concentration gradient and the negative membrane potential favor entry of Na+ ions Na+ inflow is large • Consequently, if those two ions are allowed to move through a same channel, much more Na+ would enter than K+ would leave the cell. The net entry of positives charges would cause a depolarization. ii) Through relative effects of permeability changes. • For instance, suppose that without the nicotinic channels PK/PNa = 100. This ratio would necessarily decrease with the opening of channels equally permeable to Na+ and K+ (at the extreme case, this ratio would be equal to 1 as PK = PNa). Since the membrane potential depends on the relative ion permeabilities, an increase in Na+ permeability would cause a depolarization.

  44. Summary of the effects of changing ion gradients and permeabilities (1): Excitability depends on Na voltage-gated channels (2): A change in permeability will cause a small and variable change in AP duration, depending on the amplitude of the altered action potential and the kinetic properties of the voltage-gated channels. (1): Assuming gradient and permeability changes offset each other perfectly (2): Assuming chloride equilibrium potential equals resting potential (3): For subthreshold depolarisations only. If the depolarisations become too large, inactivation of the voltage-gated Na channels would lead to a reduced excitability.

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