Lecture4-Action Potential (1)

8/12/2019 Lecture4-Action Potential (1)

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Review from Last Lecture Na+and Cl-ions are more concentrated in the extracellular

environment whereas K+is more concentrated in the intracellular

environment at resting potential.

The resting potential can be calculated or measured using an

intracellular probe.

Permeability and unequal distribution of K

+

ions is the majormechanism for establishing the interior negative resting potential of

a neuron although Na+are slightly permeable and contribute by

depolarizing the resting potential. The contribution of Na+ to resting

potential is variable between neurons and organisms.

Permeability of Cl-can alter the resting potential transiently but has

little effect on steady state potentials.

Leaky potassium channels are responsible for the permeability of

K+.


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What are these K+ Channels I Keep

Hearing About?

Resting potential suggested to Hodgkins, Huxley, Katz and others that thereare ion-specific pathways (channels) through the lipid bilayer that can becontrolled or gated.

For the establishment of resting potential, K+channels are by far the mostimportant.

The first gene for a K+channel was isolated from Drosophila melanogaster

in 1987. It was termed Shaker due to the observed phenotype of a mutateversion.

Based on DNA sequence homology nearly 100 distinct K+have beenidentified in a number of different organisms from bacteria to humans. Thestructure and kinetic characteristics of some have been determined byheterologous expression inXenopusoocytes and two have beencrystallized and the structures elucidated.

K+ channels are constructed of four identical polypeptide chains each ofwhich have 2 to 6 transmembrane helical regions spanning the membrane.

Two of the transmembrane helices are connected by a polypeptide loopregion that confers ion selectivity.


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Structure of Bacterial K+Channel


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Proposed Mechanism of K+Selectivity

and Flux based on Structure Narrow channel formed by the peptide loop allows the selective

passage of dehydrated K+ions. Large ions such as Cs+cannotpass. Small ions such as Na+ cannot span the selectivity filter andand are unstable.

The selectivity filter can hold up to 4 K+ions. Electrostatic repulsionbetween these ions may help to speed the ion flux.

There is a water filled cavity connected to the interior of the cell.Negative charges in the protein allows dehydration of the K+ions.

Again distance may dictate which ions can be dehydrated as aprerequisite for movement through the filter.


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Biology 4822

Formation of Action Potentials


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Measuring Potentials in the Cell

The triangle symbol in electronics represents an amplifier. Theupside down Devo hat is ground.


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Transmission of Information Down a

Neuron Luigi Galvani (1791) demonstrated that placing a battery across a

motor neuron stimulated contraction of frog muscle. In contrast to a

resting state this is an action state elicited by a imposing a new

voltage. This is an Action Potential.

A change in voltage (V) will cause a ions to move (current, I) through

a membrane if the membrane is permeable to the ion (conductance,

g). The relationship is Ohms law, I = gV, where I is in amperes (1A

= 1 C/sec), g is in seimens (g =1/W=A/V) and V is in volts.

A change in current will result in a change in voltage and a change

in voltage will result in a change in current if conductance is greater

than zero.


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The Effect of Changing in Current on the

Membrane Potential A change in current across the membrane results in the rapid

change in membrane potential from negative to positive

(depolarization) followed by a reestablishment of resting potential.

This is the action potential.

In the presence of a constant current flow, a continuous train of

action potential with identical peak membrane potentials is

observed.


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A Closer Look at an Action Potential As the current increases inside the cell a threshold voltage is

reached and the membrane further depolarizes in the absence of anincrease in current. This is the rising phase

The overshoot is depolarization above 0 Volts.

Repolarization of the membrane to resting potential is the falling

phase.

Repolarization below the resting potential is undershoot orrefractory period.


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How Can the Membrane Potential Become

Positive During the Action Potential? During the rising phases of an action potential, the interior of the cell goes from net

negative to net positive. Based on the model derived last lecture for resting potential,this can occur by by the movement of negative ions (Cl-) out of the cell or the

movement of positive ions (Na+or K+) into the cell.

During the failing phase of an action potential, the interior of the cell goes from net

positive to net negative. Based on the model derived last lecture for resting potential,

this can occur by by the movement of negative ions (Cl-) into the cell or the

movement of positive ions (Na+

or K+

) out of the cell.

IntracellularExtracellular

460 mM Na+

10 mM K+ 400 mM K+

540 mM Cl-

0 mM A-

50 mM Na+

60 mM Cl-

510 mM A-


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What Ion(s) are Required for Nerve

Conduction? In the absence of an input of energy, only the movement of Na+

downs its gradient would cause depolarization. (Check Ekfor eachion from last lecture)

E. Overton (1902) demonstrated that external Na+ions are requiredfor nerve and muscle function.

If the depolarization is due to an increase in the permeability of Na+

across the membrane, than the magnitude of the overshoot shouldrequire Na+ in the external media and the magnitude of the peakovershoot should be proportional to the external Na+ concentration(Hodgkin and Katz, 1949).


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Examining Current During an ActionPotential

The relationship between current and voltage is I = gV. If the voltage is changed and held constant, the generated current can be

examined. This technology was developed by Cole etal(1940) and is

termed Voltage Clamp Experiments.

Set a command voltage that is compared to actual voltage. Inject current to

maintain constant voltage. Record required current.


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Voltage Clamp Experiment Results of clamping voltage at -130 mV(A) or 0 mV (B).

Hyperpolarization of the membrane does not result in a significant

current flow.

Depolarization results in a rapid inward current flow (negative by

convention) followed by a slower delayed outward current flow.


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Clamping at Different Voltages:Effect on

Current

If the transient movement of one ion is responsible for depolarizationthan the current should be zero at Eion.

