Introduction to cardiac

electrophysiology

2.

Dr. Tóth András

2018

Topics

• Ion channels

• Local and action potentials

• Intra- and extracellular propagation of

the stimulus

4 Ion channels

4.1 Basic features

Ion channel

Protein (or protein komplex) located within the plasma

membrane, acting as a pore and facilitating selective

transport of ions. Following its activation an ion channel

does generate transmembrane electric current. This current

may flow either „inward” or „outward” direction.

Failure or abnormal function of an ion channel complex

(most typically as a consequence of genetic disorders) may

lead to severe malfunction at the organ level. Indeed, ion

channel related disorders – “channelopathies” – are being

identified as the background pathomechanisms of an

increasing number of diverse human diseases.

Basic features of ion channels 1.

Motto: For normal cell function precisely controlled and fully synchronized

transport of a wide variety of positive and negative ions is essential.

Ion channels are present within the plasma membrane of all cell types and intracellular

organelles. Inside an ion channel there is a narrow tunnel that allows only ions of certain

size and/or charge to pass through (selective permeability). A given ion channel may either

be “gated” (regulated) or “nongated” (non-regulated).

Compared to „other types of ion transport proteins” ion channels have two important,

distinctive features:

(1) The rate of ion transport through a channel is very high (often > 106 ion/s).

(2) The direction of the passage of ions through the tunnel is determined by

their electrochemical gradient (a function of the concentration gradient and

membrane potential). For the "downhill" ion transport no metabolic energy

(ATP, co-transport or active transport) is needed.

Since they are able to regulate influx or outflux of ions, ion channels are often

direct or many times indirect (!!!) targets for a wide variety of drugs.

Basic features of ion channels 2.

Motto: Some ion channels are fully regulated (i.e. „gated” channels), others are

pracically nonregulated (i.e. „nongated” – or „leakage” – channels).

„Gated” ion channel – the opening (and sometimes closing) of these channels is

triggered by the income of a specific stimulus. This specific stimulus may be:

(1) change of membran potential (voltage) (voltage gated ion channels)

(2) binding of a specific ligand or signalling molecule (ligand gated channels)

(neurotransmitter, hormon, local hormon) to the channel protein

(3) mechanical stimulus (deformation, pressure, stretch);

(4) light energy (photon);

Since a ”gated” ion channel is regulated – it only opens following the income of the

adequate trigger signal. The closing, however, may be both controlled or spontaneous.

„Nongated” (or leakage) ion channel – is always open (or leaking) and facilitates the

passage of one or more ion types in direction of their electrochemical gradient.

A ”nongated” channel is nonregulated, as well, (i.e. further to the electrochemical

potential the transport flux is solely a function of the temperature.

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

Principal structure of an ion channel

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

narrowest point and is selective for its specific ion (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).

Ion channels are involved in a huge variety of cellular functions, many of which has

critical importance: e.g. chemical signaling, transcellular transport, pH regulation,

regulation of cell volume, etc.

Their significance is especially prominent in the nervous system (e.g. generation

and propagation of the nerve impulses, signal propagation through the synapses

via transmitter-activated ion channels, etc. (Most animal venoms and toxins –

produced by spiders, snakes, scorpions, sea snails, bees, etc. – are acting by

modulating ion channel conductance and/or kinetics.)

In addition, ion channels are key components of a wide variety of physiological

mechanisms: e.g. excitation-contraction coupling in cardiac, skeletal and smooth

muscles, epithelial transport of nutrients and ions, activation of the immun system,

insulin release from the beta-cells of the pancreas, etc.

Not surprisingly, many ion channels are among the most favoured targets

of the pharmaceutical research.

Biological roles

There are over 300 types of functional ion channels in a living cell.

Their classification can be accomplished in several different ways: by the

nature of the „gating” mechanism, the species of ion passing through the

pore, or the structure and/or localization of the channel proteins.

A further heterogeneity arises in the cases when channels with different

constitutive subunits give rise to a single, specific current.

Absence or mutation of one or more of the contributing types of channel or

regulatory subunits may result in loss of function, and potentially underlie

multiple diseases.

Diversity

Q:

What is the principal difference between

membrane receptor and ion channel?

Q:

Are there membrane receptors, which also

serve as ion channels?

