Lecture 7

Question 1

What is myotonia

Answer 1

Hyper-excitability of skeletal muscle, delayed relaxation, muscle stiffness, lots of action potentials or longer action potentials dependent on the channel type mutated.

Question 2

What gene is mutated in myotonia congenita

Answer 2

CLCN1 coding the CLC1 gene - loss of function mutation

Question 3

Which gene is mutated in paramyotonia/ K aggravated myotonia

Answer 3

SCN4A codes for Nav1.4 and is a gain of function mutation

Question 4

What is the CLC1 channel important in maintaining

Answer 4

Maintain the resting membrane potential in skeletal muscle cells

Question 5

What is the Nav1.4 channels role in skeletal muscle

Answer 5

Action potential firing and skeletal muscle contraction

Question 6

What happens as a result of Nav1.4 mediated depolarisation

Answer 6

spreads down the T tubule, activate voltage dependent L-type Ca channels which lead to Ca influx into the cell and Ca release from stores via ryanodine leading to contraction of myofilaments.

Question 7

What is the Nernst for Cl

Answer 7

-70mV

Question 8

What are the two types of myotonia congenita

Answer 8

Thomsen
autosomal dominant, infancy and adulthood, mild symptoms, warm up phenomenon (the more they try to use their muscles the more relief they get from their symptoms), some muscle weakness and hypertrophy
Becker
Autosomal recessive, earlier onset, more severe, warm up, some muscle weakness, more hypertrophy.

Question 9

What is mexiletine

Answer 9

Use dependent Nav channel inhibitor - sub threshold dosage means there is a dampening down of sodium channel conductance

Question 10

Does CLC1 have more recessive or more dominant mutations and where are they found

Answer 10

More recessive which are found throughout the protein. Dominant mutations also spread throughout the protein

Question 11

Why is CLC1 an unusual chloride channel

Answer 11

Double barrel Cl channel - CLC1 forms heteromers, one channel has two pores each with a small gate blocking Cl entry. There is also a large gate that is capable of blocking both pores. For any current to pass the large gate must be open, small gates regulate size of the current.

Question 12

How were CLC1 tail currents used to investigate WT CLC1 channel Po

Answer 12

IV curve at different potentials, Use tail currents at same driving force and plot graph (Po/G Vs mV) Calculated Vo and found that half the channels were open at -20mV.

Question 13

What percentage of CLC1 channels are open in physiological conditions in skeletal muscle at resting Vm

Answer 13

15%

Question 14

Which mutant CLC1 channel has a large effect on voltage dependence.

Answer 14

I290M - At resting potential essentially no CLC1 channels are open therefore not contributing to the resting potential making it more positive so more likely for an action potential to fire/ propagation/ muscle contraction

Question 15

Why are studies of the I290M CLC1 mutant channel alone not representative of its effect in human patients

Answer 15

Because it is a dominant mutation. Only one copy of the faulty CLC1 channel being made, 1 healthy copy also being made. 50/50 mix.

Question 16

What was the result of a study using a 50/50 mix of WT and I290M mutant CLC1 channels on voltage dependence

Answer 16

Vo is +25mV, at resting potential range, around 0 channels are open which is unexpected (expect 7.5%)

Question 17

What impact does the I290M mutant CLC1 channel have on WT channels

Answer 17

Has a dominant negative inhibitory effect on the WT subunits within the channel

Question 18

What is the recessive mutation of CLC1 studied called

Answer 18

E291K

Question 19

How does the recessive mutant CLC1 channel differ to the dominant ones

Answer 19

mutant subunits don’t make up the channels, only the WT ones make it that far - but only 50% will do this. 50% at the membrane is sufficient to maintain a normal resting potential.

Question 20

What is the result of a full mutant with recessive mutations of CLC1

Answer 20

Complete loss of Cl current

Question 21

What is K aggravated myotonia

Answer 21

When patients have too much K in their plasma (banana) triggers myotonic symptoms

Question 22

What is paramyotonia congenita

Answer 22

Autosomal dominant, neonatal to infancy, appears during exercise, worsens with activity, cold sensitive, no hypertrophy.

Question 23

Where are the Nav1.4 mutations found

Answer 23

Spread across the protein. Cluster in the S4 region of the 4th subunit

Question 24

How was Na content in vivo investigated

Answer 24

Magnetic resonance imaging of Na23 in muscle.
Compared leg Na content in paramyotonia patients vs 10 normal. Na was measured before and after cooling. Also looked at Vm and intracellular Na on muscle biopsies.

Question 25

What changed between paramyotonia patients compared control in terms of Na content

Answer 25

intracellular Na content rose in PM patients after cooling and after exercise.

Question 26

What changed between paramyotonia patients compared control in terms of Vm

Answer 26

Membrane potential became significantly more positive than the volunteers. -40 as oppose to -75

Question 27

What are the characteristics of the WT Nav1.4 IV curve and patch clamp curve

Answer 27

Fast activation to peak conductance, followed by fast inactivation which is then followed by slow inactivation.

Question 28

What is the effect of the F1705I Nav1.4 mutation on activation

Answer 28

No impact on activation

Question 29

What did the tail current protocol of the F1705I mutant show

Answer 29

Delay in inactivation, taking longer than it should. Activation is the same but it takes a more positive membrane potential before inactivation begins, therefore channels are staying open for longer - extended Ca release and extended muscular contraction

Question 30

Explain the tail current protocol for measuring inactivation

Answer 30

Pre clamp t a number of pre potentials, allow for activation/ inactivation by waiting around 300ms. At this point clamp all the prepotentials to a common potential, therefore any current generated is only due to the number of open channels left, independent of the driving force. Measure the current here for each prepotential and plot each as a fraction of the max current that was recorded. If the current is 0 then all channels were closed at the point of clamping to the same potential. The max current represents when the most channels were open at the point of clamping to the same potential and analysis of the changes in voltage dependent inactivation can be performed.