Bioelectricity week 3

Bioelectricity -- Week 3
Bioelectricity week 3: A) Passive and Active simulations. Wow! B) Channels Wow! Train system: Build the engine

Engine has to be built within the membrane, 100 Angstroms! Really tiny.

Bioelectricity Week3_2
Observe passive & active simulations. Observe channels.
1. Introduction to week 3 2. Why are passive and active so different? 3. The simulation set-up 4. The Passive simulation 5. The Active Simulation 6. Where does the active response come from? 7. Problem session , passive v active 8. Channels: Experimental isolation of a channel 9. Channels: Observed currents, voltage step 10. Channels: Probability of being open 11. Problem session, Channel probabilities 12. Week 3 conclusions

Train system: Build the engine.

Week 3_2: Why are the passive and active responses to a trans-membrane stimulus so different? Stimulus means a current of a fixed magnitude is forced through the patch.

Week 3_2: Why are the passive and active responses to a trans-membrane stimulus so different?
Stimulus means a current of a fixed magnitude that is forced through the patch.

Response means the voltage across the membrane that occurs during or after the stimulus. Passive response means the response for membrane patch that is passive. Passive means that properties do not change as time goes by, or as the voltage changes changes. Most ordinary material is passive, for example, wood or plastic.

Bioelectricity 3_2: Why are the passive and active responses to a trans-membrane stimulus so different?
Active response means the response from living tissue, in this case tissue that follows the Hodgkin-Huxley model for nerve membrane. so different means that the trans-membrane voltage, following the very same stimulus pulse, is so different from passive. But how? Well you
will just have to wait and see

NC woods, broken tree from storm

Bioelectricity Week3_3
Observe passive & active simulations. Observe channels
1. Introduction to week 3 2. Why passive and active so different? 3. The simulation set-up 4. The Passive simulation 5. The Active Simulation 6. Where does the active response come from? 7. Problem session , passive v active 8. Channels: Experimental isolation of a channel 9. Channels: Observed currents, voltage step 10. Channels: Probability of being open 11. Problem session, Channel probabilities 12. Week 3 conclusions

Train system: Build the engine.

Simulation of Patch, Equations-1

Simulation Equations Im = Ic + Ir Ic = Im Ir

Simulation of Patch, Equations-2

Simulation Equations Im = Ic + Ir Ic = Im Ir Cm dVm / dt = Im Ir

Simulation of Patch, Equations-3

Simulation Equations Im = Ic + Ir Ic = Im Ir Cm dVm / dt = Im Ir Im is Istim and then zero

Simulation of Patch, Equations-4

Simulation Equations Im = Ic + Ir Ic = Im Ir Cm dVm / dt = Im Ir Im is Istim and then zero Ir is Vm / Rm (Ohms law)

Simulation of Patch, Equations-5

Simulation Equations
Im = Ic + Ir Ic = Im Ir Cm dVm / dt = Im Ir Im is Istim and then zero Ir is Vm / Rm (Ohms law)

So, dVm = ( Istim Vm/Rm) *dt / Cm

Simulation of Patch, Equations-6

Simulation Equations
Im = Ic + Ir Ic = Im Ir Cm dVm / dt = Im Ir Im is Istim and then zero Ir is Vm / Rm (Ohms law) So, dVm = ( Istim Vm/Rm) *dt / Cm

Computation: Using discrete steps dt is a time step, and dVm is the resulting trans-membrane voltage change.

North Carolina woods edge

Bioelectricity Week3_4
Observe passive & active simulations. Observe channels
1. Introduction to week 3 2. Why passive and active so different? 3. The simulation set-up 4. The Passive simulation 5. The Active Simulation 6. Where does the active response come from? 7. Problem session , passive v active 8. Channels: Experimental isolation of a channel 9. Channels: Observed currents, voltage step 10. Channels: Probability of being open 11. Problem session, Channel probabilities 12. Week 3 conclusions

Train system: Build the engine.

