Medical physics 2.

Electric properties of the living stuctures -2

Ferenc Bari
professor

Department of Medical Physics and Informatics

Faculty of Medicine
University of Szeged

Szeged, February 24. 2022.

What will be in focus on the current lecture?

2. What signals does the researcher measure (extra- and intracellular, transepithelial
potential, field potential, etc.) and why?

3. Bioelectricity as an integral feature of living organisms

4. Electrical characteristics of cells and tissues (voltages, currents, how and how to
measure them)

5. Physical (mathematical) modeling of cellular and tissue-level electricity: the Nernst

equation, the Goldman equation, linear cable theory, and the Hodgkin-Huxley model of
action potential generation and propagation
 Composition of extra- and intracellular fluids

Arrows indicate chemical gradients

-why don’t move the ions at steady state
(equilibrium, resting potential etc.)?
Electrical field –potential difference
counteracts
(U= E x d)
 The resting potential in cells are normally more negative inside than outside. This varies from
-9mV to -100mV. Excitable tissues of nerves and muscles cells have higher potentials than other
cells (epithelial cells and connective tissue cells).
Dead cells do not have membrane potentials
A cell is “polarized” because the interior
(ICF) side of the membrane is relatively
more negative than the exterior (ECF).
In a mammalian cell at resting potential, the
concentration of K+ is highest inside the
cell, while the concentration of Na+ is
highest outside the cell
Sodium-potassium pumps use the energy of
ATP to maintain these K+ and Na+ gradients
across the plasma membrane
These concentration gradients represent
chemical potential energy

That can vary a lot.

 Diffusion potential
Resting membrane potentials often arise as
"diffusion potentials" of certain ions across the
membrane (the classical case is the K+ -
diffusion potential). The size and polarity of the
diffusion potential depends on the electrolyte
tested, on the concentration difference across
the membrane, and on the nature of the
membrane's permeability. If the membrane is
It lasts for ever (if the electrochemical equilibrium is achieved)
permeable to all ions (as is the case with
cellophane), the diffusion potential is initially
maximum, but progressively declines as the
ions diffuse across the membrane and the
concentration gradient is reduced V

This type is the simplest

It is transient, since Cl¯and H+ have
different motility.
+ After a certain time it will be in equilibrium
And diffusion potential disappears

Cl- H+
 Nernst Equation

under standard conditions, the cell

k BT Co RT Co potentials of electrochemical cells can be
U   ln   ln determined with the help of the Nernst
q Ci F Ci equation. The Nernst equation is often
used to calculate the cell potential of an
electrochemical cell at any given
Co
At 300 K get U  60 mV log temperature, pressure, and reactant
concentration.
Ci
• F = 96,400 Coulomb/mole
•Simplest equation for membrane potential – one ion
 The Nernst equation describes the diffusion potential at
equilibrium

RT Cout
U =  ln
zF Cin

U: The equilibrium diffusion potential

R: Gas constant (8.31 J  K-1mol-1)
T: Absolute temperature (K)
F: Faraday constant (96500 Coulomb/mole)
z: Number of charges of single ion (1 with K+ and Na+, 2 with Ca++, -1 with Cl-
)
ln: Natural logarithm (base: e = 2.71828)
Cin, Cout Inside and outside concentrations of the diffusible ion species
 What if there are multiple diffusible ions? (1)

Intra- and extracellular cation concentrations:

Inside Outside Equilibrium potential

Na+ 15 mM 150 mM +60 mV
K+ 150 mM 6 mM -90 mV

Actual resting membrane potential: -70 mV

How is it possible?
K+ can almost freely move but Na+ can’t
 What if there are multiple diffusible ions? (2)

RT Cout
Nernst equation: U =  ln
zF Cin

Goldman equation (special case for Na+ and K+):

RT PK  [K+]out + PNa  [Na+]out

U =  ln
F PK  [K+]in + PNa  [Na+]in
PK represents conductivity (1/R) for K+ ions
PNa represents conductivity (1/R) for Na+ ions

This is a simplified model (two ions) if PK is lot higher than PNa we neglect Na+ role (Nernst equation)
The Goldman equation and the role of ion channels

RT PK  [K+]out + PNa  [Na+]out

U =  ln
F PK  [K+]in + PNa  [Na+]in

P = Permeability – this is where the ion channels come in

In more general form (where Cl- also plays a role)

