STUDY GUIDE: EXCITATION & HODGKIN-HUXLEY

QUESTIONS (Lectures 1-4):

1. The voltage-clamp experiment shown below shows the set of currents evoked as a cell was stepped from -60 mV (near rest) to command potentials (Vc) ranging from -110 mV to +60 mV. Answer the following questions based on the information contained in the figure.

  • What type of current is evoked at Vc = -110 mV? Think about the parallel conductance model
  • Explain why inward current increases in magnitude from Vc from -40 to -10 mV.
  • Explain why inward current decreases in magnitude from Vc from -10 to +50 mV
  • What is spike threshold? Make a case for why you think this voltage corresponds to threshold
  • What is sodium's Nernst potential?
  • Assume the late (steady-state) current is the "delayed rectifier" potassium current and that the K+ reversal potential is -80 mV. Why is there no apparent inward delayed rectifier K+ current at Vc = -110 mV?
    • 050203_HHtraces

    2. What is the difference between inactivation and deactivation of an intrinsic current? How would you implement a voltage-clamp protocol to test whether a newly identified inward current undergoes either process.

    3. Below are two INa traces at different Vc: -35 mV and +34 mV. Which trace is which and how do you know?

    050203_2traces


    4. How do you measure steady-state inactivation?

    5. How do you measure the time-dependence of de-inactivation?

    6. During action potentials, when does membrane potential get closest to ENa and why at that time?

    7. During action potentials, when does membrane potential get closest to EK and why at that time?

    ANSWERS
    1. The voltage-clamp experiment:

  • At -110 mV the only current flowing is leakage current, which is a K+ dominated current. Note, the leakage current is distinct from the delayed rectifier K+ current that activates during depolarizations.
  • Because INa is activating as Vc depolarizes from -40 to -10 mV, i.e., m increases at higher voltages because of its steady-state voltage-dependence of activation (m_infinity).
  • At voltages exceeding -10 mV, Na+ channels are already fully activated (m is nearly equal to 1), but driving force is decreasing from -10 to +50 mV.
  • Threshold is approximately -30 mV, where the early inward current first appears. only a little inward current is needed at threshold because a little depolarization via a little inward current generates a positive feedback cycle in which depolarization evokes more inward Na+ current, causing more depolarization, and so on. So threshold corresponds to the onset of the early inward Na+ current.
  • Delayed rectifier K+ current does not activate at hyperpolarized membrane potentials, i.e., the n-gates do not open at these voltages.

    • 2. Both processes describe a decrease (or shut down) of an intrinsic current. For voltage-dependent currents, inactivation occurs when the stimulus voltage is maintained, whereas deactivation occurs when the stimulus voltage is removed. To test whether a newly identified current deactivates, clamp the membrane voltage to a level that activates the current in question and then restore the resting voltage again, which should deactivate the current. Then repeat the stimulus voltage command and maintain that voltage. Does the current shut off? if so, it is inactivating.

    3. The black trace was evoked at -35 mV and the red trace was evoked at +34 mV. Although the current magnitudes are nearly the same, the trace at -35 mV does not fully inactivate (note that the black trace maintains -75 pA for the duration of the trace whereas the red trace does decline fully to 0 pA). Also the red trace activates and inactivates much faster than the black trace, which is consistent with faster kinetics at the higher voltage (+34 mV).

    4. Use a two-pulse protocol. The pre-pulse spans a range of voltages over which the current goes from fully de-inactivated to fully inactivated. Then a test pulse at a fixed level is used to evoke the current following each pre-pulse. The evoked current after each pre-pulse is divided by the maximal evoked current. This fraction of current still available after each prepulse is plotted versus the pre-pulse potential to yield the "h_infinity" or steady-state inactivation curve.

    5. Again, use a two-pulse protocol. The two test-pulses are identical in magnitude and duration. The first test pulse should evoke the current, which activates and then fully inactivates by the end of the test-pulse. The second test pulse is delivered after a time interval. Presumably, the current will de-inactivate (to some extent) during that interval. Run the two-pulse protocol with many different time intervals and on the y-axis plot the fraction of current evoked by the second test pulse versus the time interval on the x-axis. This plot will show how long it takes to de-inactivate the current. Curve fitting will allow you to precisely determine the parameter "tau_h".

    6. Vm gets closest to ENa at the peak of the action potential, when INa is fully activated and not yet inactivated, but IK has not yet activated. In other words, Na+ is the dominant influence on Vm, thus Vm approaches ENa.

    7. Vm gets closest to EK at the nadir of the action potential, when INa has fully inactivated (thus no longer influences Vm), and IK has also fully activated. At the end of the action potential IK is still active because its kinetics are slow and even though Vm has decreased, the n-gates are still (briefly) in their permissive state and the K+ current continues to flow, which causes Vm to approach EK. After a little more time, the n-gates deactivate IK and Vm returns to the resting state, which is more depolarized than EK.


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