Action Potentials

Purves et al., Chapters 3 & 4 (review figure from chapter 2)

Summary

Fig. 3.8 A p. 48
what we know about K+ and Na+ permeability during the passing of the action potential
Na+ conductance goes up then down early
K+ conductance goes up then down but much later.
There is something wrong with this figure: K+ conductance is much higher than Na+ conductance during the resting potential.

Bernstein knew about selective K+ permeability
Thought it was lost during the action potential (actually Na+ permeability increases)

See pdf
Cole and Curtis used the AC Wheatstone bridge to show that the resistance decreased during the action potential
R1 & R2 divide one path, Rv (variable) and Ru divide the other
Galvanometer between two nodes
Ru = Rv x R2/R1
Now it is easy to realize that the Goldman is like the Nernst equation where the relative permeabilities of Na+ and K+ change

Fig. 2.3 DEF p. 28
An action potential is non-decremental

Alumnus research in neuroscience

Joel Geerling, a chemistry major, took this class (and also graduated) in 2000. He went to Wash U for an MD-PhD. Related to the importance of sodium (covered throughout this course and in this outline) is the hormone aldosterone from the adrenal cortex and its regulation of sodium in the kidney. It is well-known, especially by athletes, that a sodium deficiency leads to increased sodium appetite, Joel's work, in the 4 papers referenced below, addresses this issue at the level of the brain. They illustrate the importance of understanding techniques such as confocal microscopy, as well as brain anatomy.
JCGeerling et al., Aldosterone target neurons in the nucleus tractus solitarius drive sodium appetite. J. Neurosci 26, 411-417, 2006
JCGeerling et al., Aldosterone-sensitive neurons in the rat central nervous system, J Comp Neurol, 495, 515-527, 2006
JCGeerling & ADLoewy Aldosterone-sensitive neurons in the nucleus of the solitary tract: Bidirectional connections with the central nucleus of the amygdala, J. Comp Neurol, 497, 646-657, 2006
JCGeerling & ADLoewy Aldosterone sensitive neurons in the nucleus of the solitary tract: Efferent projections, J Comp Neurol 497, 223-250, 2006
NOW (2012), Joel is doing a neurology residency at Beth Israel / Harvard in Boston

Passive spread of potential along axon

Fig. 2.3 ABC p. 28
If there were not something very special (voltage gating that activates Na+ channel), passive voltage spread would be decremental
Current down along the axon gets smaller because it leaks through the membrane resistance and capacitance.
At any place along the axon, a spike would depolarize the axon to threshold for a spike a certain distance ahead of it, and that distance depends on the square root of the radius.
Spike at one place would also depolarize the axon behind it to threshold, but it does not generate a retrograde action potential because of the refractory period, explained (below) by the inactivation of the Na+ channel.

Fig. 2.2 p. 27 (again)
TERMS: threshold, generator potential, all-or-none, refractory, unidirectional
NOTE also the membrane acting as a low pass filter

Fig B Box 2A p. 31 shows exponential decay and space constant (lambda)
Fig C Box 2A p. 31 shows exponential charging of capacitance and time constan (tao)

Cable equation
see pdf

Personal reflection. My fellow graduate student, Paul Kottler, and I took Warren Dennis's course (Physical chemistry of cell systems) together; we also studied for our PhD exam together. My mentor, Jerry Wasserman, had been famous for asking a question about the speed of the action potential and asking it each subsequent year because he was never satisfied with the answer. We resolved to retire this question since we knew that our coverage on the cable equation was much more than Wasserman would expect. When I found out that I passed, I asked Wasserman how he liked my answer and he replied that "it was ok, a little theoretical." So Paul and I decided we had done our duty for posterity, retiring the question, by answering it with a derivation that involved differential equations.

Summary:
(1) an action potential at one place depolarizes the membrane ahead of it to threshold.
(2) the spread is passive.
(3) current down the axoplasm leaks out through membrane resistance and capacitance.
(4) solving, space constant varies with square root of radius, time constant independent of radius.
(5) that is why invertebrates use giant axons for fast propagation.
(6) myelinated axons also have faster propagation for larger axons.