U 
   
k BT  Pk K  o  PNa Na  o  PCl Cl  i 
ln 
 
q 
  
  
 Pk K i  PNa Na i  PCl Cl o   
If the membrane was permeable for only a certain ion (K+,
Na+, Cl- or Ca2+) there was a stable equilibrium-membrane
potential for each

- 60 mV 140 mM
EK= log10  - 90 mV
+1 4 mM

- 60 mV 15 mM
ENa= log10  + 60 mV
+1 140 mM

- 60 mV 4 mM
ECl= log10  - 80 mV
-1 103 mM

- 60 mV 10-7 M
ECa= log10  + 120 mV
+2 10-3 M
 The Goldman equation and the role of ion channels –how was it
measured- Voltage clamp method

RT PK  [K+]out + PNa  [Na+]out

E =  ln
F PK  [K+]in + PNa  [Na+]in

change
don’t change

The voltage clamp is an experimental method used by electrophysiologists to measure

the ion currents through the membranes of excitable cells, such as neurons, while holding the
membrane voltage at a set level. A basic voltage clamp will iteratively measure the membrane
potential, and then change the membrane potential (voltage) to a desired value by adding the
necessary current. This "clamps" the cell membrane at a desired constant voltage, allowing the voltage
clamp to record what currents are delivered. Because the currents applied to the cell must be equal to
(and opposite in charge to) the current going across the cell membrane at the set voltage, the recorded
currents indicate how the cell reacts to changes in membrane potential. Cell membranes of excitable
cells contain many different kinds of ion channels, some of which are voltage-gated. The voltage clamp
allows the membrane voltage to be manipulated independently of the ionic currents, allowing
the current–voltage relationships of membrane channels to be studied.
What is Um when multiple channels are activated?

In addition to K+ and Cl- permeabilties, Na+ (and Ca2+) conductivity are playing
role in setting the membrane potential

13
 The Nobel Prize in Physiology or Medicine 1963

Alan Lloyd Hodgkin Andrew Fielding Huxley

Sir Bernard Katz

The Nobel Prize in Physiology or
Medicine 1970
 Goldman-Hodgkin-Katz equation: For a muscle cell

RT PK [ K  ]o  PNa [ Na  ]o  PCl [Cl  ]i

Um  ln Applies only when Um is
F PK [ K  ]i  PNa [ Na  ]i  PCl [Cl  ]o
not changing!
For T=37°C:
Resting potential

PK [ K  ]i  PNa [ Na  ]i  PCl [Cl  ]o (simulation)

Um  61  log
PK [ K  ]o  PNa [ Na  ]o  PCl [Cl  ]i
 Donnan equilibrium- Donnan potential

The usual cause is the presence of a different charged substance that is unable to pass through the membrane and
thus creates an uneven electrical charge. For example, the large anionic proteins in blood plasma are not
permeable to capillary walls. Because small cations are attracted, but are not bound to the proteins, small anions
will cross capillary walls away from the anionic proteins more readily than small cations.
On the side of negatively charged proteins, a higher cation concentration and a lower concentration of the small-
molecule permeable anion are formed relative to the opposite side, in the form of an equilibrium state. -10 - -15
mV potential difference is created. (The side of proteins is the more negative.)
 Electrical Model

Um 
 gV i i g ClU Cl  g KU K  g NaU Na  g CaU Ca (from Kirchoff’s laws)

g i g Cl  g K  g Na  g Ca
g is like conductance (=1/R) and like permeability
This equation is equivalent to Godman-Hodgkin-Katz equation.

Please consider that Ca2+ conductance also plays role (in case of cardiac muscle cells it
is crutial!!
 Example squid axon

[K]in =125mM
IK = gK (Um – UK), Um = -60 mV
IK = gK (-60 – (-75)) mV = gK(+15 mV).
Um = -60 UK = -75 g always positive.
DU = Uin – Uout

[K]out = 5 mM positive current = positive ions flowing out of the

cell.
Um not sufficient to hold off K+ flow so ions flow
out. When Um = Uk then no flow.

