BioNB 442: Lab 8

Voltage Clamp

Introduction.

You will build a slightly improved active model cell, record from it, then voltage clamp the model in order to measure conductance changes.

• Circuit 1: Build the following circuit. The LM358 datasheet is here. The H11F1 is a device which couples input to output using only light and has an LED at the input and a light-controlled resistor as its output.
  
  On the model, you can directly monitor the sodium conductance by connecting the junction of the H11F1 input LED and 62 ohm resistor to another channel of the USB-6008. The display should look something like the one below. Where the red channel is proportional to the conductance and blue is the membrane voltage. In a real cell you cannot monitor conductances directly, but rather you have to measure currents and voltages and compute conductaces as current/voltage.
  
• Circuit 2: In a real cell you need to hold the voltage constant in order to understand the shape of the conductance because the conductance mechansim is affected by voltage and is obscured by the simultaneous changes in voltage and current which occur during an action potential. To do this we need to build a voltage clamp. A simple version is shown below. Note that only one opamp has been added to the circuit, and one resistor value changed.
  
  
  A typical recording is shown below. Current is calculated as
  ((command voltage)-(membrane voltage)) /75 ohms
  Where the 75 ohms is the resistor coupling the clamp driver to the cell. You can see the capacitative spike caused by the clamp charging the membrane capacitance, followed by the transient inward sodium current.
  

Assignment

1. Refering to Circuit 1 above, build the circuit, then use the physiological stimulator program described on the Matlab soft instruments page to produce pulses on the sound card output (winsound 0). The middle ring of the stereo phone jack (Channel 2, see lab 1) carries the pulses. Apply those to the model cell as shown above. Set the stimulus length to 10 mSec. You should be able to stimulate action potentials in the model and record them using gPRIME connected to the USB-6008. What is the membrane time constant of the model cell (computed and measured)? Why is the rise time of the action potential faster than the computed time constant? What is the resting potential and action potential overshoot. Explain these values based on the circuit.
2. Refering to Circuit 2 above, add the clamp driver to Circuit 1. Download lab8niVclamp.m and show that you can clamp your model cell using this code to generate the command pulses and to record two voltages as shown above. Modify the program to produce a series of voltage steps ranging from 2.5 to zero volts (-2.5 to 0 after inversion). The steps should start at 2.5 volts for 2 seconds, jump to the first step voltage for 100 mSec, then back to 2.5 volts for 2 seconds, then jump to the second step voltage for 100 mSec, and so on. The voltage and current traces should be stacked on the same plots for comparison, something like the image below. The traces here were lowpass filtered, and the linewidth was set to 2. What can you say about the voltage dependency and dynamics of the transient current? How about the steady-state current?
   
   

Your written lab report should include:

1. A printout of all data screens you use for measurements.
2. A commented listing for the voltage clamp programs.