Coding
frequency of action potentials, not size since they are all-or-none
sometimes action potentials come in bursts
or at beginning of depolarization because of "adaptation"

Hodgkin Huxley experiments

Fig. 3.10 p. 50
Propagation of action potential (spike) shown with opening and closing of Na+ and K+ channels drawn in

Note relative opening and closing of channels
"sodium pump" already established the ion gradients
oscilloscope essentially graphs voltage as a function of time
action potentials can also be listened to on a loud speaker
activation, inactivation, voltage gating

Fig. Box 3A p. 42
general recording "geometry" -
differential amplifier compares 2 voltages and puts out current
operational amplifier is a differential amplifier to clamp voltage
space clamp - really just do whole axon at once

Fig. 3.2 p. 44
voltage clamp data
voltage clamp - change voltage then pump and monitor current needed to keep it there
I - t curves

Fig. 3.3 p. 44
divide into early and late components as I - V curve
Ohm's law: E=IR, thus, the axes of an I-V curve are reversed and the slope is
conductance = 1/R in units of Siemens (formerly "mho")
NOTE: iNa = gNa(V-VNa) - driving potential

Fig. 3.4 p. 45
experiment with low Na+ to show early current is Na+

early fast sodium tetrodotoxin (TTX) sensitive (see box C, Chapter 4, on toxins)
TTX from puffer fish, puffer fish is a delicacy in Japan, but careful preparation is importantto prepare sushi, best if enough TTX left to make mouth numb
saxitoxin from dinoflagellates (red-tides are "blooms" and filter feeding shellfish can become poisonous
Other experiments (e.g. dose of TTX) show few sodium channels, works only if applied to outside of axon

late slow potassium TEA (tetraethyl ammonium) sensitive

Channels

Fig. 4.3 p. 61
In summary, resting potential is based on predominant K+ permeability
then Na+ channels activate
then Na+ channels inactivate
then a late K+ channel activates

GENERALIZATION - action potential is based on Na+ and K+
there are MANY other channel types

Figures Box 4C p. 63
for 20 years they have been studied by "heterologous expression" in cells like Xenopus oocytes
inject exogenous mRNA into clawed African frog egg

Channels are at a low "concentration" (except in post-synaptic membrane).
It takes little tetrodotoxin to block action potential so they are proteins that are not highly expressed.

Thus, channel mutants might be lethal, so they used tricks to get genes like conditional mutants (like temperature sensitive with permissive and restrictive temperatures)

Fig. 4.5 ABCD p. 4.5
KV2.1 (A) is like "delayed rectifyer" K+ channel of action potential. "Rectifier" means that it only allows current in one direction. KV4.1 and HERG have inactivation.

One famous conditional channel mutant in Drosophila, ether-a-go-go, shakes under ether anesthesia. A hunan homologue was found, HERG = human ether-a-go-go-related-gene. HERG inactivates so quickly that it only opens after voltage is over. Contributes to long action potential in cardiac muscle. Long QT syndrome is sometimes mutation of HERG, QRS in EKG (electrocardiogram) is ventricular depolarization, T is repolarization.

A lot of work was done with KV4.1 (B)

Fig. 4.6 Ct o F p. 67
K+, A-type conductance, not at all like K+ channel of action potential
sea slug Anisodoris, Drosophila fruit fly - Shaker mutant
Tetramer makes channel, each component crosses membrane 6 times with hydrophobic domains, S1-S6, inactivation is "stopper" on chain at N-terminal, voltage gating is S4 with + charged arginines or lysines every 3 or 4 amino acids, rotates and moves. Pore is between S5 and S6, not so hydrophobic.

Fig. 4.8
Human K+ channel, includes detailed structure and model
tetramer
shows where K+ is when it is opened

Fig. 4.6G p. 67
This figure shows a 12-transmembrane protein for the Cl- channel
A famous Cl- channel is the cystic fibrosis transmembrane conductance regulator (CFTR)
cystic fibrosis is most common genetic disorder in Caucasians (1/2000), lungs fill up with thick mucus. One presumes the channel is two components.