• Remember electric field points in direction of [K]in =125 mM

force on positive test charge
• E always points from higher potential (for ions)
ds
E E Do Soma 3
(Resting
[K]out = 5 mM Potential)
In an ‘average’ resting animal cell:
- outward K + current is affected by:

-  large concentration difference

-  high K + permeability
-  negative membrane potential

- - inward Na + current is affected by:

-  large concentration difference
-  low Na + permeability
-  negative membrane potential

- In case of the two currents are equal Stable membrane potential

Electrogenic pumps also generate membrane potential

The pump has three effects:

(1) it makes the sodium concentration high in the extracellular
space and low in the intracellular space;
(2) it makes the potassium concentration high in the intracellular
space and low in the extracellular space;
(3) it gives the intracellular space a negative voltage with
respect to the extracellular space (10-20 mV)
 How can we describe one cell’s electric properties
Extracellular lead

Intracellular lead
 There are multibarrel electrodes for complex
investigations

Reimann F , Gribble F M Diabetes 2002;51:2757-2763

Copyright © 2011 American Diabetes Association, Inc.

How to explain electronic vs action potential

• Electrotonic and action potentials

elektrotonic
action
There are several types of action potentials
Some important differences
Electrotonic potential
Graded potentials are changes in membrane potential that are confined to a
relative small region of the plasma membrane
The size of a
graded potential
(here, graded
depolarizations)
is proportionate
to the intensity
of the stimulus.
Graded potentials can be: EXCITATORY or INHIBITORY
(action potential (action potential
is more likely) is less likely)

The size of a graded potential is proportional to the size of the stimulus.

Graded potentials decay as they move over distance.
 Action potential conduction

• Propagation
• Depolarized to threshold
• Sodium channels open
• Influx of Na+
• Positive charges coming in
depolarize the membrane just
ahead to threshold
• Next population of sodium
channels open
 Action potential conduction

• Propagation of the action potential

• Orthodromic
• Action potential travels in one direction - down axon to the axon terminal
• Antidromic (experimental)
• Backward propagation is possible if the initiation of AP occurs in the middle of axon
• Cannot turn back on itself
• Refractory (inactivated sodium channels)
• Typical conduction velocity: 10 m/sec (varies between 0,5-100 m/s)
• Factors Influencing Conduction Velocity
• Depends on how far the depolarization ahead of the action potential spreads
• The spread depends on resistance of space
• Path of the positive charge
• Down the inside of the axon
• Across the axonal membrane - leakage
• Axonal excitability
• Axonal diameter (bigger = faster)
• Number of voltage-gated channels
• Neural pathway that are specially important for survival have evolved unusually large axons -
squid giant axon
 Action potential conduction
• Factors Influencing Conduction Velocity

• Layers of myelin sheath insulate the

leakage of charges and facilitate
current flow down the inside of axon

• Nodes of Ranvier
• Every 0.2-2.0 mm
• Place of AP generation
• Place of voltage-gated sodium
channels

• Saltatory conduction
• AP travels by leaping
 Graded potentials

(Local response, local excitation, local potential)

• Not “all-or-none”
• Electrotonic propagation:
spreading with decrement
• Summation: spatial & temporal
During action potencial the conductivity
of the cell membrane is altered
 The action potential, in reality
• The Voltage-Gated Sodium Channel
• Generalized epilepsy with febrile seizures (channelopathy)
• Caused by a single amino acid change in the extracellular region of one
sodium channel (out of many)
• Slowed inactivation prolongs action potential
• Toxins as experimental tools

• Puffer fish toxin: Tetrodotoxin (TTX)

Toshio Narahashi
Clogs Na+ permeable pore by binding tightly
Blocks all sodium-dependent action potentials
• Red Tide toxin: Saxitoxin

Na+ Channel-blocking toxin

Produced by marine protozoa, Gonyaulax dinoflagellate, typical
shellfish prey
Occasional blooming of the dinoflagellates cause red tide
• Structural studies and physiological studies
 The action potential, in Reality
• Voltage-Gated Potassium Channels
• According to Hodgkin and Huxley’s experiments, falling phase
cannot be explained solely by the inactivation of gNa
• Existence of potassium gate was also proposed
• open in response to depolarization
• Potassium gates open slowly (need about 1msec after
depolarization)
• Delayed rectifier
• Potassium conductance serves to rectify or reset
membrane potential
• Function to diminish any further depolarization
• Four separate polypeptide subunits join to form a pore
• Key Properties of the Action Potential
• Threshold
• Rising phase
• Overshoot
• Falling phase
• Undershoot
• Absolute refractory period
• sodium channel de-inactivation
• Relative refractory period
- potassium channel closure (hyperpolarization)
Ionic basis of action potential
 New technology: The patch clamp technique

The voltage clamp technique

shown before was adequate for
large currents, but produced
large ‘background noise’