Sketch

Simulation of Passive Patch

Simulation of Passive Patch

Stimulus

Simulation of Passive Patch,

Response during stimulus

Simulation of Passive Patch,

Response after stimulus

(not zero immediately, but falling exponentially)

Simulation of Passive Patch, Results Summary

Simulation of Passive Patch, Conclusions 1

That is, what happens? The stimulus causes an abrupt rise in trans- membrane voltage Vm The change in Vm lasts after the stimulus pulse ends. After the stimulus Vm returns slowly to its baseline value The energy in the stimulus pulse is dissipated in the resistance.

Simulation of Passive Patch, Conclusions 2

Talk about what happens Loosely, one puts the pulse in. That changes Vm markedly at the start of the stimulus, but Vm decays until the resistance uses up the pulses energy. Vm is a distorted version of the stimulators current pulse. Vm is technically interesting but otherwise unsurprising.

North Carolina meadow, August 2012

Bioelectricity Week3_5
Observe passive & active simulations. Observe channels
1. Introduction to week 3 2. Why passive and active so different? 3. The simulation set-up 4. The Passive simulation 5. The Active Simulation 6. Where does the active response come from? 7. Problem session , passive versus active 8. Channels: Experimental isolation of a channel 9. Channels: Observed currents, voltage step 10. Channels: Probability of being open 11. Problem session, Channel probabilities 12. Week 3 conclusions

Train system: Build the engine.

Active simulation (only tissue changes)

Same sketch Same mathematical model, except Rm active. Everything just the same except that now the tissue is active.
Im = Ic + Ir Ic = Im Ir Cm dVm / dt = Im Ir Im is Istim and then zero Ir is Vm / Rm (Ohms law) So, dVm = ( Istim Vm/Rm) *dt / Cm

Active tissue - stimulus

Passive

Active

Active tissue response during stimulus

Passive

Active

Active tissue response following stimulus, going where?

Passive

Active

Active tissue response again

vm and time axes longer than before

Passive

Active

Comparison of Active and Passive

There is a difference during the stimulus, but in this case it is a small difference. There is a after the stimulus is over.

huge huge huge difference in what happens

Comparison of Active and Passive Vm Responses (to the very same stimulus)
After the stimulus: The vm curve for passive tissue decays back to the baseline. Vm for active tissue gets bigger and bigger

--- 10x--- and the

wave form goes on several milliseconds coming back to and even below the original vm baseline. Wow!

for a long time

Striking a match
The difference is like what happens if one strikes a mat With passive tissue, the match flames but then gradually dies.. With active tissue, it is as if one strikes the match and then waves it over a puddle of gasoline. Match --- Flame BOOM

North Carolina tree

Bioelectricity Week3_6
Observe passive & active simulations. Observe channels
1. Introduction to week 3 2. Why passive and active so different? 3. The simulation set-up 4. The Passive simulation 5. The Active Simulation 6. Where does the active response come from? 7. Problem session , passive v active 8. Channels: Experimental isolation of a channel 9. Channels: Observed currents, voltage step 10. Channels: Probability of being open 11. Problem session, Channel probabilities 12. Week 3 conclusions

Train system: Build the engine.

Where does the AP come from?

This is a mystery.
( Not a mystery to everyone but a mystery to us at this point in this course ) Stimulus is zero Cm is charged, but discharging through Rm? So how can Vm be getting bigger?

At the end of the stimulus:

Im = Ic + Ir Ic = Im Ir Cm dVm / dt = Im Ir Im is Istim and then zero Ir is Vm / Rm So,
dVm = ( Istim Vm/Rm) *dt / Cm

Where does the AP come from? algorithm?

This is a mystery .
So how can Vm be getting bigger?

Im = Ic + Ir Ic = Im Ir Cm dVm / dt = Im Ir Im is Istim and then zero Ir is Vm / Rm So, dVm = ( Istim Vm/Rm) *dt / Cm

After the stimulus Im=0. Which ions are moving? Why?

What does ions have to do with it? The model of the membrane must be defective, or at least insufficient.

A clue
A very substantial clue is given by examining the behavior of channels, as is done in the next lectures.

North Carolina pine trees

Bioelectricity Week3_7
Observe passive & active simulations. Observe channels
1. Introduction to week 3 2. Why passive and active so different? 3. The simulation set-up 4. The Passive simulation 5. The Active Simulation 6. Where does the active response come from? 7. Problem session , passive v active 8. Channels: Experimental isolation of a channel 9. Channels: Observed currents, voltage step 10. Channels: Probability of being open 11. Problem session, Channel probabilities 12. Week 3 conclusions

Train system: Build the engine.