Fig. 4.6A p.67
Sodium channel, now diverse (human 10 genes)
electric eel Electrophorus electricus 600 V
Huge - 1820 amino acids - "pseudotetramer"
S4 - gating - positively charged (basic) arginine (R) or lysine (K)

Fig. no longer in book
rotation of S4
Pore between 5 & 6 (not hydrophobic)

Fig. 4.1AB p. 4.1
Patch clamp
low current (1-2 pA)
low conductance - 10 pS
stopper to inactivate.

There are different types of Na+ channels, and some are targets of local anesthetics benzocaine and lidocaine.

Potassium channels (lots of them, 100)
Leak (resting potential) 20 pS
Delayed rectifier (repolarization of action potential) 10 pS
Anomalous rectifier - maintain depolarization - cardiac, fertilization
HERG human ether-a-go-go related gene

Calcium channels
16 genes
Ca2+ regulation by parathormone, calcitonin and vitamin D important
Ca2+ channel in synaptic terminal vesicle release - very important, also many others
Ca2+ channel is receptor for IP3 (inositol trisphosphate "second" messenger) on smooth endoplasmic reticulum
Ca2+ channel in muscle sarcoplasmic reticulum
Ca2+ channel in t- (transverse-) tubule in muscle

Box 4B p. 62
Toxins
Tetrodotoxin puffer fish (saxitoxin dinoflagellates) block Na+ channel
scorpions
and many others

Fig. Box 4D p. 70
genetic diseases of channels
myotonia (stiffness from too much excitation) from Cl- channel defect

Fig. Box 4D p. 70
paralysis from Ca2+ channel defect
CSNB from Ca2+ defect Congenital (i.e. genetic) stationary (as opposed to degeneration) night blindness (would affect rods)

Fig. Box 4D p. 70
myotonia, paralysis or stiffness from Na+ channel
Long QT syndrome from Na+ or K+ channel defects EKG (electrocardiogram) has PQRST waves, P from atrial depolarization, QRS from ventricualr depolarization and T from ventricular repolarization
K+ channel from HERG = human ether-a-go-go(EAG) related gene EAG - Drosophila twitch under ether anesthesia

Fig. 4.7 p. 68
Structural studies on bacterial K+ channel - it takes a lot of protein to do X-ran crystallography

Fig. 4.7 p. 68
selectivity by pore size
interesting that non-hydrated ion passes.
Hydrated - size is inverse
Li > Na > K > Rb > Cs (lyotropic series)

Fig. 4.6 (again) p. 67
There are a lot of configurations of channels

Exam questions from 2005 - 2012 relating to this outline

Discussing passive spread of a decremental potential, the current spreading down the axoplasm gets smaller with distance from a place where a stimulus is applied. What happened to the current that was no longer going down the axoplasm?

it leaked through the axon membrane's resistance and capacitance

What does the space constant and the time constant have to do with why squids have giant axons?

cable equation's space constant varies with the square root of the radius while the time constant is independent of the radius, and the space constant over the time constant of passive spread ought to predict the action potential speed

Loss of what hormone, from an adrenalectomy, would make a rat crave salt?

aldosterone

The lecture and figures refer to a sodium channel that is about to be depolarized to threshold by an upstream action potential as "closed." Right after the action potential has passed, what term do we use to describe the status of the sodium channel?

inactivated

Why would long QT syndrome limit a person's ability to respond to stress?

inability to have shorter action potential in the ventricualr myocardium would limit how fast the pulse can get

Why, when the voltage of the squid giant axon is clamped at +65 mV (inside positive), does the early current flow out instead of in?

despite the fact that sodium is going up against its ion gradient, the driving potential, the voltage minus the sodium equilibrium potential, would drive current that direction

A family of "I-T curves" were used by Hodgkin and Huxley to generate two "I-V curves." Say something about these curves.

the "family" was for different voltage clamp levels, giving the V on tie I-V curves. early and late times were chosen tor the two I-V curves. the early curve showed the negative conductance region representing the soduim channel activation

"The space clamp meant that voltage clamping was done along the whole axon." Why would this be more useful than firing a real action potential?

realistic (measurable, not infinitessimal) currentscould be measured

How much current should be carried by sodium at the sodium equilibrium potential?

none

What are the differences in how four components form a channel for Shaker vs. Electrophorus?

