Lecture 7 Flashcards
What is myotonia
Hyper-excitability of skeletal muscle, delayed relaxation, muscle stiffness, lots of action potentials or longer action potentials dependent on the channel type mutated.
What gene is mutated in myotonia congenita
CLCN1 coding the CLC1 gene - loss of function mutation
Which gene is mutated in paramyotonia/ K aggravated myotonia
SCN4A codes for Nav1.4 and is a gain of function mutation
What is the CLC1 channel important in maintaining
Maintain the resting membrane potential in skeletal muscle cells
What is the Nav1.4 channels role in skeletal muscle
Action potential firing and skeletal muscle contraction
What happens as a result of Nav1.4 mediated depolarisation
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.
What is the Nernst for Cl
-70mV
What are the two types of myotonia congenita
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.
What is mexiletine
Use dependent Nav channel inhibitor - sub threshold dosage means there is a dampening down of sodium channel conductance
Does CLC1 have more recessive or more dominant mutations and where are they found
More recessive which are found throughout the protein. Dominant mutations also spread throughout the protein
Why is CLC1 an unusual chloride channel
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.
How were CLC1 tail currents used to investigate WT CLC1 channel Po
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.
What percentage of CLC1 channels are open in physiological conditions in skeletal muscle at resting Vm
15%
Which mutant CLC1 channel has a large effect on voltage dependence.
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
Why are studies of the I290M CLC1 mutant channel alone not representative of its effect in human patients
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.
What was the result of a study using a 50/50 mix of WT and I290M mutant CLC1 channels on voltage dependence
Vo is +25mV, at resting potential range, around 0 channels are open which is unexpected (expect 7.5%)
What impact does the I290M mutant CLC1 channel have on WT channels
Has a dominant negative inhibitory effect on the WT subunits within the channel
What is the recessive mutation of CLC1 studied called
E291K
How does the recessive mutant CLC1 channel differ to the dominant ones
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.
What is the result of a full mutant with recessive mutations of CLC1
Complete loss of Cl current
What is K aggravated myotonia
When patients have too much K in their plasma (banana) triggers myotonic symptoms
What is paramyotonia congenita
Autosomal dominant, neonatal to infancy, appears during exercise, worsens with activity, cold sensitive, no hypertrophy.
Where are the Nav1.4 mutations found
Spread across the protein. Cluster in the S4 region of the 4th subunit
How was Na content in vivo investigated
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.
What changed between paramyotonia patients compared control in terms of Na content
intracellular Na content rose in PM patients after cooling and after exercise.
What changed between paramyotonia patients compared control in terms of Vm
Membrane potential became significantly more positive than the volunteers. -40 as oppose to -75
What are the characteristics of the WT Nav1.4 IV curve and patch clamp curve
Fast activation to peak conductance, followed by fast inactivation which is then followed by slow inactivation.
What is the effect of the F1705I Nav1.4 mutation on activation
No impact on activation
What did the tail current protocol of the F1705I mutant show
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
Explain the tail current protocol for measuring inactivation
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.