Early inward current is zero at ~52 mV, approximately that of ENa+.

The later outward current increases with increasing depolarization.


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Clamping in the Absence of External Na+ Removal of external Na+causes a loss and slight reversal of inward current but does

not effect late outward current. Na+is responsible for inward current.

Another ion is responsible for the outward current.

Following the release of K+by neurons loaded with radioactive K+ indicated the

outward current is a K+flux.


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Calculating Membrane Conductance (g) for Na+

and K+during the Action Potential

Remember Ohms law, I ion= gion(Vm-Eion) Set Vmwith voltage clamp.

Calculate EK+and ENa+by ionic composition on each side of the

membrane as in last lecture.

Determine INa+and IK+by current measurements during

depolarization in the presence and absence of external Na+,

From these measurements, gNa+and gK+were calculated at differing

Vmvalues.


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A Model for the Generation of an Action

Potential Na+and K+conductance change over time during an action potential.

During initial depolarization, K+that are open at resting membrane potentialmust shut.

There is a rapid activation of Na+conductance at a set Vm. This leads torapid depolarization by Na+influx.

This is followed by a slower activation of K+conductance. This lead torepolarization by K+efflux more slowly over time.

Na+conductance is deactivated at peak depolarization. This inactivation canbe mimicked by a short depolarizing prepulse prior to extendeddepolarization leading to a diminishment of Na+influx. In contrast, A shorthyperpolarizing prepulse actually enhances Na+influx during depolarization.

This strongly suggests that there are ion-specific channels that arecontrolled by the voltage differences in the membrane.

Are there current changes in the membrane during depolarizationresponsible for the gating of channels?


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Gating Current Buried in Capacitative

Current

Na+

gating occurs very quickly and the signal is buried in thecapacitative current.

Give two identical voltage steps of opposite polarity, any change in

current symmetry is due to movement of a charge in the membrane

supporting the idea that Na+channels are sensing the Vmat

threshold and opening. This asymmetry is observed and the mean time of charge

movement is 0.5 msec.


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Single Channel Patch Clamp Recording


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Recordings of Current from Single Na+

Channels Seven separate single Na+ channel recordings from patch clamp of

squid axon when depolarized to -10 mV. Some channels do not open during depolarization.

Most channels open only once. The majority between 1-2 ms. This

is slower than gate current (0.5 ms).

The mean inward current is 1.5 pA and the mean opening time is 1

ms.

The probability of opening decreases with increasing time of

depolarization.


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Sum of the Microscope Currents

Capitulated Macroscopic Current The sum of hundreds of single channel recordings given the same

kinetics of the observed macroscopic current.


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Probability of a Na+ Channel Opening is

Related to MembraneVoltage Depolarization does not determine when a particular channel will

open, how long it will stay open nor how many times it will open.

It determines a probability of opening but the actual openings are

random events.


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Structure of a Voltage-gated Na+Channel

Ten Na

+

channel genes have been identified. They have 4 separate domains each consisting of 6 transmembrane

helices.

In each domain is a peptide (P) loop region proposed to form the ionselectivity filter.

Each domain has a voltage sensor which is a helical region in which one

side is lined with positive amino acids. It is proposed that a change inmembrane potential will cause a translation of the helix opening the poreduring depolarization.


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Inactivation of Voltage-gated Na+

Channel

Prepulse experiments suggests that inactivation is a distinct mechanismfrom activation.

Proteolytic treatment to the internal surface of squid axons abolishedinactivation. External treatment of the axon has no such effect.

Mutagenesis of amino acids residues of a loop region between domains IIIand IV abolishes inactivation.

It is proposed that this section of the polypeptide blocks the pore afterdepolarization. This is the Ball and Chain model.


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Model for Voltage-Gate Na+Channels


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Various Voltage-gated K+ Channels


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Single Channel Recording for a Voltage-

Gated K+Channel


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Action Potentials Can Have Many Forms (A) squid axon (B) myelinated axon of frog motor neuron (C) cell

body of frog motor neuron (D) cell body of interneuron of guinea pig(E) Purkinje neuron of guinea pig

Ch i I C t ti D i


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Change in Ion Concentration During an

Action Potential Start at -73 mV and depolarize to +40 mV in Squid Axon

Qinitial= (1 x10-6C/V cm2)(73 x 10-3V) = 7.3 x 10-8C/cm2(6.25 x1018ions/C) = 4.6 x

1011ions/cm2

Qfinal= (1 x10-6C/V cm2)(40 x 10-3V) = 4.0 x 10-8C/cm2(6.25 x1018ions/C) = 2.5 x

1011ions/cm2

Total Ions of Na+Membrane/cm2after depolarization = Qinitial+ Qfinal= 7.1 x 1011

Na+ions/cm2

If we assume a 1 cm length of squid axon with a 0.05 cm radius Surface area = 4(3.14)(0.05 cm)2 = 7.9 x 10-3 ml = 0.31cm2

Mol of Na+in = [(7.1 x 1011ions/cm2)(0.31 cm2)]/6.02 x 1023ions/mol = 3.7 x 10-13mol=0.4 pmol

Volume = (3.14) (0.05 cm)21 cm = 8 x 10-3ml = 8 x10-6L

[Na+] increase = 3.7 x 10-13mol/8 x 10-6L = 5 x 10-8M = 0.05 M

The original [Na+] = 30 mM = 30,000 M. This is a change of 0.0002 %. The neuron can form action potential repeatedly. In addition, the Na+/K+ pump is

activated by depolarization resetting the Na+levels rapidly.

What is the percent change if the neuron has a 1 m diameter and is a 100 m long?

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Lecture4-Action Potential (1)