W7L1 Flashcards
What is responsible for the peak amplitude of an AP?
Hodgkin & Katz’s hypothesis : the peak of action potential is Na+ mediated
- started with injecting 100% Na+
- then decrease percentage: 50% Na+ with 50% choline, 30% Na+, 0%Na+
- at 50% Na+, there is still AP but peak amplitude is reduced
Peak of action potential is predicted by Nernst equation, i.e., peak is mediated by Na+ permeability
Nernst Equation
ENa = 60 log [Nao/Nai] in mV at 27oC
ENa = 60 log Nao - 60 log Nai
Plot ENa versus log Nao will give straight line of slope 60mV per 10 fold change of Na0
Y = m X - b
ENa = Y
log Nao = X
Sequential Conductance Changes
First, there is fast Na+ conductance to reach the peak. Second, there is delayed K+ conductance to cause hyperpolarization
Action potential Em varies in time
Calculate potential using Goldman equation
- At rest
- PK=1
- PNa= 0.04
- PCl=0.45
- Em= -60mV - At peak
- PK=1
- PNa= 20
– At peak, the potassium permeability is the same, but permeability of sodium is hugely increased (500 Volt increase).
- PCl=0.45
- Em= +42mV - AHP (after hyperpolarization phase)
- PK=1.8
- PNa= 0
- PCl=0.45
- Em= -70mV
The Em is similar to the nernst equation for the molecule with the highest permeability per state (rest, peak or AHP) (potassium for rest and AHP, sodium for peak)
Ions in squid
- K+
- Intra(mM) = 400mM
- Extra(mM) = 20mM
- Eion = -75 - Na+
- Intra(mM) = 50mM
- Extra(mM) = 440mM
- Eion = 55 - Cl-
- Intra(mM) = 52mM
- Extra(mM) = 560mM
- Eion = -60
Voltage Clamp study of action potential - Squid
Voltage clamp was developed in 1940s by Kenneth Cole
Hodgkin & Huxley used this technique to study squid giant axon
If squid don’t have big axon, they escape from predators slower and will die via natural selection
Voltage Clamp by two electrodescan be done in giant squid axon
- put 2 electrodes, inject current, measure voltage
- initially, had inward current, which is followed by outward current
Voltage Clamp: K+ current alone
Do this by…
- Replaces Na+ with impermeable ion (e.g., choline+ in1940s)
- Or block Na+ channel with tetrodotoxin (Puffer fish toxin)
Results:
- the current is not activating at the beginning anymore, there is a delay. It does not activate until at least half a millisecond later
- eventually reaches a steady state level at 2-3 milli seconds
- overall: delay in activation
- the current remains constant, there is NO inactivation
I_K = delayed rectifier current
Voltage Clamp: isolating Na+ current
Do this by…
- Subtract K+ current recorded with zero [Na+]o from total current
- OR block K+ current with tetraethylammonium (TEA)
Characteristics of Na+ Current
initially, quick to rise (inward current), which is activation occuring in the first 0.5 ms
then there is delary, which is inactivation within a few ms
Conductance changes during an AP
First, fast Na+ conductance
Second, delayed K+ conductance
- potassium delayed rectifier
AP rise phase
Positive feedback loop; it is self generation and rapid
- initial trigger (membrane depolarization that exceeds threshold)
- depolarization
- Na+ channel open
- Inward Na+, current I_Na
- then goes back to step 2
AP repolarization phase
2 components:
1. Conductance of potassium is a delayed negative feedback
- Na+ channel inactivation
Depolarization leads to…
1. K+ channel to open
- Outward K+ current (I_K)
- Hyperpolarization
After hyperpolarization (AHP)
potassium conductance is elevated…so still lots of K+ moving out of the cell, contributes to very negative membrane potential
sodium channel inactivation contributes to this too
Absolute and Relative Refractory Periods
Absolute
- Na+ ch. inactivation
Relative
- K+ ch. open
Na+ channel inactivation
Dependent on III and IV area of the subunit
Not ball and chain
The III-IV loop is for inactivation
Channel blockers for studying AP
- Block K+ channels (prolongs AP, does not really impact peak)
- Tetraethylammonium (TEA)
- 4-aminopyridine (4-AP) - Blocks Na+ channels (decreases AP peak)
- Tetrodotoxin (TTX)