4.2 Experimental techniques

Bert Sakmann – Nobel laureate – developer of the “patch clamp” technique

Principles of the “patch clamp” technique

Needed:

Electrode + pipette

Special pipette solution

Gigaseal (R > 1GOhm)

Voltage command

Current measurement

Steps of “patch clamp” recording

“Patch clamp” configurations

Common investigations:

Single channel current

Current of a channel type or a

group of channels

Special pipette solution –

modification of the IC milieau

Automated “patch clamp” measurement

4.3 Principles of regulation

Simple, “dual-state” ion channel – background channels

spontaneously oscillate between open and closed states

State diagram of a komplex “multistate” ion channel

4.4 Biophysical properties

Determination of “single channel” current

1. A single channels is in either open or closed

state.

2. The open state of a channel is short lived

compared to the time course of the

macroscopic current.

3. The length & latency of the open state is highly

variable, it may happen that the channel

does not open, at all.

4. The probability function of channel opening

correlates with the shape of the

macroscopic current curve.

5. A channel may open several times during a

single cycle if there is no inactivation.

Integrated (macroscopic) current of two groups of voltage dependent

channels following a voltage command: 1) Na+; 2) delayed K+

The Na+ channel spontaneously inactivates, the K+ channel not

Sum of 300 recordings

„macroscopic

Na+ current

Determination of the mean open time

Modulation of the channel can be followed in change of the

mean open time

Current-voltage relationship characteristic for the „inward” and

„outward” rectifying channels

Current-voltage functions of two groups of ion channels

(Na+ & K+) and their combination

The K+ current shows linear relationship – no voltage-inactivation

The Na+ current is nonlinear because of the voltage inactivation

Current-voltage relationship of two types of ion channels -

determined in isolated atrial cardiomyocytes

Principles of activation/inactivation kinetics

Working structural model of slowly inactivating Kv4 type

K+ channels

4.5 Classification of ion channels

Voltage-gated ion channels

Their opening (and often closing) is driven

by changes in membrane potential, i.e

the equilibrium state of their spontaneous

oscillation (probability of the open/closed

states) is voltage dependent.

Classification by gating mechanism 1

Functions

They have a crucial role in excitable tissues

(neurons, muscles, glands) by ensuring

excitability and stimulus-propagation.

Depending on circumstances voltage-gated

ion channels allow rapid and synchronized

depolarization in response to voltage stimuli,

but can also contribute to repolarization or

hiperpolarization.

Major subfamilies

- Voltage-gated Na+ channels (Nav)

- Voltage-gated K+ channels (Kv)

- Voltage-gated Ca2+ channels (Cav)

- Voltage-gated H+ channels (Hv)

- Hyperpolarization-activated channels

- Transient receptor potential (TRP)

channels

Ligand-gated ion channels

Also known as ionotropic receptors - open in

response to specific ligand molecules binding

to a channel protein: e.g. „neurotransmitter-

gated”, „G-protein-dependent”, „modulated”

(kovalent modification – phosphorilation, etc.)

channels. Binding of the ligand causes a

conformation change and subsequent ion flux

across the channel.

These channels are typically involved in initialization of impulses (i.e. early

phase of depolarization of excitable cells – nerves, muscles, glands).

Depending on circumstances they may initiate depolarization, repolarization

or hyperpolarization, as well.

Examples: cation-permeable “nicotinic” acetylcholine receptor, ATP-gated „P2X”

receptors, ionotropic glutamate-gated receptors, anion-permeable GABA receptor.

Sometimes 2-nd messenger-activated channels are also mentioned here, though

ligand and 2-nd messenger are different categories.

Classification by gating mechanism 2

Classification by gating mechanism 3

„Modulated” channels may also be voltage-

gated, however, covalent modification (e.g.

phosphorylation) also effectively modulates

the probability of the open/closed states.

Probability of the open and closed states of

„G-protein gated” channels is modulated via

binding of a G-protein, activated by activation

of a receptor (e.g. muscarinic Ach receptor)

Other gating mechanisms

Include activation/inactivation from the inside of

the membrane by e.g. second messengers.

Certain ions may also be considered as second

messengers causing direct activation of the

channel (e.g.: inward rectifying K+ channels,

Ca2+-activated K+ channels, two-pore-domain K+

channels

Classification by gating mechanism 4

Mechanosensitive ion channels (1)

Opening of these channels is triggered by

mechanical stimulus (stretch, shear,

pressure, displacement, vibration)

Light-gated channels (2)

These channels (e.g. channelrhodopsin) are

directly opened by the action of light.

Temperature gated channels

Some TRP receptors (TRPV1, TRPM8) are

opened either by hot (e.g. the capsaicine

receptor) or cold temperatures.

Cyclic nucleotide-gated channels

Two families: 1) the cyclic nucleotide-gated

(CNG) channels (cAMP and cGMP binding),

and 2) the hyperpolarization-activated, cyclic

nucleotide-gated (HCN) channels.