4 proteins for shaker, 4 domains of one protein in electrophorus

At one time they thought that the positively charged amino acids in S4 lined the channel, but eventually they agreed that S4 did (what?)?

sensed toe voltage for gating to cause a conformational change for activation

Aldosterone sensitive neurons mediate an animal's specific appetite for what important substance relevant to action potentials?

sodium

"The time constant is independent of radius." Then why are giant axons faster?

the space constant relates to the square root of the radius

Current goes down the axoplasm from the location where the action potential is located. The amount of current gets smaller as a function of distance from the action potential. Why does it get smaller?

it leaks out through membrane resistance and capacitance

In a Voltage clamp experiment, on the I-t curve, there is an early inward current unless the voltage is clamped (at about what level?).

the sodium eauillibrium potential (+55 mV)

Why was it useful to obtain conditional mutants (like temperature- or ether-sensitive mutants), rather than ordinary mutants, to isolate genes like shaker and ether-a-go-go in Drosophila?

channelopathy mutants would be lethal

Why would long QT syndrome interfere with the body's response to stress?

can't get the heart beats short enough to speed up the heart rate

What part (domain) of the Shaker protein is responsible for inactivation?

a stopper (ball of amino acids) at the N-terminal end

Although it is thick mucus in the lungs that is life-threatening in cystic fibrosis victims, it is a channel that is deficient. What kind of channel?

a chloride channel

What causes the S4 transmembrane span of the sodium channel to rotate?

detection of the depolarizing voltage

Why was Electrophorus electricus useful in channel research?

sodium chasnnels were so plentiful in the electric organ that they could be isolated and characterized

Parathormone, calcitonin, and (what other hormone?) help to maintain appropriate levels of blood Ca2+?

vitamin D

How did the electric eel Electrophorus electricus assist in the isolation of a channel?

sufficient concentration of sodium channel to allow characterization

In theory, and in data, what is the direction and amount of Na+ current when the voltage in the axon is clamped to the Na+ equilibrium potential?

none

Paving the way for the Nobel Prize winning Hodgkin and Huxley work, what could you conclude from the Cole and Curtis finding that the AC bridge went out of balance as the
action potential goes by?

conductance increased

In terms of amino acid sequence, how does the S4 of the voltage-gated Na+ channel differ from the typical transmembrane alpha helix?

charged arginines or lysines every 3 or 4 amino acids

To hold the voltage of a squid giant axon at a clamped level of 0 mV, Hodgkin and Huxley had to pump current out through the membrane at 0.5 ms (fairly early) to compensate for
what?

sodium current

What does tetrodotoxin block?

the sodium channel

"Long QT syndrome can be caused by a mutation of HERG." Translate.

genetic long myocardial action potential

How does the space constant of an axon relate to the axon's size?

with square root of radius

Some potassium channels do show inactivation. What part of the molecule is responsible?

stopper on the N-erminus

CSNB is a channelopathy. S=stationary (not progressive degeneration). NB=night blindness (affecting rod photoreceptors). Why is the term C=congenital applied?

it's genetic

An action potential depolarizes the axon ahead of it to threshold, and that is why the action potential propagates. It would also depolarize the axon behind it. Why does it not cause a
backward action potential?

refractory potential, inactivation

Selecting for conditional channel mutants, like temperature sensitive mutants, has been especially useful in Drosophila. Why not just isolate regular mutants?

they might be lethal

Why would two resistors in series connected to a battery be called a Voltage divider?

