Lecture 2- Voltage Gated Na+ Channel Flashcards
What are 2 electrodes inserted into?
Squid giant axon
What are the 2 types of electrodes?
- Recording voltage difference across membrane
2. Intracellularly injecting current
What are electrodes connected to?
Feedback circuit
What does feedback circuit do?
Compare the measured voltage across the membrane with voltage desired by the experimenter
What happens if 2 values differ?
Current is injected into axon to compensate for the difference
What is continuous feedback cycle?
Voltage is measured and current injected effectively clamps the membrane at a voltage
What happens in a voltage clamp?
The membrane potential is maintained constant or held at a command potential
What is the purpose of making axons bigger?
Conduct impulses more quickly
What does amplifier receive?
Input from inside/outside of axons
What does amplifier detect?
Changes in potential across membrane
What does Na+ channels confer?
Excitability
What depends on sodium channel ?
Nerve cells
Muscle cells
Heart cells
What is present at internode?
Transmembrane current
What current is present at node?
Inward current
What allows activation of the next node?
Current that flows in a circuit
What are necessary to propagate impulse?
Local circuit
What do Na+ channels exhibit?
Voltage- dependent activation
Clamp steps
Start from a negative baseline
Step more positive
What does voltage clamp allow?
Separation of membrane ionic and capacitance currents
What is the whole cell patch clamp arrangement?
Studying the properties of small patch of membrane
What is the process of whole cell patch clamp arrangement?
A glass pipette with a very small opening is used to make tight contact with a very small area of neuronal membrane
2. small suction happens to back of pippete
3. Seal between pipette and membrane become so tight - no ions can flow between pipette and membrane
4. All ions gang flow when single ion channel open flow into pipette
5.
How is the electrical current measured?
Electronic amplifier connected to the pippete
What is depolarising pre-pulse?
Electrical stimulus that causes potential difference measured across membrane to become more positive or less negative
What happens when DPP is short?
Threshold will decrease
What happens when DPP is at a negative value?
All the Na+ channels are available
What happens when DPP is depolarised?
All the Na+ channels are inactivated
What does depolarisation activate?
Available sodium channels
What can be plotted?
Availability of sodium channels
What is conductance ?
How easily current can flow across the membrane
Na+ channels have a similar structure comprising?
Alpha subunit and beta subunits
Alpha subunit
260 Kda
Beta subunit
33-36 Kda
What does alpha subunit have ?
4 repeat domains each containing 6 membrane spanning segments (s1-s6)
What is S4 segment?
Positively charged
Act as voltage gate changing shape when activated
Opening and closing the channel (voltage sensor)
How is inactivation brought?
IFM motif on the linker between domain 3-4
What is IFM motif?
Hydrophobic
When the channel is opened it can be blocked by IFM loop
Giving rise to inactivation
Where are P loops located?
Domain 5 and 6
Response for ion selectivity
What are Beta subunits responsible for?
Neuronal excitability
Cellular adhesion
What does potassium channels have with sodium channel?
Structural homology
What do potassium channels form?
Homo and Hetero multimers
What is the crystal structure for Na+ channel?
Arcobacter butzleri
When do local anaesthetics target sodium channel?
when they are in conformational state
Inactivated
What do local anaesthetics do?
Put sodium gabbros into sleep state preventing them from working
What happens at a negative potential?
All the channels are available
What happens at a positive potential?
All the sodium channels can be put into the inactivated state
What happens in the or sender of LA?
The sodium availability curve shifts left
Less sodium channels available to be activated
What does beta1-subunit accelerate?
Sodium current inactivation in Xenopus oocytes
Cell lines that do not express beta-subunits
What do Beta subunits have?
Large IG loops
What do Beta subunits interact with?
Intercellular and extracellular proteins
Controlling sodium channel localisation
Control of channel density
Where are sodium channels concentrated?
At nodes and initial segments
The very beginning of the axon
What does Beta subunit change?
Rate of inactivation
What does Beta Increase?
Rate of inactivation
What are Na+ channels?
Targets for toxins and drugs
Neurotoxin binding site 1
TTX
STX
Micro-conotoxin
Neurotoxin binding site 2
Veratridine
Batrachotoxin
Neurotoxin binding site 3
Alpha-scorpion toxin
Sea anemone toxin
Neurotoxin binding site 4
Beta-scorpion toxin (outside)
What is tetrodotoxin?
Guaninium group
Mimics sodium paralytic poison found in puffed fish (Californian)
Salamander and a South American fro
May be made by symbiotic bacteria
What are the steps that lead to action potential?
