The skeletal muscle chloride channel, myotonia congenital and fainting goats

Question 1

What is skeletal muscle made up of ?

Answer 1

elongated fibres that have to contract quickly

Question 2

What is the transverse tubule?

Answer 2

invaginations from the sarcolemma around contractile apparatus and sarcoplasmic reticulum (calcium release sites): excitation-contraction coupling
- allows action potentials to be propagated round and through and into the skeletal muscle to cause rapid spread of excitation through the muscle

Question 3

in skeletal muscle how much of membrane conductance is chloride ions ?

Answer 3

70-80%

Question 4

What would happen if potassium was important for maintaining the resting membrane potential ?

Answer 4

it would build up in the extracellular space during sustained activation which would make the Ek less negative causing collapse of the potassium gradient
- therefore voltage gated chloride channels are important because they become more as depolarisation occurs

Question 5

What is the resting membrane potential close to ?

Answer 5

it is close to both Ek and Ecl

Question 6

What is chloride conductance required for ?

Answer 6

it is required for depolarisation and stabilising the membrane potential
- this acts as a safety mechanism to prevent depolarisation as a result of potassium accumulation in the T-tubule system - this is only used in skeletal muscle

Question 7

What gene is defective in myotonic goats and what does it cause?

Answer 7

CLCN-1 it is a loss of function mutation
- goats have difficultly relaxing skeletal muscle
- they faint when they are startled or excited
- muscle becomes deeply tendorised - good for burgers
the symptoms are analogous to humans

Question 8

What did Adrian and Bryant do in 1974?

Answer 8

intracellular recording from goat skeletal muscle
- isolated skeletal muscle from normal goats and myotonic goats
- normal goats= as depolarising current increases there was a greater number of action potentials and at the highest depolarisation action potentials still occurred even after the stimulation had stopped
removal fo Cl- from the bathing saline of muscle fibres caused the normal goats to act like myotonic goats
therefore this implied that the myotonic goats must handle cl- differently

Question 9

What happened when WT and A855P mutant CLC-1 was injected into oocytes?

Answer 9

2 electrode voltage clamp electrophysuilogy was carried out

• at rmp there is about 30% less activity in cl-channels in myotonic gene compared to wt
• this will alter ap threshold firing and also affect r repolarisation

Question 10

What happens in human myotonia?

Answer 10

delayed relaxation after voluntary contraction
increased excitability
repetitive action potentials

Question 11

What are the 3 human myotonia diseases?

Answer 11

thomson’s disease = autosomal dominant myotonia congenita
backer’s disease= recessive generalised myotonia congenita
myotonic dystrophy = DNPK- not caused by mutations in CLCN1 but a knock on effect from another gene

Question 12

What can placing the hand of a patient with myotonia congenita into cold water do ?

Answer 12

can evoke a myotonic episode

Question 13

How many people worldwide are affected by myotonia congenita?

Answer 13

1 in 100,000

Question 14

What sort of a mutation is caused by myotonia congenita?

Answer 14

loss of function mutation

Question 15

What are the characteristics of myotonia congenita?

Answer 15

muscle stiffness and an inability of the muscle to relax after voluntary contraction

Question 16

What gene is affected in myotonia congenita?

Answer 16

loss of function mutation in the CLCN1 gene which encodes CLC-1

Question 17

Where is CLC-1 predominantly expressed and what are its main functions ?

Answer 17

skeletal muscle where it contributes to RMP and reduced excitability

Question 18

How do we inherit the CLC-1 gene?

Answer 18

both maternal and paternal inherited CLCN1 genes are inherited and co-expressed in skeletal muscle cells
- the channel is a dimer with each subunit having its own conducting pore

Question 19

What happens if you inherit a WT form and a dominant mutant form of the gene ?

Answer 19

25% of the channels will be both WT
25% of the channels will be both M
50% of the channels with have a WT and a M subunit
- if the protein defect persists in the mixed dimer the mutation will be dominant and therefore 25% of the channels will be normal and 75% of the channels will be defective

Question 20

What happens if you inherit a WT form and a recessive mutant form of the gene ?

Answer 20

25% of the channels will be both WT
25% of the channels will be both M
50% of the channels will have a WT and a M subunit
- if protein function of the mixed dimer is normal the mutation is recessive and 1 mutant allele is not sufficient to cause disease
- 75% normal channels and 25% defective channels

Question 21

What happens if the mutation prevents the protein formation (e.g. nonsense)?

Answer 21

50% normal levels of channels

- depending on the physiology the phenotype may be normal or affected

Question 22

What happens if you have 2 mutant recessive alleles?

Answer 22

it gives a 100% defective CLC-1 channel

Question 23

How do you determine if you have a recessive or dominant form of the disorder?

Answer 23

it is dependent upon the effect the mutation has on the protein

Question 24

What does the type of inherited disorder depend on ?

Answer 24

• what the mutation does to the protein, particularly in a multimer
• how much loss of protein can be tolerated until disease arises- need at least 25% of normal functioning protein

Question 25

What was shown when both WT and F307S were injected into oocytes?

Answer 25

when these genes were together or if it was just F307S the currents were shifted to the right
- this indicates that the mutation is dominant

Question 26

What was shown when both WT and 1556N were injected into oocytes?

Answer 26

only the 1556N produced currents that were shifted to the right indicating that 1556N is a recessive mutation
- WT expression alonside 1556N is sufficient to rescue the response

Question 27

What is another dominant mutation in Thomson’s disease ?

Answer 27

G230E mutation = glycine to glutamate mutation

• didn’t cause a LOF and didn’t change the voltage dependence but it changed the ion selectivity
• it increased the amount of sodium that can pass through the channel- this causes depolarisations and further contraction

Question 28

What is the most common form of adult onset muscular dystrophy ?

Answer 28

defect in the DMPK= dystrophia myotonia protein kinase
DM1= myotonic dystrophy type 1
caused by CTG expansion in the 3’UTR region of DMPK
- this causes the mRNA to be overly long= pathogenic mRNA

Question 29

What happens to the pathogenic mRNAs?

Answer 29

they accumulate in the nucleus and interfere with splicing of pre-mRNAs including CLC-1
- splicing in the nucleus doesn’t work properly due to large mRNA molecules

Question 30

What does mis-splicing of CLC-1 introduce?

Answer 30

introduces a premature stop codon, loss of CLC-1 protein and myotonia

Question 31

What is DMD?

Answer 31

duchenne muscular dystrophy- muscle degeneration

- not caused by CLC-1

Question 32

What should have been spliced out which causes myotonic dystrophy ?

Answer 32

an extra exon, exon 7a should have been spliced out = it causes a frame shift error leading to a premature stop codon

Question 33

What are morpholine oligos?

Answer 33

they are synthetic ribonucleotde analogues- they are not digested by nucleases not evoke an immune response

Question 34

Why might gene therapy be useful for myotonic dystrophy ?

Answer 34

using the morpholino oligos with a complimentary sequence to exon 7a you can bind it to the extra exon and therefore block it joining the mRNA sequence so it produces normal coding for CLC-1

• It anneals to pre-mRNA or mRNA to block protein interaction
  e. g.in ribosome or spliceosome

Question 35

What was the outcome of testing the morpholino oligos in transgenic mice overexposing human skeletal actin long repeat?

Answer 35

• its an expanded CUG repeat = model of myotonic dystrophy
• recorded currents and they were small in this model but they the antisense morpholino the currents returned to WT levels

Question 36

What did immunohistochemical staining show about the antisense morpholino treated mice?

Answer 36

it demonstrated greater staining of CLC-1 in the mice treated with antisense morpholino