The skeletal muscle chloride channel, myotonia congenital and fainting goats Flashcards
What is skeletal muscle made up of ?
elongated fibres that have to contract quickly
What is the transverse tubule?
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
in skeletal muscle how much of membrane conductance is chloride ions ?
70-80%
What would happen if potassium was important for maintaining the resting membrane potential ?
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
What is the resting membrane potential close to ?
it is close to both Ek and Ecl
What is chloride conductance required for ?
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
What gene is defective in myotonic goats and what does it cause?
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
What did Adrian and Bryant do in 1974?
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
What happened when WT and A855P mutant CLC-1 was injected into oocytes?
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
What happens in human myotonia?
delayed relaxation after voluntary contraction
increased excitability
repetitive action potentials
What are the 3 human myotonia diseases?
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
What can placing the hand of a patient with myotonia congenita into cold water do ?
can evoke a myotonic episode
How many people worldwide are affected by myotonia congenita?
1 in 100,000
What sort of a mutation is caused by myotonia congenita?
loss of function mutation
What are the characteristics of myotonia congenita?
muscle stiffness and an inability of the muscle to relax after voluntary contraction
What gene is affected in myotonia congenita?
loss of function mutation in the CLCN1 gene which encodes CLC-1
Where is CLC-1 predominantly expressed and what are its main functions ?
skeletal muscle where it contributes to RMP and reduced excitability
How do we inherit the CLC-1 gene?
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
What happens if you inherit a WT form and a dominant mutant form of the gene ?
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
What happens if you inherit a WT form and a recessive mutant form of the gene ?
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
What happens if the mutation prevents the protein formation (e.g. nonsense)?
50% normal levels of channels
- depending on the physiology the phenotype may be normal or affected
What happens if you have 2 mutant recessive alleles?
it gives a 100% defective CLC-1 channel
How do you determine if you have a recessive or dominant form of the disorder?
it is dependent upon the effect the mutation has on the protein
What does the type of inherited disorder depend on ?
- 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
What was shown when both WT and F307S were injected into oocytes?
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
What was shown when both WT and 1556N were injected into oocytes?
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
What is another dominant mutation in Thomson’s disease ?
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
What is the most common form of adult onset muscular dystrophy ?
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
What happens to the pathogenic mRNAs?
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
What does mis-splicing of CLC-1 introduce?
introduces a premature stop codon, loss of CLC-1 protein and myotonia
What is DMD?
duchenne muscular dystrophy- muscle degeneration
- not caused by CLC-1
What should have been spliced out which causes myotonic dystrophy ?
an extra exon, exon 7a should have been spliced out = it causes a frame shift error leading to a premature stop codon
What are morpholine oligos?
they are synthetic ribonucleotde analogues- they are not digested by nucleases not evoke an immune response
Why might gene therapy be useful for myotonic dystrophy ?
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
What was the outcome of testing the morpholino oligos in transgenic mice overexposing human skeletal actin long repeat?
- 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
What did immunohistochemical staining show about the antisense morpholino treated mice?
it demonstrated greater staining of CLC-1 in the mice treated with antisense morpholino