Recording of AP in sensory neuron
Due to stimulus, gradual activation of sodium channel
depolarisation
Action potential
Action potential is around …
25mm long
If there are nodes at 1mm interval, over how many will by involved simultaneously in propagating a single impulse?
20
In the frogs node of Ranvier what channels give rise to an outward current?
Sodium channel
Kinetically fast potassium channel
What does mammalian node of Ranvier do not have?
Fast potassium channels
What does biological membrane provide?
Large capacities
Taken to be 1mFcm-2
What does intermodal membrane provide?
Capacity about 1000 times more than a node of Ranvier
In parallel, what is the total capacity?
100 + 100 + 100 + 100 = 400pf
In series, what is the total capacity?
1/(1/100 + 1/100 + 1/100 + 1/100)
How does the node repolarize?
Myelin has a low capacitance
Barrett and Barrett resistance make myelin a poor insulator because here are current pathways across it
Axolemma
Large capacitance
What happens when DAP occurs?
Current flow across the node
What happens when sodium channel activate?
They charge the internode generating DAP
Current flows in a circuit
Includes a part that goes through and underneath the myelin
Myelin sheath is what?
Leaky
What does myelin provide?
Low capacity sheath
What has a high capacity?
Single intermodal axon membrane
If myelin was a good insulator, why wouldn’t the axon work?
It would not have a resting potential
The action potential at the node couldn’t repolarise
Gigohm resistance
> 1 x10^9
Across a whole myelin sheath (over 100 stacked lamellae)
Close to 50 megaohms
How are axons ensheathed?
Sequentially by single Schwann cells
What does Schwann cells produce?
Basement membrane that included laminin
What is laminin?
A matrix protein that is essential for normal nerve development, function and regeneration
In the CNS, what do oligodendrocytes do?
Wrap axons with myelin
Each oligodendrocyte can interact with more than one axon
What is present underneath the myelin?
Juxta paranode
Consist of fast potassium channels
At the internodal axon
Bigger diameter than at the node
Why does the internodal axon have a bigger diameter than at the node?
Neurofilaments that are phosphorylated in the internode
Make internodal axons thicker/fatter at the node
Alpha subunits
Tethered in position in the node
Beta subunits
Interact with other cell-adhesion molecules
What do Glial CAMS recruit?
Axons CAMs at point of contact
What is expressed by Schwann cells that interact with neurofatin 106?
Gliomedin
What is present at the end of myelin (NF-155) that binds and contacts with Casper?
Paranodal loop
What are Axonal CAMs?
Attachment sites for cyto-skeletal proteins
What channel is the most important for conducting impulses down the axon?
Sodium channel 6, Nav 1.6
Present at the node of Ranvier
Allows conduction of impulses over long distances
What is the major sodium channel found in the brain and is found near the initial segment?
Sodium channel 2
Nav 1.2
Involved in initiating AP close to the cell body before it’s propagated away
TTX-s
Sodium channel sensitive to/ blocked by TTC at nanomolar concentration
TTX-r
resistant to TTx
Famously resistant is cardiac sodium channel 5
What is the sensitivity to TTX determined by?
Amino acid sequence in the P loop
What does TTx Contain?
Guanidinium which is believed to mimic sodium
Positively charged
What is TTX made by?
symbiotic bacteria
NTBS1
Interacts with the pore loop for TTX and saxitoxin
STX (marine toxin, super anaesthetic) - found in algae in a red tide
Algae fed on by shellfish become concentrate with STX
Toxic to eat
NTBS2
Batrachotoxin
Frogs produce this and on feathers in the rubbish bird
Open sodium channels at rest
NTBS3
Alpha scorpion toxin
Prevent sodium channel inactivation
NTBS4
Beta scorpion toxin
Activated the sodium channel at rest
What does mutation in beta subunit cause?
Major effects on how the channels work in the brain
Whole cell patch clamp arrangement
Signal amplifier used
Single wire passes current and measures potential
Respond to command voltage and passes current into neuron
Can measure potential across membrane
Sits inside blunt microelectrode
Can make a seal - slight patch of membrane broken by suction
Solution inside cytoplasm = solution inside electrode
Feedback amplifier
B1
SCN1B
Both CNS and PNS
Epileptic phenotype
Dravet syndrome model
B2
SCN2B
Both CNS and PNS
Reduced excitability and optic nerve function
B3
SCN3B
Known PNS
KI shows important cardiac effects
B4
SCN4B
Both CNS and PNS
Persistent Na* current in purkinje neurons
