Part 2 molecular and functional characterisation of
voltage-gated Sodium channel
Molecular characterisation of voltage-gated sodium channels
Electric organs of the electric eel Electrophorus electricus are an enriched source of voltage-
gated sodium channels
Electric organs are modified muscle embryologically as derived from muscle
Example of comparative approach
Using affinity chromatography for purification voltage-gated Na channels
Purification of voltage-gated Na channels from
Electrphorus electric organs using TTX for affinity
chromatography yielded approx. 260 kDa protein that is
now known as the alpha sub0unit
In the affinity chromatography colum, there are
immobile beads and tagged to them are tetrodotoxin
Na channels bind to TTX (Na channel blocker) and
become immobilised (cant move) and all the other
proteins pass through, which can be thrown away.
Therefore, TTx binds to the Na channels and purifies
them from the mixture .
Now most proteins in the mixture will fow out but not
the Na channel.
Therefore, add extra TTX to compete with the voltage-
gated Na channel and then the Na chennels will be
eluted off into test tube. Shown by the last tube in the
image
Sequencing the alpha-subunit of the electrophorus
voltage-gated sodium channel
Once the Na channel was purified and isolated, can use
molecular biology techniques to sequence the Na ion
channel
1820 aa divided into 4 homologous domains
Hydropathy analysis indicated 6 membered-spanning helices in each domain.
Hydropathy scale which is based on the hydrophobic and hydrophilic properties of the 20 aa
Aa in the protein was sequenced
Found out that there us a sequence similarity between the 4 homologous domains
When have a long seq look for a pattern
Linear representation of the protein
In the membrane, proteins are 3D in structure
, Voltage-gated Na channels in the brain
Voltage-gated Na channels in the mammalian brain are
comprised of an alpha subunit and2 beta subunits
It is the alpha subunit that forms the pore of the ion channel.
There are beta subunits associated with the voltage-gated Na
channel but we will not focus on them.
The beta subunit helps in regulating the alpha subunit.
The voltage-gated Na channel superfamily
The N and C terminus are located interellularly
Topology of the voltage-gated Na channel shown schematically
S1,2,3,4,5,6, are helices passing through the membrane.
Relating structure to function in voltage-gated sodium channels
Properties of voltage-gated sodium channels:
1. Voltage-sensitive (mechanism of activation
2. Selective permeability to sodium ions
3. Inactivation
1. Voltage sensitive- stimulated to open by a change in membrane potential
How ca a protein be voltage sensitive
voltage-gated Sodium channel
Molecular characterisation of voltage-gated sodium channels
Electric organs of the electric eel Electrophorus electricus are an enriched source of voltage-
gated sodium channels
Electric organs are modified muscle embryologically as derived from muscle
Example of comparative approach
Using affinity chromatography for purification voltage-gated Na channels
Purification of voltage-gated Na channels from
Electrphorus electric organs using TTX for affinity
chromatography yielded approx. 260 kDa protein that is
now known as the alpha sub0unit
In the affinity chromatography colum, there are
immobile beads and tagged to them are tetrodotoxin
Na channels bind to TTX (Na channel blocker) and
become immobilised (cant move) and all the other
proteins pass through, which can be thrown away.
Therefore, TTx binds to the Na channels and purifies
them from the mixture .
Now most proteins in the mixture will fow out but not
the Na channel.
Therefore, add extra TTX to compete with the voltage-
gated Na channel and then the Na chennels will be
eluted off into test tube. Shown by the last tube in the
image
Sequencing the alpha-subunit of the electrophorus
voltage-gated sodium channel
Once the Na channel was purified and isolated, can use
molecular biology techniques to sequence the Na ion
channel
1820 aa divided into 4 homologous domains
Hydropathy analysis indicated 6 membered-spanning helices in each domain.
Hydropathy scale which is based on the hydrophobic and hydrophilic properties of the 20 aa
Aa in the protein was sequenced
Found out that there us a sequence similarity between the 4 homologous domains
When have a long seq look for a pattern
Linear representation of the protein
In the membrane, proteins are 3D in structure
, Voltage-gated Na channels in the brain
Voltage-gated Na channels in the mammalian brain are
comprised of an alpha subunit and2 beta subunits
It is the alpha subunit that forms the pore of the ion channel.
There are beta subunits associated with the voltage-gated Na
channel but we will not focus on them.
The beta subunit helps in regulating the alpha subunit.
The voltage-gated Na channel superfamily
The N and C terminus are located interellularly
Topology of the voltage-gated Na channel shown schematically
S1,2,3,4,5,6, are helices passing through the membrane.
Relating structure to function in voltage-gated sodium channels
Properties of voltage-gated sodium channels:
1. Voltage-sensitive (mechanism of activation
2. Selective permeability to sodium ions
3. Inactivation
1. Voltage sensitive- stimulated to open by a change in membrane potential
How ca a protein be voltage sensitive
