Bioelectricity
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Overview
❒
Preliminary notions – electrical potentials in
bicompartimental systems
❒
Resting membrane potentials
❒
Local and action potentials
❒
Propagation of action potentials
❒
Synapses
❒
Electrophysiological research methods
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Preliminary notions
Electrical potentials in
bicompartimental systems
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
General considerations
❒
Differences in ionic concentrations between
different compartments lead to electric
phenomena:


❒
Electrical potential differences between the
compartments
Ionic currents (if the sepparating membrane is
permeable and allows transport)
Such conditions are found in any living cell and are
responsible for the electrical phenomena occurring
at this level
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Case I – Planck-Henderson equation
❒
Diffusion potentials
❒
Two compartments with KCl (C1 > C2)
❒
PK > PCl
❒
❒
1
2
Between the two compartments a
difference in the electrical potential is
established = diffusion potential
The diffusion potential decreases in
time, due to ion transport through the
membrane; can reach 0 after a long
enough time
uCl −u K RT C 2
 E= E 1− E 2 =
⋅
ln
uCl u K zF C 1
UMF Carol Davila
Dept. of Biophysics
●
●
uCl – mobility of Cl- ions
uK – mobility of K+ ions
Bioelectricity (AP)
2008-2009
Case II – Nernst potentials
❒
Selectively permeable membrane (for K+only)
❒
Two compartments with KCl (C1 > C2)
❒
PCl = 0
❒
❒
1
2
At equilibrium, the voltage difference
between the compartments equals the
Nernst potential of K+
(an osmotic flow of water can also
occur, from the right compartment
towards the left one)
1
RT [ K ]1
 E=
ln 1
zF [ K ]2
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Case III – Donnan equilibrium
❒
Selectively permeable membrane (impermeable
for protein anions)
❒
❒
❒
❒
1
Left: KCl (PK = PCl ≠ 0) and protein
anions (non-diffusable; PA=0)
Right: water
Between the two compartments a
Donnan equilibrium is reached
Osmotic flow of water towards the left
compartment
2
1
−1
RT [ K ]1
RT [Cl ]1
 E=
ln 1 =−
ln
F
F
[ K ]2
[Cl −1 ]2
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Case III – Donnan equilibrium
❒
❒
❒
❒
1
2
❒
UMF Carol Davila
Dept. of Biophysics
Consequence:
1
K+ and Cl- diffuse towards the right
compartment
K+ is attracted to the protein anions →
[K+]1 > [K+]2
Cl- is rejected by the protein anions →
[Cl-]1 < [Cl-]2
At equilibrium, the electrochemical
(Nernst) potential of the two species is
equal
−1
[ K ]1 [Cl ]2
1 =
−1
[ K ]2 [Cl ]1
➔
Different concentrations of
the two species in the two
compartments
Bioelectricity (AP)
2008-2009
Case IV
❒
Selectively permeable membrane (impermeable
for protein anions and Na+)
❒
❒
❒
1
❒
❒
UMF Carol Davila
Dept. of Biophysics
PA = PNa = 0
K+ si Cl- will diffuse until a Donnan
equilibrium is reached
The equilibrium potential will be equal
to the Nernst potential of K+
2
No osmotic flow, if the number of Na+ ions compensates the number of
protein anions
This is a similar situation to many living cells (ex: the muscle cell, where
under resting conditions PCl = PK = 1 and PNa = 0.04 - neglectible)
Bioelectricity (AP)
2008-2009
Resting membrane potentials
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
General cosiderations
❒
❒
Due to constant ionic gradients between the inside
and the outside of a living cell, a voltage difference
is established between the two compartments =
Resting potential
The resting potential can be measured
electrophysiologically (with intracellular electrodes)
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
The Goldman-Hodgkin-Katz
equation
❒
The resting potential of a cell can be calculated
based on the Goldman – Hodgkin – Katz
equation
x
−y
∑
P
[C
]

P
[
A
]i −cel
RT
C
e−cel
A
 E=
ln
x
−y
F
∑ P [C
∑P [A ]
]

