Notes for Session 4a,4b  (2/2/99 and 2/4/99)

Electrically excitable Na channels

The voltage-gated Na channel is of course the basis of the nerve impulse.  There is actually a family of about a dozen such channels, and individual members are expressed in nearly every neuron but also in cardiac muscle, skeletal muscle, pancreas, and several other cells types.  All these cell types do indeed fire stylized all-or-none action potentials.

The gene we'll use today, called the rat IIA Na channel, was cloned about 10 years ago.   Look here for an overview of Na channel structure, the gating mechanism, and some phosphorylation sites.

First, we'll record the classical Na current waveforms first measured accurately by Hodgkin and Huxley.  For various reasons, our records will be less pretty; but later on in the period, you can try to speed up the voltage clamp for prettier records.  For the moment, set up the two-electrode voltage clamp and use the "I-V" protocol.  Aim for records like these,  and analyze them to produce similar current-voltage relations.

You can also study inactivation with a variable prepulse like the one you used for Shaker K channels.
You can also study the time course of  recovery from inactivation with another protocol.

Use variable Na (10% to 100%) to show shifts in the reversal potential and to get currents that are smaller but may challenge the voltage-clamp circuit less.  You can also fiddle in various other ways to speed up the voltage clamp.  You can use very low-resistance (~ 0.5 megohm) pipettes, if they don't kill the cell.  You can set a grounded piece of aluminum folder, held with modeling clay between the current and voltage electrodes, to stop direct capacitive coupling between the two.

The professional traces have no capacitive transients.  How do they do that?  Look in the CLAMPEX help file under "P/N subtraction", and write a nice protocol file for yourself to do this.

Action Potentials

Now you are ready to test the oocytes that are expressing both Na and K channels.  You want to measure real trains of real action potentials in response to sustained depolarizing pulses (0.5 seconds long).  Use the Gene clamp in current-clamp mode.  Use all your knowledge gained so far:

Remember that Na channels inactivate if the resting potential is not nice and neagtive (how negative?  Use your data!).
Remember that an unclamped membrane has a very long time constant.
Plot the firing frequency versus injected current.

Are you ready to use a Hodgkin-Huxley simulator to predict your firing freqencies on the basis of the voltage-clamp data?