1

2

Sodium channels

Voltage-gated Na+ channels (Nav)

Epithelial Na+ channels (ENaC)

Potassium channels

Voltage-gated K+ channels (Kv)

Ca2+-activated K+ channels (BKCa, SK, etc.)

Inward-rectifier K+ channels (Kir)

„Two-pore-domain” K+ channels („leak” channels)

Calcium channels

Voltage-gated Ca2+ channels (CaV)

Classification by transported ion type 1

Proton channels

Voltage-gated H+ channels (Hv), with strong pH dependence

and tiny conductance

Cloride channels

Poorly-understood superfamily of anion channels with at least

13 members. (pl. ClC, CLIC, Bestrophin, CFTR). Nonselective

for small anions. Since cloride is the most abundant transported

anion, they are called cloride channels.

Non-selective cation channels

These channels are permeable to several cations (mainly Na+,

K+ and Ca2+). A number of TRP (transient receptor potential)

channels are also classified into this group.

Classification by transported ion type 2

Based on less common features, e.g number of transmembrane pores or

transient behaviour of response.

Two pore channels

Small (2 members) cation-selective ion channel family, containing two

KV-style 6-TM domains, probably forming a dimer in the membrane.

Tranzient receptor potential (TRP) channels

This family, containing at least 28 members has extremely diverse

activation properties. Some are probably constitutively open, others

show voltage, [Ca2+], pH, redox state, osmolarity or stretch dependence.

Their selectivity is also greatly variable, some are Ca2+-selective, others

are less selective cation channels.

6 subfamilies: canonical (TRPC), vanilloid (TRPV), melastatin (TRPM), polycystin (TRPP),

mucolipin (TRPML), and ankyrin transmembrane protein 1 (TRPA) .

Further ways of classifications

Example: superfamilies of K+ channels

Q:

How is that possible, that Na+ ions may pass an

ion channel, but K+ ions not?

How is that possible, that K+ ions may pass an

ion channel, but Na+ ions not?

Ion Atomic radius (Angstöm) Energy of hydration (kcal/mol )

Na+ 0.95 -105

K+ 1.33 -85

Help

4.6 Structure

Representative examples of ion channel “superfamilies”

S4 helices are the “voltage-sensors” of voltage-gated channels –

amino acid homology is extensive

2D model for the voltage dependent Na+ channel

2D model for the voltage dependent Ca2+ channel

• a) Conventional „gating” model:

The voltage sensor (S4 segment) moves

across the plasma membrane

• b) Novel „paddle” model:

The voltage sensor S4 and S3 segments

compose a „hairpin” outside of the

channel. Activation of the channels is

induced by the rotation of these paddles.

• c) The „transporter” model:

This new model is based on the

assumption that the charges present on

the voltage sensor (S4) do not move

accross the membrane, insteed they

rotate around their longitudinal axes,

shifting the „gating” charges from

extacellular to intracellular position.

Suggested models of activation mechanism of voltage

dependent ion channels

Contains at least 9 members. Is largely responsible for creation and propagation of

action potentials.

The pore-forming α subunits are very large (up to 4,000 amino acids) and consist

of four homologous repeat domains (I-IV) comprising 6-TM segments (S1-S6)

each. This α subunits also coassemble with auxiliary β subunits, each spanning

the membrane once.

Voltage gated sodium channels (Nav)

The Cav family contains 10 members. They are known to play a crucial role in

excitation-contraction coupling in the muscles, as well, as their fast neuronal

excitation with transmitter release.

The structure of the α1 subunits have a resemblance to α subunits of the Na+

channels. They are also known to coassemble with other (α2δ, β, γ) subunits .

Voltage-gated Ca2+ channels (Cav)

This family contains almost 40 members, which are further divided into 12

subfamilies. These channels are known mainly for their role in repolarizing the

cell membrane following action potentials.

The α subunits (homologous to the sodium channels) have 6-TM segments, and

also assemble as tetramers to produce a functioning channel.

Voltage gated potassium channels (Kv)

Functional model of a voltage-gated K+ channel

"Birth of an Idea",

2007

1.50 m x 0.90 m x 0.90 m

Steel, glass, wood

Sculpture by Julian Voss-

Andreae based on potassium

channel

Photo by Dan Kvitka

Sculpture commissioned and

owned by Roderick MacKinnon

Please use only with link to

www.JulianVossAndreae.com

4.7 Structure-function relation

Model of the function of the S4 helix as „voltage sensor”

A total of 6 charges should relocate in the membrane to open the channel

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

„A few” cardiac ion channels

Cardiac action potentials and major ion currents involved

Q:

Which are the most important properties

of the ion channels?