C
i−cel
A
e−cel
∑
UMF Carol Davila
Dept. of Biophysics

C – cation species

A – anion species

PC – cation permeability

PA – anion permeability
Bioelectricity (AP)
2008-2009
The Goldman-Hodgkin-Katz
equation
1
−1
[ K ]i−cel [Cl ]e−cel
= −1
=30
1
[ K ]e−cel [Cl ]i−cel
[ Na1 ]e−cel
[ Na1 ]i−cel
=14.5
PR= E K =−84 mV
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
The equivalent electrical circuit of
the cell membrane
gK, gNa, gCl – K, Na, Cl channel
conductances
● E , E
K
Na, ECl – Nernst potentials
for K, Na, Cl
● C
m – membrane capacitance
● E
m – electromotive tension of
the membrane (resting potential)
●
2
Cm = 1 μF/cm
● The conductance of an ion
channel is constant, and is a
characteristic of the channel
●
UMF Carol Davila
Dept. of Biophysics
E K E Na E Cl


R
R Na RCl g K E K  g Na E Na g Cl E Cl
E m= K
=
1
1
1
g K  g Na g Cl


R K R Na R Cl
Bioelectricity (AP)
2008-2009
Local and action potentials
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
General considerations
❒
❒
❒
Excitability threshold = the minimal intensity of
a stimulus capable to induce an electrical response
of a neuron
Firing threshold = the minimal value of
membrane depolarization to be reached, in order to
trigger an action potential
The thresholds are influenced by:


UMF Carol Davila
Dept. of Biophysics
The type of neuron (different types of neurons will
have different depolarization thresholds)
Ionic concentrations of Na+, K+, Ca2+
Bioelectricity (AP)
2008-2009
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
General considerations
❒
Local potentials (LP) and action potentials (AP) are
temporary changes in the membrane
potential (depolarization), which propagate
through the cell membrane
➔
Local potentials – lower than threshold depolarization
➔
Action potentials – higher than threshold depolarization
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Local potentials
❒
Lower than threshold depolarization
❒
Decremental (electrotonic) propagation
❒
❒
Their amplitude is proportional to the intensity of
the stimulus (modulation in amplitude)
Occur in the dendrite and neuronal body
membrane
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Action potentials
❒
❒
❒
Are triggered by an over-threshold
depolarization

High intensity stimulus

Sumation of local potentials
Constant amplitude for each type of neuron
(approx. 120 mV)
Propagation speed constant (characteristic of the
nervous fiber)
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Action potentials
❒
Non-decremental propagation
❒
Modulated in frequency
❒
Occur in the axon (nervous fibers)
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Action potentials
Phases of an AP:
❒
❒
❒
❒
Latency (between
stimulus and
depolarization)
Pre-potential (local
potential)
Peak:

Ascendent phase

Descendent phase
Post-potential
(hyperpolarization)
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Action potentials
❒
Refractory period – the interval in which the
neuron is non-responsive to stimuli

Absolute refractory period:
➔
➔

Relative refractory period (decreased excitability):
➔
➔
➔
UMF Carol Davila
Dept. of Biophysics
Ascendent phase of the AP
Part of the descendent phase
Pre-potential
The rest of the descendent phase
Post-potential
Bioelectricity (AP)
2008-2009
Action potentials
❒
Neuronal accomodation = the increase of the
excitability threshold, as a response to repetitive
stimulation

Fast / slow

Involved mechanisms:


UMF Carol Davila
Dept. of Biophysics
Receptor desensitization (fast)
Plasticity (up/down-regulation, other mechanisms) (slow)
Bioelectricity (AP)
2008-2009
Molecular events
❒
❒
Ionic channels involved in an AP:

Na+ channels

K+ channels

(Ca2+ channels)
In resting state:

Passive Na+ and K+ flow through leak channels


UMF Carol Davila
Dept. of Biophysics
K+ channels >>> Na+ channels → PK >>> PNa; PR ≈ EK
The ion concentrations are maintained stable
through the activity of the Na/K pump
Bioelectricity (AP)
2008-2009
Alpha subunit of a Na channel
Closed and open K channel
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Molecular events – Na and K channels
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Molecular events
❒
Local potentials
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Molecular events
❒
Action potentials
UMF Carol Davila
Dept. of Biophysics
The stimuls opens the Na
channels
● When the firing threshold is
reached, an avalanche
opening of Na channels leads
to positivation of the
membrane potentials
● The Na channels become
inactive after a certain time
period
● The depolarization opens the
K channels and the potential
starts to decrease
● The K channel have a slow
kinetic – they remain open for
a while after the potential
reaches resting state –
responsible for the
hyperpolarizing post-potential
●
Bioelectricity (AP)
2008-2009
Gary Matthews, Blackwell Publishing
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Propagation of action potentials
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
General considerations
❒
❒
Any LP / AP leads to a local change in charge
distribution on the membrane
As a result, electrical currents appear between the
depolarized areas and the neighbouring, nondepolarized regions:


