introduction to electrophysiologyweb.med.u-szeged.hu/phcol/jegyzet/ta_1e06c.pdf · before...
TRANSCRIPT
Introduction to
electrophysiology
Dr. Tóth András
Topics
• Transmembran transport
• Donnan equilibrium
• Resting potential
• Ion channels
• Local and action potentials
• Intra- and extracellular propagation of the stimulus
Level of significance
• “Entry” level (even under 6)
• “Student” level (for most of you)
• “Gourmand” level (only for the pros)
1. Transmembran transport
Major types of transmembran transport
1
dx
dcA
JD
x
cDAJ
dx
dcDAJ
=
∆∆
−=
−=J: net rate (flux) of diffusion
A: area
dc/dx: concentrationgradient
D: diffusion coefficient
(D: cm2/s)
Fick’s first law of diffusion
2
ηπ r
kTD
6=
Diffusion of solutes as a consequence of the random
thermal (Brownian) motion of the particles
Stokes–Einstein
equation
Einstein relation____
(∆x2) = 2 Dt
3
Time required for diffusion as a function of diffusion
distance
4
Fick’s law for membrane
x
DK
x
cDAJ
x
cDAJ
∆=
∆∆
−=
∆∆
−=
β
β
Diffusion across a semipermeable membrane
ββββ: partition coefficient
K: permeability coefficient
5
Osmotic motion across a semipermeable membrane
6
Definition of the osmotic pressure
ΦΦΦΦ: osmotic coefficient
ΦΦΦΦic: osmotically effective concentration - osmolality
van’t Hoff’s Law
π= iRTm
π= iRTc
π = RTΦic
Φic = ∆Tf /1.86
I.e.: 154 mM NaCl solution
ππππ = 6.42 atm
Φ Φ Φ Φic = 0.286 osmol/L
7
Mechanism of facilitated diffusion
8
Principle of transport of ions across ion channels
9
The principle of function of the Na+/K+–ATPase
10
Secondary active transport processes
11
Transport via proteins shows saturation kinetics
Michaelis-Menten
equation
Vmax: maximal rate of
transport
Km: concentration of
the substrate for which the rate of
transport is equal
to Vmax/2
12
2. Ionic equilibrium
[ ][ ] ( )BA
B
A
o
EEzFX
XRT
zFECRT
−+=∆
++=
+
+
ln
ln
µ
µµ
Electrochemical potential (difference)
13
Nernst equation
[ ][ ] ( )
( ) [ ][ ]
[ ][ ]B
ABA
B
ABA
BA
B
A
X
X
zF
RTEE
X
XRTEEzF
EEzFX
XRT
mEquilibriu
+
+
+
+
+
+
−=−
=−−
−+=
ln
ln
ln0
[ ][ ] lg60
B
A
X X
XmVE
+
+
−=+
For monovalent cations
Z = 1
14
A B
0.1 M
K+
0.01 M
K+
EA – EB = -60 mV
Examples of uses of the Nernst equation 1.
0.1 M
HCO3-
EA – EB = +100 mV
A B
1 M
HCO3-
Is there equilibrium in any of the two cases?
15
A B
0.1 M
K+
0.01 M
K+
EA – EB = −−−−60 mV
Examples of uses of the Nernst equation 2.
A B
At –60 mV the K+ is in electrochemical equilibrium
across the membran
No electric force !!!
+++++++
–––––––
16
1 M
HCO3-
0.1 M
HCO3-
A B
0.1 M
K+
0.01 M
K+
EA – EB = −−−−60 mV
Examples of uses of the Nernst equation 3.
EA – EB = +100 mV
A B
At –60 mV the K+ is in electrochemical equilibrium
across the membran
No electric force
At the given membran potential the HCO3
- is not in electrochemical equilibrium
Electric force: +40 mV
+++++++
–––––––
––––––––
++++++++
17
1 M
HCO3-
0.1 M
HCO3-
A B
[K+] = 0.1 M
[P-] = 0.1 M
[K+] = 0.1 M
[Cl-] = 0.1 M
A B
[K+] =
[Cl-] =
[P-] = 0.1 M
[K+] =
[Cl-] =
Initial state
Before Gibbs-Donnan equilibrium is established
1. The principle of electroneutrality should be preserved !!!
2. The electrochemical potential should be zero for each diffusible ion !!! (Not for the undiffusible ion !!!)
Equilibrium?
18
A B
[K+] = 0.1 M
[P-] = 0.1 M
[K+] = 0.1 M
[Cl-] = 0.1 M
A B
[K+] = 0.133 M*
[Cl-] = 0.033 M*
[P-] = 0.1 M
[K+] = 0.066 M*
[Cl-] = 0.066 M*
Initial state Equilibrium state* (!?)
