The skeletal muscle chloride channel, myotonia congenital and fainting goats Flashcards Preview

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Flashcards in The skeletal muscle chloride channel, myotonia congenital and fainting goats Deck (36)
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1
Q

What is skeletal muscle made up of ?

A

elongated fibres that have to contract quickly

2
Q

What is the transverse tubule?

A

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

3
Q

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

A

70-80%

4
Q

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

A

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

5
Q

What is the resting membrane potential close to ?

A

it is close to both Ek and Ecl

6
Q

What is chloride conductance required for ?

A

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

7
Q

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

A

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

8
Q

What did Adrian and Bryant do in 1974?

A

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

9
Q

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

A

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
10
Q

What happens in human myotonia?

A

delayed relaxation after voluntary contraction
increased excitability
repetitive action potentials

11
Q

What are the 3 human myotonia diseases?

A

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

12
Q

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

A

can evoke a myotonic episode

13
Q

How many people worldwide are affected by myotonia congenita?

A

1 in 100,000

14
Q

What sort of a mutation is caused by myotonia congenita?

A

loss of function mutation

15
Q

What are the characteristics of myotonia congenita?

A

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

16
Q

What gene is affected in myotonia congenita?

A

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

17
Q

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

A

skeletal muscle where it contributes to RMP and reduced excitability

18
Q

How do we inherit the CLC-1 gene?

A

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

19
Q

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

A

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

20
Q

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

A

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

21
Q

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

A

50% normal levels of channels

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

22
Q

What happens if you have 2 mutant recessive alleles?

A

it gives a 100% defective CLC-1 channel

23
Q

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

A

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

24
Q

What does the type of inherited disorder depend on ?

A
  • 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
25
Q

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

A

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

26
Q

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

A

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

27
Q

What is another dominant mutation in Thomson’s disease ?

A

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
28
Q

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

A

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

29
Q

What happens to the pathogenic mRNAs?

A

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

30
Q

What does mis-splicing of CLC-1 introduce?

A

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

31
Q

What is DMD?

A

duchenne muscular dystrophy- muscle degeneration

- not caused by CLC-1

32
Q

What should have been spliced out which causes myotonic dystrophy ?

A

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

33
Q

What are morpholine oligos?

A

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

34
Q

Why might gene therapy be useful for myotonic dystrophy ?

A

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
35
Q

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

A
  • 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
36
Q

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

A

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