UMF Carol Davila
Dept. of Biophysics
The resting potential of the depolarized area will be reset
The vicinity areas become themselves depolarized => AP
propagation
Bioelectricity (AP)
2008-2009
Types of LP/AP propagation
❒
Propagation:

Decremental
➔
➔
The amplitude of depolarization decreases with distance
The membrane propagation constant = distance where the
amplitude decreases to half

Rm
d=
Ri

❒
Rm – membrane resistivity
Ri – intracellular fluid
resistivity
Non-decremental
➔
➔
UMF Carol Davila
Dept. of Biophysics
❒
Recurrent propagation
Saltatory conduction
Bioelectricity (AP)
2008-2009
Recurrent propagation
❒
Characteristic for non-myelinated fibers
❒
Slow conduction (0.5 – 2 m/s)
❒
Local Herman
currents
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Saltatory conduction
❒
Characteristic for myelinated fibers
❒
Fast propagation (→ 120 m/s)
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Gary Matthews, Blackwell Publishing
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Wikimedia Commons
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Synapses
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
General considerations
❒
❒
Synapse = specialized structure for impulse
transmission between neurons; accounts for
unidirectional transmission
Clasification of synapses:


UMF Carol Davila
Dept. of Biophysics
Chemical synapses (the majority of synapses in the
human nervous system)
Electrical synapses
Bioelectricity (AP)
2008-2009
Chemical synapses
Structure:
❒
Presynaptic level


Axon terminal (pre-synaptic
bouton)
Contains neurotransmitter
molecules
❒
Synaptic cleft (20-50 nm)
❒
Post-synaptic level


UMF Carol Davila
Dept. of Biophysics
Post-synaptic neuron
membrane
Membrane receptors for
neurotransmitters
Bioelectricity (AP)
2008-2009
Chemical synapses
❒
Synaptic transmission:






UMF Carol Davila
Dept. of Biophysics
An AP at the axon terminal level opens the voltage-dependant
Ca2+ channel existing at that level
Ca2+ influx determines conformational changes of the SNAP proteins
and fusion of vesicles with the neuronal membrane, and the
neurotransmitter is released in the synaptic cleft
The neurotransmitter molecules diffuse through the synaptic space
and bind to specific receptors on the postsynaptic membrane
The postsynaptic receptors are chemically activated by the
neurotransmitters and open their ion channels, leading to local
depolarization of the postsynaptic membrane (local potential)
If depolarization reaches the firing threshold, an AP will propagate
throug hthe postsynaptic neuron
The neurotransmitter molecules dettach from the receptors and:
➔
Are reuptaken by the presynaptic neuron
➔
Are degraded by enzymes existing in the synaptic cleft
➔
Diffuse outside the synaptic cleft
Bioelectricity (AP)
2008-2009
Gary Matthews, Blackwell Publishing
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Chemical synapses
❒
Quantum transmission:


1 vesicle – approx. 104
molecules of neurotransmitter
Increased stimulus intensity →
increased number of released
vesicles
http://www.colorado.edu


❒
1 released vesicle → local potential of small amplitude (EPSP/IPSP =
excitatory/inhibitory post-synaptic potential)
The response of the postsynaptic neuron is the summation of al
EPSPs/IPSPs → modulation of the intensity of the response
Synaptic delay 0.5 – 5 ms
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Chemical synapses
❒
The chemical synapses can be:

Excitatory
➔
➔

Inhibitory
➔
➔

Ionotropic postsynaptic receptors – Cl- channels
Activation leads to postsynaptic hyperpolarization
Modulatory
➔
➔
UMF Carol Davila
Dept. of Biophysics
Ionotropic postsynaptic receptors – Na+ channels
Activation leads to postsynaptic depolarization
Metabotropic postsynaptic receptors
Activation leads to metabolic changes of the postsynaptic
neurons, which influence their activation threshold, the level of
receptor expression etc
Bioelectricity (AP)
2008-2009
Chemical synapses
❒
❒
❒
The type of synapse is given by the type of
postsynaptic receptors
A neurotransmitter can have both excitatory and
inhibitory effects, depending on the the type of
receptor to which it binds
A neuron can express more than one type of
receptors; the final activity depends on all the
expressed receptors
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Gary Matthews, Blackwell Publishing
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Chemical synapses
❒
Examples:

Excitatory synapses:
➔
➔

Inhibitory synapses:
➔
UMF Carol Davila
Dept. of Biophysics
Nicotinic synapses (acetylcholine) – CNS, neuromuscular
junction
Glutamate synapses – CNS
GABA-ergic synapses
Bioelectricity (AP)
2008-2009
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Electrical synapses
❒
❒
Specialized structures where the electric
impulses can travel directly from the
presynaptic to the postsynaptic neuron
Structural aspects:



Very small synaptic cleft (2
nm!)
Contiguity between the preand postsynaptic membrane
ion channels
No neurotransmitters
involved
Wikimedia Commons
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Electrical synapses
❒
Functional characteristics:



UMF Carol Davila
Dept. of Biophysics
Practically instantaneous transmission (no synaptic
delay)
Transmission is bidirectional (!!!)
Due to fast transmission, they can electrically
synchronise a large mass of cells
Bioelectricity (AP)
2008-2009
Electrophysiological reasearch
methods
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
General considerations
❒
❒
The electrophysiological methods allow
measurements of cell electrical potentials or
transmembrane currents through ion channels, by
using electrodes
Examples:
UMF Carol Davila
Dept. of Biophysics

TEVC

The patch-clamp method
Bioelectricity (AP)
2008-2009
General considerations
❒
Basic principles:



UMF Carol Davila
Dept. of Biophysics
They use “glass electrodes”: capillary glass tubes
(pipettes) with a sharp tip of very small diameter, filled
with conductive solutions; a chlorinated silver
electrode is introduced in the pipette, connected to the
electronic measurement circuits
We measure voltage differences between the
glass electrodes and reference electrodes
Signals are filtered and amplified
Bioelectricity (AP)
2008-2009
TEVC
(two-electrodes voltage clamp)
❒
❒
Uses two intracellular electrodes:

The current electrode

The voltage electrode
Principle:



UMF Carol Davila
Dept. of Biophysics
The voltage electrode measures the membrane potentials
(compared to a reference)
Any transmembrane current (through ion channels) leads to
changes of the membrane potential; its value is maintained
constant by injection in the cell of an equal current of
opposite sign, through the current electrode
The injected current necessary to maintain a constant
membrane voltage is therefore equal to the current through
the membrane ion channels
Bioelectricity (AP)
2008-2009
The patch-clamp technique
❒
❒
❒
❒
Newer method (historically)
Principle: one measures the voltage between a glass
electrode attached to the cell / intracellular and a
reference electrode in the cell bath solution
A high resistance contact is established between the
electrod and the cell membrane = Gigaseal (> 1
GOhm), preventing current leak
We can measure the electrical activity of the entire cell
(Whole-cell) or of a single / a few ion channels
(Single-channel)
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Molecular Devices, The Axon CNS Guide
to electrophysiology and biophysics
laboratory techniques
 1993-2006 by Molecular Devices
Corporation.
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
The patch-clamp technique
❒
Configurations:

Cell-attached

Whole-cell

Excised patch:
➔
➔
➔
❒
Inside-out
Outside-out
Perforated patch
Either the voltage (voltageclamp) or the current
(current-clamp) can be
maintained constant during
measurements
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
The patch-clamp technique
❒
Whole-cell recordings
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
The patch-clamp technique
❒
Single-channel recordings
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
The patch-clamp technique
❒
Single-channel recordings
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009
Supplementary reading
❒
Purves, Dale; Augustine, George J.; Fitzpatrick, David; Katz, Lawrence C.; LaMantia, Anthony-Samuel;
McNamara, James O.; Williams, S. Mark – Neuroscience - Sunderland (MA): Sinauer Associates, Inc.; c2001
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=neurosci.TOC&depth=2
❒
http://hyperphysics.phy-astr.gsu.edu/hbase/electric/bioelcon.html#c1
❒
http://highered.mcgraw-hill.com/sites/dl/free/0072437316/120060/ravenanimation.html
UMF Carol Davila
Dept. of Biophysics
Bioelectricity (AP)
2008-2009