Lecture 15

Question 1

What are the three characteristics of Voltage-gated Na+ channels that can be identified with patch-clamp method?

Answer 1

1) Open with little delay
2) Stay open for about 1 minute
3) Cannot be open again by depolarization.

Question 2

What is the structural correlate of absolute refractory period?

Answer 2

Inactivation of voltage-gated Na+ channels.

Question 3

Describe the transition from one state to another in Voltage-Gated Na+ channels:

Answer 3

Na+ channels open rapidly from a resting state in response to membrane depolarization, however if depolarization is maintained Na+ channels exit open state and enter the inactivated state.

Question 4

How many gates do Na+ channels in axons have? Name them:

Answer 4

2, activation and inactivation gates.

Question 5

What is the main feature of Na+ entry into the cell?

Answer 5

It is characterized by a positive feedback loop and requires intervention to stop. Inactivation gates close in delayed response to depolarization, interrupting the escalating positive feedback loop.

Question 6

What is the amount of Na+ current correlated to?

Answer 6

The amount of Na+ current is proportional to the umber of open channels and is dependent upon the magnitude of depolarization.

Question 7

What determines the number of Na+ channels available at any given membrane potential?

Answer 7

The process of inactivation determines the number of Na+ channels available to open at any given membrane potential.

Question 8

Describe the functions of local anaesthetics:

Answer 8

Prevent action potential propagation of nerve axons by blocking voltage-gated Na+ channels.

Question 9

Describe the structure of local anaesthetics:

Answer 9

They are small lipid soluble molecules and as such they cross nerve sheath and cell membrane to reach the site of action. They consists of an aromatic group linked by an amide or ester bond to a basic side chan, and at physiological pH they are mostly charged (+).

Question 10

Name three clinically useful local anaesthetics:

Answer 10

1) procaine
2) lignocaine
3) bupivacaine

Question 11

Why are permanently charged derivates of LAs such as QX 314 ineffective?

Answer 11

Because they cannot penetrate nerve cell membrane.

Question 12

What is the blocking action of LAs dependent upon?

Answer 12

It is dependent on the Na+ channel being in the open state and the drug only blocks it from the inside (use-dependent block). Block is also voltage-dependent (depolarization enhances block). LAs also appear to enhance the Na+ inactivation process, stabilising it.

Question 13

Describe tetrodotoxin:

Answer 13

Is a naturally occuring, virulent poison that blocks nerve conduction and cause death by respiratory paralysis. It is found in certain integral organs of the pacific puffer fish (ovaries, liver).

Question 14

How does TTX act?

Answer 14

It blocks voltage-activated Na+ channels of nerves and skeletal muscle in nanomolar range, but cardiac Na+ are much less sensitive (micromolar range). It blocks Na+ channels from outside of the channel and has no effect when applied from the inside. This suggests that it binds to amino acid residues associated with the outer mouth of the channel.

Question 15

What is the peculiarity of one particular residue on the P-loop of Na+ channels?

Answer 15

If it mutates the gluatamate (E) to glutamine (Q) (negative to neutral) nM sensitivity is lost and 1 μM TTX does not cause block. Other residues in the P loop also contribute.

Question 16

Describe saxitoxin:

Answer 16

Has properties similar to those of TTX and blocks Na+ channels at the same time. One of a group of toxins produced by a dinoflagellates. If ingested by humans can cause paralytic shellfish poisoning.

Question 17

Describe μ-Conotoxin:

Answer 17

Constituents of venom of the group of predatory molluscs, the cone shells (conus). Injected into prey via harpoon-like disposable tooths, prey are paralysed and die. They are small positive charged peptides and one in particular μ-conotoxin GIIA selectively blocks skeletal muscle Na+ channels (little effect on neuronal Na+ channels).

Question 18

What are the three neurotoxins that modulate Na+ channels to keep it open for longer periods, leading to repetitive firing of the neuron?

Answer 18

• Batrachotoxin
• Pyrethrins
• b-scorpion toxin
• sea anemone and a-scorpion toxins

Question 19

Describe batrachotoxin:

Answer 19

Is secreted by the skin of Columbian poison frogs. It inhibits inactivation and shifts the activation voltage to more negative potentials so channels stay open for longer. It enters the cell and acts internally. Also found in the skin and feather of a bird genus (Pitohui).

Question 20

Describe pyrethrins:

Answer 20

Natura insecticides produced by plants. Non-toxic for mammals but have rapid effects on insect Na channels- prolong activation and inhibit inactivation. Similar to DDT.

Question 21

Describe b-scorpion toxins:

Answer 21

Binds to the outer side of the IIS4 voltage sensor. Toxin binding alone has no effect on activation but when the channel is activated by depolarization, the bound toxin enhances activation by negatively shifting the voltage dependence (activation at more negative potentials).

