Voltage-Dependent Ion Channels 1: All About Sodium
Channels!
The ACTION POTENTIAL!
Occurs in neurons, and other excitable tissues such as cardiac and
skeletal muscle
An influx of sodium ions is followed by an efflux of potassium, producing
the characteristic, reproducible shape of the action potential
depolarisation curve
How is this rapid and reproducible pattern of ion fluxes achieved?
PUMPS versus CHANNELS!
Initial question addressed by
researchers was the mechanism by
which ions are transported across a
membrane, as the lipid bilayer is
impermeable to charged molecules. 2
options:
ION TRANSPORTERS (PUMPS):- pick up
an ion, undergo a conformational
change and release the ion to the
other side of the membrane. Requires
a large amount of energy (ATP) as
ions are transported against their
concentration gradient, and it is a relatively slow process. This mechanism
is not fast enough to produce the rapid rise in ion flux required for the
action potential.
ION CHANNELS:- create a water-filled pore through the membrane, via
which ions flow down their electrochemical gradient (balance of
concentration and charge). A fast process (sufficient to produce the action
potential), but ions can only flow down their gradient rather than against.
CHANNEL PROPERTIES required for action potential!
2 types of channels: one for sodium and one for potassium.
ION SELECTIVITY:- one channel only recognises a particular ion. Sodium
flux happens first, in response to modest depolarization, and potassium
ion channels open later to return membrane potential to resting state.
VOLTAGE DEPENDENCE:– opening of channel only happens at particular
membrane potential (depolarization of the membrane from -80mV to -
55mV means that inside of cell becomes slightly more positive and
stimulates opening of sodium channels. Potassium channels only open
when membrane is much more depolarized). These ion channels must be
able to detect change in membrane potential.
INACTIVATION MECHANISM:- ion channels must also be able to shut down
– opening must be discrete to give electrical signals their short, sharp
characteristics and action potentials their characteristic shape.
This lecture focuses on sodium channels…
PURIFICATION and CLONING of a SODIUM CHANNEL!
, 30-40 years ago, biochemists started to try and identify these ion
channels by purifying the channel proteins. This was successfully
completed for sodium channels much earlier than potassium, because of
two tools:
1. Rich source of sodium channels available (tissues of ELECTRICAL EEL) –
have series of electroplaques along their surface that can produce an
electrical discharge, like synapses that make electrical signals but the
signal is instead discharged. These electroplaques are full of ion
channels to create sodium currents.
2. A potent inhibitor to use as a tag: TETRADOTOXIN from the Puffer fish:-
Also need a mechanism to detect protein of interest as it is purified –
useful tag for sodium channel was toxin called tetrodotoxin in puffer
fish.
These 2 natural products were
used to purify the sodium
channel. Protein was
solubilized from membrane
environment and purified by
membrane fractionation and
chromatography. Pure sodium
channel tracked using toxin as
tag.
Cloning the channel was
achieved by taking some of the
protein and microsequencing
(in 1980s this was laborious) –
allowed probe to be designed
to pull out corresponding cDNA
for the whole of that sodium
channel.
Sodium channel sequence turned out to be enormous protein. 2000 amino
acids, 208kDA, glycosylated (a lot of sugars attached, typical of protein
expressed on cell surface)
EXPRESSION OF CLONED NA+ CHANNEL PROTEIN:-
confirms the cloned cDNA encodes a voltage-dependent
Na+ channel. Achieved by expressing the cDNA sequence
in toad oocytes (useful for this purpose because large
eggs, can be seen with naked eye, easy to inject mRNA
run off from cDNA). Egg translates this message into a
protein expressed in the cell membrane.
Electrophysiological recording from these cells utilised to
see if that mRNA gives a sodium channel: top trace is
experimenter depolarizing membrane by applying voltage
to membrane, producing a response current which
persists, declines and shuts down.
:-
This current response could be blocked by the puffer fish toxin
Channels!
The ACTION POTENTIAL!
Occurs in neurons, and other excitable tissues such as cardiac and
skeletal muscle
An influx of sodium ions is followed by an efflux of potassium, producing
the characteristic, reproducible shape of the action potential
depolarisation curve
How is this rapid and reproducible pattern of ion fluxes achieved?
PUMPS versus CHANNELS!
Initial question addressed by
researchers was the mechanism by
which ions are transported across a
membrane, as the lipid bilayer is
impermeable to charged molecules. 2
options:
ION TRANSPORTERS (PUMPS):- pick up
an ion, undergo a conformational
change and release the ion to the
other side of the membrane. Requires
a large amount of energy (ATP) as
ions are transported against their
concentration gradient, and it is a relatively slow process. This mechanism
is not fast enough to produce the rapid rise in ion flux required for the
action potential.
ION CHANNELS:- create a water-filled pore through the membrane, via
which ions flow down their electrochemical gradient (balance of
concentration and charge). A fast process (sufficient to produce the action
potential), but ions can only flow down their gradient rather than against.
CHANNEL PROPERTIES required for action potential!
2 types of channels: one for sodium and one for potassium.
ION SELECTIVITY:- one channel only recognises a particular ion. Sodium
flux happens first, in response to modest depolarization, and potassium
ion channels open later to return membrane potential to resting state.
VOLTAGE DEPENDENCE:– opening of channel only happens at particular
membrane potential (depolarization of the membrane from -80mV to -
55mV means that inside of cell becomes slightly more positive and
stimulates opening of sodium channels. Potassium channels only open
when membrane is much more depolarized). These ion channels must be
able to detect change in membrane potential.
INACTIVATION MECHANISM:- ion channels must also be able to shut down
– opening must be discrete to give electrical signals their short, sharp
characteristics and action potentials their characteristic shape.
This lecture focuses on sodium channels…
PURIFICATION and CLONING of a SODIUM CHANNEL!
, 30-40 years ago, biochemists started to try and identify these ion
channels by purifying the channel proteins. This was successfully
completed for sodium channels much earlier than potassium, because of
two tools:
1. Rich source of sodium channels available (tissues of ELECTRICAL EEL) –
have series of electroplaques along their surface that can produce an
electrical discharge, like synapses that make electrical signals but the
signal is instead discharged. These electroplaques are full of ion
channels to create sodium currents.
2. A potent inhibitor to use as a tag: TETRADOTOXIN from the Puffer fish:-
Also need a mechanism to detect protein of interest as it is purified –
useful tag for sodium channel was toxin called tetrodotoxin in puffer
fish.
These 2 natural products were
used to purify the sodium
channel. Protein was
solubilized from membrane
environment and purified by
membrane fractionation and
chromatography. Pure sodium
channel tracked using toxin as
tag.
Cloning the channel was
achieved by taking some of the
protein and microsequencing
(in 1980s this was laborious) –
allowed probe to be designed
to pull out corresponding cDNA
for the whole of that sodium
channel.
Sodium channel sequence turned out to be enormous protein. 2000 amino
acids, 208kDA, glycosylated (a lot of sugars attached, typical of protein
expressed on cell surface)
EXPRESSION OF CLONED NA+ CHANNEL PROTEIN:-
confirms the cloned cDNA encodes a voltage-dependent
Na+ channel. Achieved by expressing the cDNA sequence
in toad oocytes (useful for this purpose because large
eggs, can be seen with naked eye, easy to inject mRNA
run off from cDNA). Egg translates this message into a
protein expressed in the cell membrane.
Electrophysiological recording from these cells utilised to
see if that mRNA gives a sodium channel: top trace is
experimenter depolarizing membrane by applying voltage
to membrane, producing a response current which
persists, declines and shuts down.
:-
This current response could be blocked by the puffer fish toxin
