Part 1 - Voltage Clamp of K+ Current ( IK )

Collected Data : https://docs.google.com/spreadsheets/d/1UArGLeBwguDwpFXvkdP19wLGHYiIc84Duz7DeYf39ck/edit?usp=sharing

  • Determine how IK varies with stimulus voltage

  1. Choose tutorial "Voltage Clamping a Patch"

  2. Click on "Start the Simulation"

  3. Click on VClamp

  4. Click on Membrane Current Plots

  5. Increase Total ms to 12 ms

  6. Increase Stimulus Duration ( for testing level ) to 10 ms

  7. Remove Na+ conductance , by setting to 0.0 S/cm^2

  1. Vary the test amplitude of stimulation ( change voltage , mV in Stimulus Control window ) , starting at −100 mV and stepping in 10 mV increments to +100 mV. Measure the largest current for each voltage step ( check beginning and end of testing level ). Left clicking on trace to get a reading for x,y ( y is the amplitude in mA/cm2 ). Plot the current vs the voltage step, on the graph provided ( next page )

    Vm ( mV )I ( mAcm2 )
    -100-0.00800178
    -90-0.00450186
    -80-0.000954157
    -700.00274448
    -600.0137072
    -500.0762199
    -400.25128
    -300.56275
    -200.971343
    -101.42375
    01.88685
    102.34651
    202.79808
    303.24056
    403.67417
    504.09954
    604.51741
    704.92852
    805.33359
    905.73329
    1006.12823

    image-20250212152710086

    • we get the conductance value for free from the slope

    g=0.0342 nAcm2mV=0.0342 109 A( 102 )2 m2103 V=0.0342 105 Am2103 V=0.0342 102AVm2=0.000342 AVm2
    • Siemens ( S ) is AmperesVolts

    • so here I guess its Siemensm2

    • now to find approximated nernst value for sodium :

    y=( 0.0342x )+( 2.28 )
    • let y=0 :

      0=( 0.0342x )+( 2.28 )
      x66.6667 mV
  2. Calculate chord conductance ( G ) for these K+ currents , then construct a plot of GK versus Vm

    I=g( ( Vm )( Eion ) )
    • solving for g :

    g=I( Vm )( Eion )
    Vm ( mV )g ( 102 AVm2 )
    -1000.0003528277261
    -900.0003550642795
    -800.0003561616275
    -700.0003748777489
    -600.0007913630853
    -500.002789791735
    -400.00673293856
    -300.01189218317
    -200.01694567436
    -100.02114867575
    00.02440281424
    100.02687223005
    200.02875104037
    300.03019502241
    400.03131724073
    500.03219845901
    600.03289671645
    700.03345429369
    800.03390259406
    900.03426521477
    1000.03456009159

    image-20250212161035292

    • the curve is sigmoidal , and you could fit it with a Boltzman equation to find the slope

      • a large slope = channel requires a broader voltage range to switch from closed to open state

      • a small slope = channel opens very quickly over a narrow voltage range

    • but we just can take gmax , like 0.03456009159 , and divide it by 2

      gmax2=0.034560091592=0.017280045795
    • now go to the G / V plot , and lookup corresponding voltage for where gmax2 is :

      • [ ?? , 0.017280045795 ]

      • from the table , its somewhere between 20 mV and 10 mV

      • the actual value we call now V1/2

      • V1/2 = the voltage at which 50% of the channels are open

        • if V1/2 is more negative , the channel activates at lower voltages

          • probably a sodium channel

        • if V1/2 is more positive , the channel requires a stronger depolarization to activate

          • probably a potassium channel

  3. Current reverses ( changes from inward to outward: negative to positive ) at the Nernst potential for the permeant ion. Locate that reversal point for the K+ current and compare with the anticipated Nernst value.