Two sources of voltage (Ohm's law) E=IR, E = IR1 + IR2, used in Wheatstone bridge

In discussing the passive properties (i.e. without an action potential), the current gets smaller with distance from the stimulus going along the axoplasm (down the inside of the axon). Why does it get smaller?

along the way, it is lost through the membrane (the membrane's resistance and capacitance)

The Shaker K+ channel is a tetramer of proteins that each cross the membrane 6 times. Why is the Electrophorus Na+ channel called a pseudotetramer instead?

because one huge molecule has 4 repeated domains each the size of one shaker channel protein

When (or why) is there a negative conductance region in the I-V curve in Voltage-clamp data?

early, when Na+ channels activate then inactivate

What would be the cause of death if you ate a puffer fish?

Na+ channel block, no action potentials

What does an alpha helix, namely S4, with positively charged (basic) amino acids [arginine (R) or lysine (K)] every 3 or 4 amino acids do for the action potential's sodium channel?

it detects Voltage to gate the activation of the channel

Cole and Curtis already knew that the conductance went up as the action potential went by. Hodgkin and Huxley went a bit further. Which conductance(s) went up?

Na+ and K+

Give the name of at least one "channelopathy" (genetic disease resulting from a mutation affecting a channel protein).

paralysis, myotonia, long QT syndrome, congenital stationary night blindness

You remove both adrenal glands of a rat and let it recover. Answer either (1) What is different about the animal's specific appetite? Or (2) This is explained by the absence of what hormone?
 
Craves salt (NaCl) b/c of loss of aldosterone
 
A certain voltage is applied at one place in an axon. On the basis of passive spread only (no action potentials) what would be the voltage (relative to the applied voltage) one space constant away? (Your answer can be very approximate.)
 
1/3 approximates 1/e
 
The voltage of an axon is clamped from the resting potential to 0 mV. At 0.7 ms (early), Answer either (1) What direction is the current? Or (2) How is this current changed if 460 mM sodium chloride is replaced by choline chloride?
 
In, there is no sodium current if there is no sodium
 
In terms of the protein, what would be the genetic explanation of permissive vs restrictive temperatures in a temperature sensitive conditional mutation?
 
A missense mutation might only disrupt function if temperature denatures the protein
 
For long Q-T syndrome, answer either (1) What cell type has a long action potential that should become shorter in strenuous exercise but does not? Or (2) The channel was first found because what conditional behavior was first noticed in Drosophila mutants?
 
Ventricular myocardial cell, shaking under ether anesthesia
 
Why do they distinguish Shaker vs Electrophorus channels as tetramer vs pseudotetramer?
 
It takes 4 shaker proteins, the electrophorus protein has all 4 domains in one big molecule
 
It seems like there ought to b e salt sensitive neurons to explain sodium appetite. However, former neuro class student Joel Geerling showed thqat neurons were sensitive to a hormone. What hormone?

aldosterone

In passive propagation (cable properties of the axon) why does the current carried down the axoplasm get smaller as you go further from the applied voltage?

leaks out through the membrane

In the classic Hodgkin-Huxley voltage clamp experiments, How did they show that the early inward current was carried by sodium ions?

replace extracellular fluie with sodium free solution

Explain absolute refractory period on the basis of a property of a channel.

an inactivated channel cannot be activated (but a closed channel can)

What would be a useful property (of a cell) when choosing a cell type for heterologous expression of a channel?

should not express the channel already, should be eukaryotic so thatpost-translational modifications wold be the same

"Human ether-a-go-go." Why would mutants be considered to be conditional when they were first found in Drosophila?

shaking only seen under ether anesthesia

How does the shaker protein detect voltage?

S4, with it's positive charges, rotates

Why would the electric organ of the electric eel be better than the squid giant axon for cloning the sodium channel?

expresses plenty of channel

Give one of the three possible membrane compartments where calcium channels would be important in muscle contraction.

pre-"synaptic" membrane, t-tbule, sarcoplasmic reticulum

Knowing about cystic fibrosis might help you to understand the channel for what neurotransmitter?

GABA


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