Gibbs-Donnan equilibrium has been attained
1. The principle of elektroneutrality is, indeed, valid !!!
2. The electrochemical potential is zero for K+ and Cl- !!!
3. * So, is there any problem ???
19
A B
[K+] = 0.1 M
[P-] = 0.1 M
[K+] = 0.1 M
[Cl-] = 0.1 M
A B
[K+] = 0.133 M
[Cl-] = 0.033 M
[P-] = 0.1 M
[K+] = 0.066 M
[Cl-] = 0.066 M
Starting state Equilibrium state
In Gibbs-Donnan equilibrium a transmembrane
hydrostatic pressure gradient is present
(There is no equilibrium between pressures !!!)
∆∆∆∆PH = 2.99 atm !!!
20
3. Resting potential
The „concentration battery”
A B
0.1 M
NaCl
0.01 M
NaCl
If the membrane is permeable for cations, but unpermeable for anions, cation current is
needed to reach equilibrium !!!
21
The „concentration battery”
A B
+
+
+
+
+
+
+
–
–
–
–
–
–
–
In case of electrochemical equilibrium
EA – EB = - 60 mV
Na+
22
0.1 M
NaCl
0.01 M
NaCl
“Measured” intra- and extracellular ionconcentrations
23
A simplified model of the resting membrane potential in
the human skeletal muscle
mV
P
mV
mV
mV
Na
90E 4)
0Prot 3)
P )2
- - 150 Prot
90- 115 3,6 Cl
100- 3,5 160 K
65 145 12 Na
E (mM) EC (mM) IC 1)
m
100K
-
-
eq
−=
=
⟩⟩
+
++
+
+
-90 mV
Cl- Na+
cc cc
cc
E E
E
K+
24
+++
+++
−−−
−=
−=
≈−=
=∆
=
KKmK
NaNamNa
ClClmCl
gEEI
gEEI
gEEI
Rg
R
UI
)(
)(
0)(
1
Conditions for the “chord conductance” equation
Theoretical estimation for the resting potential 1.
25
+
++
+
+
++
+
++++
++
++
+=
−−=−
=+
Na
NaK
Na
K
NaK
Km
KKmNaNam
KNa
Egg
gE
gg
gE
gEEgEE
II
)()(
0
++
++
+=
NaKm EEE1100
1
1100
100
+6
0
0
-70
-90
Na+
K+
Em
The “chord conductance” equation
gNa+ = 1 gK+ = 100
26
The “constant field” (Goldman-Hodgkin-Katz) equation
opClipNaipK
ipClopNaopKm
ClkNakKk
ClkNakKk
F
RTE
][][][
][][][ln
−++
−++
++++
=
Theoretical estimation for the resting potential 2.
27
Major factors affecting resting potential
C
28
Also in cardiac cells the resting potential is supposed to
be [K+] dependent
29
In cardiac cells the resting potential is, indeed, primarily
[K+] dependent
30
4. Ion channels
4.1 Experimental techniques
Major configurations of the „patch clamp” technique
31
„Single channel” current
32
Determination of the mean open time
33
Current-voltage relationship of the „inward” and
„outward” rectifying channels
34
4.2 Principles of regulation
State diagram of a simple, “dual-state” ion channel
35
State diagram of a “multiple-state” ion channel
36
Basic regulatory mechanisms of ion channels
37
„Background” channels spontaneously oscillate between
open and closed states
37
a
„Voltage-gated” channels also oscillate between the two
states, but voltage shifts the equilibrium
37
b
The open state of „neurotransmitter-gated” channels is
altered by the binding of a neurotransmitter to the
channel (e.g. nicotinic receptor)
37
c
The open state of “G-protein gated” channels is altered
by binding of activated G-protein subunits to the channel
(following receptor activation – e.g. muscarinic receptor)
37
d
„Modulated” channels may be voltage-gated, but the
ability of voltage to open the channel may be influenced
by covalent modification (e.g. phosphorilation)
37
e
4.3 Structure
Ion channel “superfamilies”
38
2D model of the Na+ channel 1.
39
2D model of the Na+ channel 2.
40
4.4 Structure-function relation
S4 helices are the “voltage-sensors” of voltage-gated
channels –amino acid homology is extensive
41
Model of the function of the S4 helix as „voltage sensor”
A total of 6 charges should relocare in the membrane to open the channel
42
Top view of the Na+ channel showing how the central ion
channel is proposed to be lined by one of the helices
from each domain
43
Functional model of a K+ channel
44
Cardiac ion channels
45
5. Local and action potentials
5.1 Local response
Local (subthreshold) response
46
Temporal summation
47
Spatial summation
48
5.2 Action potential
Responses in the membrane potential to increasing
pulses of depolarizing current
49
Action potentials from three vertebrate cell types
50
5.3 Action potentials in the heart
Ion concentrations in mammalian heart
51
“Fast” and “slow” response in the heart
52
Regional variations in the shape of the action potential of
the heart cells
53
Explanation of the kinetic differences
„Fast”
sodium
„Funny”
„Delayed
rectifier”
Calcium
„Tranzient
outward”
„Background”
Sodium
„Inward
rectifier”
Ion currents
∅∅∅∅!