‘Patch clamp’ technique has

superior signal-to-noise ratio, so
very small currents can be
measured, even down to the
current passed through a single
ion channel!
 Patch Clamping

www.essen-instruments.com/Images/figure2.gif

Invented by Sakmann and Neher [Pflugers Arch 375: 219-228, 1978]

Can be used for whole cell clamp (measure currents in whole cell, placing electrode in cell) like on left or
pulled patch as on right (potentially measure single channel).
Usually voltage clamp (command voltage or holding voltage) and observe current (I = gV). Ix = g(Vh-Vx)
where x is for each ion and Vx is Nernst potential for that ion. With equal concentration of permeable ion
on both sides, get g easily
The membrane patch is tightly sealed to the tip of
the electrode.

A few ion channels are for entering or leaving the

tip and the charge (potential is changed)

Charge/time= current
In this case in pA-s
E. Neher and B. Sakmann
Visited Szeged several times
 Ion Channels
• Can be purified
• Specific membrane spanning proteins
• High order of ionic selectivity
• Size
• Charge
• Other? These factors are not well understood
• (water sphere-interaction within the channel)
• Blockers: channel-specific drugs
• Channels are “gated”: channels are not open all the time
• Channel classes:
• Ligand-gated channels
• Voltage-gated channels
• Mechanically-gated channels
Major classes of ion channels

Mechanosensitive ion channels

Temperature sensitive ion channels

 Principal structure of an ion channel

1 – channel domains
(typically four per channel)
2 – outer vestibule
3 – „selectivity filter”
4 – diameter of the „selectivity
filter”
5 – site of phosphorylation
6 – plasma membrane

A typical channel pore is only one-two atoms wide at

the narrowest point and is selective for its specific ion
(e.g. Na+, H+, K+).
Nonetheless, several ion channels are less selective
and let pass more that one type of ion, usually with
the same type of charge: cations (positive charges)
or anions (negative charges).
 Types of ion channels

There is a thermodynamical probability

of a certain state (closed or open)
 Excitable cells: a cell in which the membrane response to
depolarisations is nonlinear, causing amplification and
propagation of the depolarisation (an action potential).
Action potential

Some of the cells (excitable cells) are capable to rapidly reverse their resting membrane
potential from negative resting values to slightly positive values. This transient and rapid change
in membrane potential is called an action potential
 Action Potentials
D. ∆ Ion conductance
1. rising phase:  in gNa
overshoot approaches ENa
(ENa is about +60 mV)

2. falling phase:  in gNa and  in gK

3. after-hyperpolarization
continued  in gK
approaches EK
(EK is about -90 mV)
 Effect of channels opening

1. When channel is closed, no current flows through channel

2. When cations (+) enter cell ("inward current"), cell depolarizes
(becomes more positive inside)
+
depolarizing Re-or hyperpolarizing
inward (+) + outward (+)
current current

The effect of the opening of a particular kind of channel on a certain action potential
depends on:

1. The permeant ion (e.g. Na+, Ca2+, K+, etc)

2. The Nernst potential for "X", the relevant ion, (EX)
3. The membrane potential (UM) when the channels open
4. When UM is negative to EX, there is inward (depolarizing) current
4. When UM is positive to EX, there is outward (repolarizing) current

48
Simple, two-state ion channel – „background” channels oscillate
spontaneously between the open and closed states
State diagram of a complex, multiple-state ion channel
 Gated Ion Channels
A. Voltage-gated Na+ channels
1. localization
a. voltage-gated
 Gated Ion Channels
A. Voltage-gated Na+ channels
2. current flow
a. Na+ ions flow through channel at 6000/sec at emf of -100mV
b. number of open channels depends on time and Vm
3 opening of channel
a. gating molecule with a net charge
b. b. change in voltage causes gating molecule to undergo conformational change
4. generation of AP dependent only on Na+
repolarization is required before another AP can occur
K+ efflux
Mechanism of voltage sensitivity

TM4 contains charged residues; these move in the

membrane when membrane potential changes
 Gated ion channels
A. Voltage-gated Na+ channels
2. current flow
a. Na+ ions flow through channel at 6000/sec at emf of -100mV

b. number of open channels depends on time and Vm

3 opening of channel
a. gating molecule with a net charge
b. b. change in voltage causes gating molecule to undergo conformational change
4. generation of AP dependent only on Na+
repolarization is required before another AP can occur
K+ efflux
Geometry of negative charges, pore size, and ion hydration work
together to provide K+ selectivity, excluding Na+