Question 22

Describe sea anemone and a-scorpion toxins:

Answer 22

Uncouple activation from inactivation by binding to a receptor site at the extracellular end of the IVS4 segment and preventing its normal gating movement. As upward movement of IVS4 is thought to initiate fast inactivation, the channel remains in an activated ‘gated’ state.

Question 23

Describe Na+ channel auxillary (β)- subunits structure:

Answer 23

There are 4 β subunit genes (β1-4) that have similar structures but are separate proteins. The extracellular domain of both proteins has an immunoglobulin-like fold.

Question 24

What is the function of Na+ channel auxillary (β)- subunits structure:

Answer 24

They modulate channel gating allowing rapid activation and inactivation. Mutation of β1 subunit is associated with epileptic seizures. The immunoglobulin domains are thought to bind extracellular proteins and be important determinants of channel localisation in cells.

Question 25

How many sodium channels α subunit genes are there in the human genome

Answer 25

9 α subunits genes.

Question 26

What are the subtypes of α subunit gnes that predominate in the CNS and what is their peculiarity?

Answer 26

Nav 1.1, 1.2, 1.3 and are sensitive to nM TTX.

Question 27

What are the two TTX-resistant isoforms of α subunit?

Answer 27

1.8 and 1.9 they are found in PNS and are expressed in small-diameter sensory dorsal root ganglion neurons including C-fibers which transmit nociceptive pain. These Na+ channels are thought to sustain repetitive firing of depolarised nerves, suggestin that changes in their expression is involved in patophysiology of chronic inflammatory pain.

Question 28

What are some evidence supporting the idea that NaV 1.8 and 1.9 are involved in the patophysiology of chronic inflammaotory pain?

Answer 28

1) 1.8 and 1.9 are increased in nerve fibers proximal to site of injury in humans.
2) Reduced expression of NaV1.8 attenuates neuropathic pain
3) Mice lacking NaV 1.8 are analgesic to noxious mechanical stimuli.

Question 29

Where is NaV1.7 expressed?

Answer 29

It is expressed selectively in dorsal root ganglion neurons, particularly nociceptive cells.

Question 30

What is the function of NaV1.7?

Answer 30

To set gain for pain in nociceptors: NaV1.7 KO ice have increased mechanical an thermal pain threshold and reduced inflammatory pain responses.

Question 31

Describe a gain of function mutation in NaV1.7:

Answer 31

It is the cause of primary erythomelalgia, an autosomal dominant, inherited disorder, severe burning sensation and redness in extremities in response to mild thermal stimuli.

Question 32

What symptoms do humans with mutations that engender loss of NaV1.7 function show?

Answer 32

They are unable to feel pain, acute or chronic.

Question 33

What interesting feature do grasshopper mouse show?

Answer 33

They preys on bark scorpion and appears immune to its toxin that induces pain in many other rodents and in humans.

Question 34

What does pain perception involves and what conclusion has been drawn from studies of the grasshopper mouse?

Answer 34

Pain perception involves TTX-sensitive NaV1.7 and TTX resistant NaV 1.8 and 1.9.
Typically venom activates 1.7 but in the case of grasshopper mouse venom binds to 1.8 and inactivates it.

Question 35

Describe the link between myotonias, periodic paralyis and Na+ channels:

Answer 35

Various mutations in the NaV1.4 Na+ channel cause episodic and transiet weakness or paralysis or relaxation defects. Mutations may affect fast inctivation gate causing channels to have slower inactivation kinetcis and faster recovery from inactivation resulting in delayed muscle relaxation.

Question 36

Why may myotonias and pariodic paralysis be aggravated by consumption of potassium rich food?

Answer 36

Missense mutations of positive charged residues in the voltage sensor lead to hypoexctiability or inexctiability and inability to depolarize muscle. Sodium channels may als open after a delay and cause late depolarization and firing.

Question 37

What are the two cardiac and neuronal sodium channelopathies?

Answer 37

Ventricular arrhythmia and Inhertied epilepsy syndrome.

Question 38

Describe ventricular arrhytmia:

Answer 38

Congenital long QT syndrome (10% of cases) and idiopathic ventricular fibirllation. Observe a prolonger ventricular action potential duration. This is due to a persistent inward Na+ current causing abnormal repolarization.

Question 39

What is a possible cause of ventricular arrhytmia?

Answer 39

Various mutation of α subunit of Nav1,5 observed.

Question 40

Describe the possible cause of inherited epilepsy syndrome:

Answer 40

Many mutations (>100) found in α subunits (Nav 1.1 and Nav 1.2) and some associated with β1 subunits.

Question 41

What are the symptoms of inherited epilepsy syndrome:

Answer 41

The mutations induce persistent sodium currents or alter voltage dependence of activation/inactivation causing enhanced excitability.