I
≈≈≈≈0
≈≈≈≈0
∃∃∃∃
„L”
„T+L”
54
The effect of tetrodotoxin on the fast response
55
6. Propagation of the stimulus
6.1 Basic principles of propagation
Potential changes recorded by an extracellular electrode
located at different distances from the current electrode
56
Maximum change in recorded membrane potential plott-
ed versus distance from the point of current passage
57
Potencial changes in a model RC-circuit
58
Electric model of the axon membrane
59
Time constant determined in a membrane
CRR im ⋅
60
Model of decremental propagation (voltage divider -
resistance ratio)
61
Length constant determined in the membrane
i
m
R
R
62
Model of conduction of the local (subthreshold) response
63
Electric model for the propagation of potential changes
64
Model of conduction of the AP in nonmyelinated fibers
65
“Saltatory” conduction of the action potential in
myelinated fibers
66
Conduction velocity of the action potential determined in
unmyelinated and myelinated fibers
67
6.2 AP propagation in heart
Structure of the electric synapse (gap junction)
MW <<<< 1500Ca2+ ↑↑↑↑pH ↓↓↓↓Em ↑↑↑↑
68
Electric model of the cardiac cells
69
Computer simulation of impulse propagation at the
microscopic level
70
The significance of gap junctions in
normal stimulus propagation in the
heart
Subcellular stimulus propagation
71
Differences in delays of intra- and intercellular activation
– single cell wide network
72
Differences in delays of intra- and intercellular activation
– multi-cell wide network
73
Impulse propagation (isochron lines) in case of normal
gap junction coupling (homogenious AP-population)
74
Impulse propagation (isochron lines) in severe gap
junction uncoupling (heterogenous AP-population)
75
In severe gap junction uncoupling propagation velocity
may decrease TWO orders of magnitude (!!!)
(from 36.7 cm/s to only 0.31 cm/s)
76
In case of normal gap junction coupling isochron lines
are relatively regularly placed, AP-population is
homogenous
77
In case of critical gap junction uncoupling action
potentials form ”clusters” with significant delays
78
Distribution of the cells forming the different clusters in
case of critical uncoupling – turn back behaviour of the
stimulus easily leading to „reentry” can be observed
79
Questions
What are the principal differences between the following iontransporters?
Sodium-calcium exchanger
Sodium-hidrogen exchanger
Calcium pump of the sarcolemma
What does equilibrium potential mean for a given ion?
How the Nernst equation can be used to analyze ion movements in case of
diffusible ions?
What will happen, if the membrane is not permeable for at least one ion?
When is Gibbs-Donnan equilibrium present across a living cell membrane?
In Fig. 22 how much Na+ has to pass the membrane to reach equilibrium?
Which are the primary conditions for establishing and maintaining steady
resting potential ?
What is the reason, for in one cell type (rbc) the resting potential equals –30 mV,
while in an other (cardiac) cell type it equals –90 mV?
What are the major factors determining the actual value of the membrane
potential?
Questions
What is the difference between a membrane receptor and an ion channel?
Are there membrane receptors, which are also ion channels?
How is possible, that Na+ ions can pass an ion channel, but K+ ions don’t?
How is possible, that K+ ions can pass an ion channel, but Na+ ions don’t?
Which are the most important properties of the ion channels?
What is the difference between electrochemical potential and membrane potential?
Which are the most important features of the local response?
Special forms of local response?
What are the major differences between local response and action potential?
What is the reason for the very different kinetic properties of the action potentials
recorded in different cell types?
How could you change the shape of the action potential?
What is the effect of tetrodotoxin on fast response?
Why is “good for us” to maintain a resting potential in the cells of our body, if it
costs such a substantial amount of energy (ATP)?
Questions
What is the explanation for the fact, that postsynaptic action potentials are
generated at the axon hillock?
Which factors determine action potential conduction velocity in myelinated fibers?
And in unmyelinated fibers?
Why is conduction velocity significantly higher in myelinated than in unmyelinated
fibers?
How would you explain the expression that cardiac muscle is “functional
syncytium”?
Where are electric synapses (i.e. gap junctions) located in the mammalian body?
Which are the major functional differences between electric and chemical
synapses?
What is the prime factor determining direction of impulse propagation in the three
dimensional cardiac muscle?
Why is the transmission of stimulus through AV node dramatically slower than in
other parts of the heart?
Is there „fast” and „slow” action potential propagation? What may be the reason?
THE END