Problem Session Examining the results

The questions ask about some important details. Look back at the graphs to answer each question.

What is the peak vm with a Passive patch? Active?

In each case measure vm against baseline. Passive tissue, during the stimulus: Passive tissue, anytime following the stimulus onset: Active tissue, during the stimulus: Active tissue, anytime following the stimulus onset:

What is the wave form duration passive? Active?

The duration has to be determined by a workable definition. Suppose we define the waveform to have ended when vm returns to about 10% of its peak value. (Give results in microseconds)
Stimulus current duration?

Passive vm duration?

Active vm duration?

NC cemetery at Orange Church

Bioelectricity Week3_8
Observe passive & active simulations. Observe channels
1. Introduction to week 3 2. Why passive and active so different? 3. The simulation set-up 4. The Passive simulation 5. The Active Simulation 6. Where does the active response come from? 7. Problem session , passive v active 8. Channels: Experimental isolation of a channel 9. Channels: Observed currents, voltage step 10. Channels: Probability of being open 11. Problem session, Channel probabilities 12. Week 3 conclusions

Train system: Build the engine.

Pull pipette
With center of a glass tube heated over flame, it can be pulled apart, with the center section necking down into a very tiny opening.

Use suction to grab tissue

Applying gentle suction, a membrane sample can be attached and pulled away from a larger membrane. If the tip is small enough, only a single channel will be contained within the membrane sample.

Apply voltage and measure current

With the pipette in another solution, a voltage step can be applied and the currents required to maintain that voltage can be measured.

NC weeds

Bioelectricity Week3_9
Observe passive & active simulations. Observe channels
1. Introduction to week 3 2. Why passive and active so different? 3. The simulation set-up 4. The Passive simulation 5. The Active Simulation 6. Where does the active response come from? 7. Problem session , passive v active 8. Channels: Experimental isolation of a channel 9. Channels: Observed currents, voltage step 10. Channels: Probability of being open 11. Problem session, Channel probabilities 12. Week 3 conclusions

Train system: Build the engine.

Channel opening, what I would have expected

Low vm, partially open Higher voltage, mostly open

But I would have been wrong

Channel opening, what actually happened, K+

Random openings, bouncing back and forth between open and closed. With low vm, not so often With high vm, more often open. Still bouncing open and closed whether vm low or high.

NC grass

Bioelectricity Week3_10
Observe passive & active simulations. Observe channels
1. Introduction to week 3 2. Why passive and active so different? 3. The simulation set-up 4. The Passive simulation 5. The Active Simulation 6. Where does the active response come from? 7. Problem session , passive v active 8 Channels: Experimental isolation of a channel 9. Channels: Observed currents, voltage step 10. Channels: Probability of being open 11. Problem session, Channel probabilities 12. Week 3 conclusions

Train system: Build the engine.

So we have to define channel openings in terms of probabilities

Amazing: critical functions of humans depend on probabilities. How can these probabilities be understood quantitatively? Analytical beginning:
If there are N channels in a patch, suppose No are open and Nc are closed. The rate of change in the number of open channels can be expressed as: dNo / dt = Nc - No Here is the rate at which closed channels open, and is the rate at which open channels close. A key point is that and are not constants. Rather they change with Vm (but not with time). Over time, the fraction of the N channels that are open will become / +

So we have to define channel openings in terms of probabilities

Analytical beginning
If there are N channels in a patch, suppose No are open and Nc are closed. The rate of change in the number of open channels can be expressed as: dNo / dt = Nc - No Here is the rate at which closed channels open, and is the rate at which open channels close. A key point is that and are not constants. Rather they change with Vm (but not with time). Conclusion: Over time, the fraction of the N channels that are open will become / +

So we have to define channel openings in terms of probabilities

Analytical beginning
Conclusion: Over time, the fraction of the N channels that are open will become / +
How do we get specific values for and ? Until now, from measuring them. We will discuss further next week.