    • looking at the table of values we collected , current reverses polarity between 80 mV and 70 mV

    • our predicted value of 66.6667 mV is somewhat near this range

  4. Describe any changes of the kinetics with voltage ( kinetics refers to the time course of the current: rate of rise and rate of fall )

    • constant slope on G / V plot between [ 50 mV , 10 mV ]

    • delay in opening , then open for long period of time

Part 2 - Voltage Clamp of Na+ Current ( INa )

  • Determine how INa varies with stimulus voltage

  1. Choose tutorial "Voltage Clamping a Patch"

  2. Click on "Start the Simulation"

  3. Click on VClamp

  4. Click on Membrane Current Plots

  5. Increase Total ms to 6 ms

  6. Increase Stimulus Duration ( for testing level ) to 4 ms

  7. Set Na+ conductance to default value

  8. Remove K+ conductance , by setting to 0.0 S/cm^2

  1. Vary the amplitude of stimulation (change voltage, mV in Stimulus Control window), starting at −100 mV and stepping in 10 mV increments to +100 mV. Measure the maximal current for each voltage step. Plot the current vs the voltage step, on the graph provided on next page.

    Vm ( mV )I ( mAcm2 )
    -100-2.57E-09
    -90-1.37E-07
    -80-6.34E-06
    -70-0.000237441
    -60-0.00635058
    -50-0.0879799
    -40-0.441195
    -30-0.947073
    -20-1.33333
    -10-1.55554
    0-1.61346
    10-1.52326
    20-1.31529
    30-1.01759
    40-0.653983
    50-0.242015
    600.2097
    700.692602
    801.20084
    901.72929
    1002.27465

    image-20250212162819707

  2. Locate the reversal potential for the Na+ current and compare with the anticipated Nernst value

    0=( 6.79103x )+( 0.22 )
    x=32.4006
    • fitted linear equation is obviously very wrong , as we don't have a straight line for the entire trace

    • looking at the collected data in the table , it crosses zero between +50 mV and +60 mV

    • the programmed ENa in the simulation is at +55.449

  3. Describe any changes of the kinetics with voltage ( also note time of peak INa )

    image-20250212163308342

    • it reaches the peak time for INa quicker and quicker as you raise Vm

  4. Repeat the stimulation sequence with both Na+ and K+ conductances at default values ( 10 ms duration testing level ). Click on Membrane Current Plots ( in Panel & Graph Manager ) and VClamp.i graph ( in Stimulus Control Panel ).

    image-20250212163601204

    image-20250212163712322

Part 3 - IK and INa During the Action Potential

  1. Choose tutorial "The Na Action Potential"

  2. Start the Simulation

  3. Click on IClamp

  4. Change total # ms to 10 ms

  5. Reset and Run

image-20250212164058004

  1. Using your results from the voltage dependence of INa and IK ( review your plots from previous pages ) , and the dependence of INa and IK on driving force  Ii=gi( VmEi )  , predict the INa and IK that occur during the action potential.

    • inward sodium current , outward potassium current

  2. Draw the time course of your predicted Na+ and K+ currents along with the aligned action potential. ( Pick key time points along the action potential to note the conductance and driving force. )

     

  1. Now open Membrane Current Plots along with Voltage vs Time Plot

  2. Change total # (ms) to 10 ms

  3. Reset and Run

image-20250212164439508

  1. Compare your prediction with the simulation and note differences. If different , redraw the currents along with the aligned action potential.

  2. Briefly explain the time course of INa and IK

    • sodium opens early and closes quickly for inactivation

    • potassium delay in opening , but then stay open for longer

  1. Now open the membrane conductance plots

  2. Assure that the axis extends to 10 ms

  3. Reset and Run

  1. Add the conductance plots to your drawing above

    image-20250212165537932

  2. Describe any differences between the time course of the conductance plots and the current plots.

    image-20250212165637681

    • the lag is much more obvious for potassium in the conductance plot

    • you can more clearly when sodium is conducting

  3. Examine the peak of the action potential and the Na+ Nernst potential , and briefly explain the relationship.

    • action potential peak = +45.3934 mV

    • sodium Nernst = +55.442

    • the action potential peak approaches the nernst values as you conduct ions of that type

  4. Examine IK and INa at the time points when Vm is not changing ( maximums and minimums ) , and Vm when either IK or INa are not changing. Briefly explain the relationship between these currents and Vm.

    • when Vm is not changing ( at min and max ) , current = zero

    • when current is not changing , the channels are inactivated or closed