A:

Integral membrane protein

Aqueous pore through the membrane

Selective permeability to one or several small ions

Single channel conductance to ion flow

Rectification - current passes more easily in one than in

other direction

Gating - oscillation between conducting and nonconducting

states

Regulation - gating is influenced by voltage, or binding a

ligand, or covalent modification

Pharmacological modulation !!!

Sites of action of clinically important drogs!!!

5 Local & action potentials

5.1 Local response

Q:

What is the difference between electrochemical

and membrane potentials?

Local (subthreshold) response

Q:

Which are the most important features

of the local response?

Temporal summation

Spatial summation

Q:

Special forms of local response?

5.2 Action potential

Responses in the membrane potential to increasing pulses of

depolarizing current

Action potentials from three vertebrate cell types

Q:

What are the major differences between

local response and action potential?

5.3 Action potentials in the heart

Ion concentrations in mammalian heart

“Fast and slow response” in the heart

Regional variability in the morphology of the action potential in

cardiac cells

Q:

What is the main reason for the very different

kinetic properties of the action potentials

recorded in different cell types?

“Explanation” of the kinetic differences in cardiac AP-s

„Fast”

sodium

„Funny”

„Delayed

rectifier”

Calcium

„Tranzient

outward”

„Background”

Sodium

„Inward

rectifier”

Ion currents

!

I

0

0

„L”

„T+L”

Q:

How could you change the shape of the

action potential?

The effect of tetrodotoxin on the fast response

Q:

What is the effect of tetrodotoxin on

fast response in cardiomyocytes?

6 Propagation of the stimulus

6.1 Basic principles of propagation

Q:

Why is it “feasible” to maintain a large resting

potential in our body cells, if it „costs” us a

large amount of energy (ATP)?

Potential changes recorded (measured) by an extracellular

electrode located at different distances from the current electrode

Maximum change in recorded (measured) membrane potential

plott-ed versus distance from the point of current passage

Potencial changes calculated for a model RC-circuit

Electric model of the axon membrane

Time constant determined in a membrane

CRR im

Model of the decremental propagation (voltage divider -

resistance ratio)

Length constant determined in the axon membrane

i

m

R

R

Model of conduction of the local (subthreshold) response

Electric model for the propagation of potential changes

Q:

What is the explanation for the well known

fact, that postsynaptic action potentials are

always generated at the axon hillock?

Model of conduction of the AP in nonmyelinated fibers

“Saltatory” conduction of the action potential in myelinated fibers

Conduction velocity of the action potential determined in

unmyelinated and myelinated fibers

Q:

Which factors determine the conduction velocity of

the action potential in myelinated fibers?

And in unmyelinated fibers?

Q:

Why is conduction velocity significantly higher in

myelinated then in unmyelinated fibers?

6.2 AP propagation in heart

Q:

How would you explain the common statement that

cardiac muscle is “functional syncytium”?

Structure of the electric synapse (gap junction)

MW 1500Ca2+

pH

Em

Q:

Where are electric synapses (gap junctions)

located in the mammalian body?

Q:

Which are the major functional differences

between electric & chemical synapses?

Electric model of the cardiac cells

Computer simulation of impulse propagation at the microscopic

level

Q:

What is the prime factor determining the direction

of impulse propagation in the three dimensional

cardiac muscle?

Q:

Why is the transmission of stimulus through

the AV node dramatically slower than its

propagation in other regions of the heart?

Q:

Are there „fast” & „slow” velocities of action

potential propagation in the heart?

What may be the reason?

The significance of the gap junction system in

physiological stimulus propagation in the heart

Bonus 4 U !!!

Subcellular stimulus propagation

Differences in delays of intra- and intercellular activation –

single cell wide network

Differences in delays of intra- and intercellular activation –

multi-cell wide network

Impulse propagation (isochron lines) in case of physiological gap

junction coupling (homogenious AP-population)

Impulse propagation (isochron lines) in severe uncoupling of the

gap junctions (heterogenous AP-population)

In severe gap junction uncoupling propagation velocity may

decrease TWO orders of magnitude (!!!)

(from 36.7 cm/s to 0.31 cm/s)

In case of normal gap junction coupling isochron lines are

relatively regularly placed, AP-population is homogenous

In case of critical gap junction uncoupling action potentials form

clusters with significant delays

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

THE END