NC trees in woods

Bioelectricity Week3_11
Observe passive & active simulations. Observe channels
1. Introduction to week 3 2. Why passive and active so different? 3. The simulation set-up 4. The Passive simulation 5. The Active Simulation 6. Where does the active response come from? 7. Problem session , passive v active 8. Channels: Experimental isolation of a channel 9. Channels: Observed currents, voltage step 10. Channels: Probability of being open 11. Problem session, Channel probabilities 12. Week 3 conclusions

Train system: Build the engine.

Problem session Number open --Q

If there are 1000 channels in a patch. Suppose our initial measurement shows 100 are open and 900 are closed. The trans-membrane voltage changes to a new value. Now is 0.8 per msec, and is 0.2 per msec. A) How many channels are expected to be open 1 msec after the initial measurement? B) How many channels are expected to be open 10 msec after the initial measurement?

Problem session Number open --A

If there are 1000 channels in a patch. Suppose our initial measurement shows 100 are open and 900 are closed. The trans-membrane voltage changes to a new value. Now is 0.8 per msec, and is 0.2 per msec.

A) How many channels are expected to be open 1 msec after the initial measurement?

Problem session Number open B1

If there are 1000 channels in a patch. Suppose our initial measurement shows 100 are open and 900 are closed. The trans-membrane voltage changes to a new value. Now is 0.8 per msec, and is 0.2 per msec.

B) How many channels are expected to be open 10 msec after the initial measurement?

Problem session Number open B2

If there are 1000 channels in a patch. Suppose our initial measurement shows 100 are open and 900 are closed. The trans-membrane voltage changes to a new value. Now is 0.8 per msec, and is 0.2 per msec.

B) How many channels are expected to be open 10 msec after the initial measurement?

NC older trees

Bioelectricity Week3_12
Observe passive & active simulations. Observe channels
1. Introduction to week 3 2. Why passive and active so different? 3. The simulation set-up 4. The Passive simulation 5. The Active Simulation 6. Where does the active response come from? 7. Problem session , passive v active 8. Channels: Experimental isolation of a channel 9. Channels: Observed currents, voltage step 10. Channels: Probability of being open 11. Problem session, Channel probabilities 12. Week 3 conclusions

Train system: Build the engine.

Week 3 in Review -1
We saw that a simulation of passive tissue produces a Vm change that decays back to baseline.

Week 3 in Review -2
We saw that a simulation of passive tissue produces a Vm change that decays back to baseline. In contrast, a simulation of active tissue produces a Vm change similar to the passive one, and then a HUGE longer change. While we can call than an action potential, we had no explanation of what made it go.

Week 3 in Review -3
Searching for clues, we examined the records of channel measurements.

Week 3 in Review - 4
Searching for clues, we examined the records of channel measurements. Amazingly, channels seem to open and close randomly!

Week 3 in Review
Searching for clues, we examined the records of channel measurements. Amazingly, channels seem to open and close randomly! But, the channel statistics depend on the trans-membrane voltage. Which means that the steady-state number open is predictable, on the average

Week 3 in Review
We saw that a simulation of passive tissue produces a Vm change that decays back to baseline. In contrast, a simulation of active tissue produces a Vm change similar to the passive one, and then a HUGE longer change. While we can call than an action potential, we had no explanation of what made it go. Searching for clues, we examined the records of channel measurements. Amazingly, channels seem to open and close randomly! But, the channel statistics depend on the trans-membrane voltage.

Wow what a week!

So next week
We move on to a way to make sense out of these mysteries See you then.

NC mixed weed and thatch

First a matter of terminology:

What is the difference between Vm and vm?

vm with a lower-case v is the trans-membrane voltage relative to the trans-membrane baseline voltage. Vm with an upper-case V is the trans-membrane voltage, absolute. Note that vm=Vm-Vr, where Vr is the resting trans-membrane Voltage.
vm is easier to measure, as it is not affected by DC drift, an issue with instrumentation. Baseline vm is by definition equal to zero. Vm is the right Vm. The baseline value will be -70mV, more or less, rather than 0 mV.
Everyone recognizes the difference between Vm and vm, but the specific notation is not always that used here.

NC grass and leaf