1. Action Potentials

2. What are they? Rapid, brief, depolarizations of the membrane potentials of excitable cells (neurons, muscle cells, some gland cells), initiated by an appropriate stimulus to a sensory receptor (chemical, physical or electromagnetic), or chemical signals released by neurons and received by other neurons, muscle cells or gland cells.

3. Characteristics of Action Potentials Show threshold for initiation Are “all-or-nothing” - their magnitude is not graded to stimulus intensity. Spread throughout the plasma membrane by a non-decremental process- the magnitude of the AP does not diminish with distance. Followed by a refractory period during which it is difficult or impossible to initiate another action potential.

4. Physiological Function Action potentials are the means of rapid (milliseconds), long-distance (up to meters) communication in the body As opposed to chemical messages - which can be long- distance, but slow (seconds to minutes) decremental electric currents - which are rapid, but can only operate over short distances (a few tens of microns)

5. We will examine the change in the voltage of a small piece of excitable membrane as we drive a current across it. Volts _ _ + + + + + + + + + - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + + + +

6. Notice that the same current strength causes a smaller voltage change for hyperpolarizing pulses than depolarizing pulses - it’s easier to depolarize the membrane than to hyperpolarize it.

7. As the strength of depolarizing current is increased, there is a sudden transition to an action potential - a threshold has been crossed. A threshold stimulus - defined in terms of current intensity and duration - is one that is able to initiate an action potential 50% of the time.

8. The next slide shows a complete action potential as it would be recorded in squid giant axon. (Not all cell types show a hyperpolarizing afterpotential as obvious as is seen here.)

10. In this set of experiments the axonal membrane is voltage- stepped to -40mV, 0 mV or +40 mV, and the resulting conductance changes for Na + and K + are plotted over time. Note that the Na + conductance is self- terminating; the K + conductance is not (at least within the timeframe shown here).

11. The next slide shows that the conductance change of the stimulated (and unclamped) cell explains the voltage change of an action potential. The conductance change is the sum of the two separate conductance (g) changes for Na + and K + that were recorded under clamp conditions. Conductance = permeability x driving force, so the conductance changes are essentially permeability changes.

13. Following an action potential, there is a brief period during which an excitable cell cannot initiate a second action potential. This is the absolute refractory period. The Refractory Period Following the absolute refractory period is a longer period during which it is more difficult to bring the membrane to threshold for a second action potential - this is the relative refractory period.

14. In the experiment shown on the next slide, a cell is given a pair of stimulus pulses with a variable time interval. As the interval is made shorter, the threshold rises, because the second stimulus starts to fall in the cell’s relative refractory period. The threshold becomes essentially infinite in the millisecond or so just after the first AP (I.e. the second stimulus comes during the absolute refractory period.

16. Questions How does depolarization cause an AP and what is responsible for threshold? What factors determine the duration of an AP? Why is the membrane refractory to a second stimulus for a time after an AP? Why is it easier to depolarize the membrane than it is to hyperpolarize it?

17. These questions can now be answered in terms of the behavior of voltage- gated channels for Na + and K +

18. Na + channels can exist in three distinct states, which form a cycle Most channels are closed, but available, at rest potential - depolarization increases open probability. At threshold the channels enter a positive feedback cycle in which. depolarization activation This positive feed back explains the rapid rise or upsweep of the spike when threshold is reached.

19. The open state or activated state of Na + channels is followed by inactivation - a closed state in which the channel cannot be reopened by depolarization. This explains the downturn of the spike before it has time to reach Ena. Inactivation is removed by some combination of repolarization and time, returning the channel to the available state.

20. The Na + channel has two gates - an activation gate in the interior of the channel and an inactivation gate suspended from the intracellular domain.

21. The experiment on the next slide shows the single- channel currents recorded from 7 individual Na + channels in response to a depolarizing voltage step. Notice how random the behavior is - the different channels open at different times, stay open for different times, and may flicker closed a time or two before each finally inactivates. The summed current from the channels is shown in the bottom trace. The Na + current that flows across a patch of membrane during an action potential is the sum of its many single-channel currents. Na + channel behavior is revealed by patch clamping

22. Recording from voltage-sensitive channels using a patch-clamp: A. the membrane surface is pulled gently to allow the glass pipette to form a tight seal with the membrane. This isolates the patch of membrane and allows the activity of one or a few channels to be detected. B. The opening of individual channels in response to depolarization is not synchronous and does not last a standard amount of time, but the cell’s total response is the sum of the individual responses (shown in 3).

23. In this slide inward currents are shown as downward deflections

24. K + channels differ from Na + channels in that they Open more slowly in response to depolarization Close (slowly) in response to repolarization Do not show time-dependent inactivation.

25. Note that the trace of the summed current rises more slowly than for the Na + channel, and that the channels continue to be active as long as the depolarization is maintained

26. Threshold can now be redefined as the stimulus intensity/duration that has a 50% probability of opening enough Na + channels to cause the inward Na + current to exceed the outward K + current. When the inward Na + current exceeds the outward K + current, the system enters the positive feedback cycle that leads to an AP

27. Three factors make threshold hard to reach Depolarization increases the driving force for K + and decreases it for Na + Depolarization opens K + channels as well as Na + channels. Open Na + channels do not stay open - they inactivate.

28. The refractory period is determined by two separate factors: Na + channel recovery from inactivation and K + channel closing. Early in the refractory period, most Na + channels are still in the inactivated state, and so not available. In the middle of the refractory period, some Na + channels have become available, but the number of open K + channels is still greater than at rest. The refractory period wanes as the last voltage-sensitive K + channels close.