How ion channels work and go wrong: structure-function studies and channelopathies

Question 1

What internal repeats do the α-subunits of Na+ channels (and also Ca2+ channels) have ?

Answer 1

4 internal repeats of 6-transmembrane domain modules. Each Na+ and Ca2+ channel has one α-subunit

Question 2

K+ channels are composed of?

Answer 2

4 smaller α-subunits, each containing 6 transmembrane domains. This allows for more variety in K+ channels, as they can be heteromultimers of different α-subunits.

Question 3

In addition to the principal α-subunits that determine all their main properties, ion channels may contain?

Answer 3

Auxiliary or regulatory β-subunits

Question 4

What is Tetrodotoxin (TTX) and what does it compete with ?

Answer 4

Tetrodotoxin (TTX) is a large molecule (molecular weight 319) with a positively charged guanidinium group that competes with the hydrated Na+ ion for entry into the channel pore from the extracellular side

Question 5

What does TTX bind to ?

Answer 5

• TTX binds extremely tightly to the Na+ channel, with ~10 nM blocking half the channels

Question 6

What can prevent TTX binding?

Answer 6

Changing a negatively-charged glutamate to a neutral glycine in the linking region between transmembrane domains S5 and S6 of repeat I prevents TTX binding

Question 7

What does the fourth transmembrane domain (S4) in every repeat (I-IV) contain?

Answer 7

Positively charged amino acid residues (arginine or lysine) at every third amino acid position

Question 8

What does the S4 domain form ?

Answer 8

An α-helix that moves through the membrane like a screw, with its position depending on the membrane potential

Question 9

What does the position of all four S4 domains determine ?

Answer 9

The position of all four S4 domains determines whether the channel is open or closed

Question 10

Explain hinged-lid model of fast inactivation ?

Answer 10

• Fast inactivation of Na+ channels does not require charged residues, but depends on 3 adjacent non-polar hydrophobic amino acid residues (isoleucine 1488, phenylalanine 1489, methionine 1490) in the region linking repeats III and IV
• These residues form a hydrophobic ‘hinged lid’ that stabilises the inactivated state of the Na+ channel by binding to the intracellular opening to the pore

Question 11

Three distinct disorders due to mutations in the α-subunit of the human skeletal muscle Na+ channel (SCN4A):

Answer 11

• Potassium-aggravated myotonia (PAM); hyperkalemic periodic paralysis (HPP); paramyotonia congenita (PC). attacks of myotonia (muscle stiffness) during cold weather
• All have dominant inheritance
• Skeletal muscle hyperexcitability (PAM, PC) or hyperexcitability and weakness/paralysis (HPP, some PC)
• Attacks caused by increased [K+] in blood plasma or cold

Question 12

Hyperkalemic periodic paralysis mutation causes incomplete inactivation. Explain this

Answer 12

• Dominant inheritance, attacks of muscle weakness and paralysis triggered by exercise, stress, fasting or eating food rich in K+ions (e.g. bananas)
• Attacks occur when [K+] in blood mildly elevated, from normal 4 mM to 5-8 mM
• Myotonia (stiff muscles) often precedes paralysis
• Inactivation is incomplete due to a point mutation in the region forming the intracellular opening of the pore

Question 13

Whether myotonia or paralysis occurs depends on?

Answer 13

Fraction of Na+ current that fails to inactivate

Question 14

Epilepsy: why is the brain prone to seizure activity?

Answer 14

1. Action potentials are regenerative events relying on positive feedback: inherently unstable
2. A single neuron can fire a train (or trains) of action potentials spontaneously, without any external stimulation (intrinsic excitability)
3. Thus, a network of excitatory neurons connected together in convergent and divergent pathways is potentially explosive:
   - Stimulation of any one cell can lead to a chain reaction due to the progressive spread of activity over a large area
4. To avoid this ‘explosion’, the brain requires at least as much inhibition as excitation, by means of inhibitory synapses

Question 15

What does the Epilepsy represent ?

Answer 15

A hyperexcitation or a failure of inhibitory regulation, either focally (e.g. motor cortex, temporal cortex) or generally (whole cortex at once)

Question 16

Now explain the ion channels and epilepsy: channelopathies?

Answer 16

1. Na+ channel inactivation too slow or incomplete in excitatory neurons
2. Reduced number of functional Na+ channels in inhibitory neurons
3. Reduced number of functional K+ channels in excitatory neurons

Question 17

Ion channel families contributing to epilepsy when mutated:

Answer 17

1. Voltage-gated ion channels:
   Na+ channels, K+ channels and Ca2+channels; hyperpolarisation-activated cyclic nucleotide-gated channels (HCN1)
2. Ligand-gated ion channels:
   - GABA receptors (inhibitory), glutamate receptors (NMDA; excitatory) and acetylcholine receptors (modulatory)