Skeletal muscle chloride channels Flashcards
What is the function of the pore gate and the common gate in ClC-1?
Pore gating - opens and closes each pore independently
Common gating - opens and closes both pores simultaneously
What is the main function of ClC-1 channel in skeletal muscle? Describe the properties of ClC-1 channel.
Name two types of myotonia caused by mutations in CLCN1 gene
Recessive generalized myotonia congenita (Becker) Dominant myotonia (Thomsen)
Draw a diagram showing tail currents and explain how they are used to construct open probability curves.
Tail current always taken at same voltage. Tail currents are a hard concept to grasp. Basically you start with a voltage you know will activate all channels. You apply that voltage to a channel and then you quickly apply a voltage below threshold. The current that is measured between these two states gives you the open probability. Because the difference between the two states gives you the current it can determine the open probability of the channel.
How many subunits form a functional ClC-1 channel and how many subunits form ClC-1 pore? How is that different from voltage gated K+ channels?
More than one subunit forms the channel pore while the channel itself is made from two subunits and are dimers, while the voltage gated K+ channel is formed via four identical protein subunits assembled around central pore. Each alpha subunit consists of six transmembrane segments and a P loop that forms the pore.
Draw a diagram showing steady-state open probability curves of WT ClC-1 and a dominant-negative mutant. Label the axis. What proportion of WT ClC-1 channels are opened at resting potential?
.01 Po at -70 mV.
What channels mediate repolarisation phase of action potential in skeletal muscle? What is different compared to repolarisation phase of action potential in a neuronal cell?
In skeletal cells the repolarisation phase is characterised by K+ and Cl- conductance.
In a neuronal cell the repolarisation phase is characterised by K+ only.
What type of response to a depolarising stimulus do you expect to see in skeletal muscle if you block 70% of muscle Cl- conductance?
A depolarisation and excitation followed by a continued depolarisation and excitation due to after discharge occuring. 60-70% of Cl- conductance needs to be blocked in order for myotonia to occur.
How does whole cell conductance depend on single channel conductance and open probability?
Whole cell conductance is the measure of ions going into, or out, of the cell. If a single channel has an extremely high conductance then it will have a direct effect on the whole cell conductance. If a cell has a high open probability with a high conductance then it will have a direct correlation on the whole cell conductance. Whole cell conductance is a measure of both inward and outward current with the open probability of multiple channels.
What types of CLCN1 mutations are always recessive?
All nonsense mutations cause recessive myotonia.
What method was used to clone human muscle Cl- channel?
CLC-1 was cloned by homology screening using cDNA of the chloride channel expressed in the electric organ of the electric ray. The electric organ express very high numbers of chloride channels (CLC-0). Together with ACh receptors they are used to generate an electric discharge (up to 200V).
Which channel has stronger voltage dependence, ClC-1 or voltage gated Na+ channel? Which parameter of Po curve can be used to compare the strength of voltage dependence of gating of different channels? Draw a diagram.
The ClC-1 channel has a stronger voltage dependence. The slope of the Po curve can be used to compare the strength of voltage dependence of gating of different channels. When the slope is steeper it means that it has a stronger voltage dependence.
What is the difference between responses of a normal and myotonic muscle to the same depolarising stimulus?
In a normal muscle the stimulus fires and the muscle cell fires undergoes excitation (depolarisation) then quickly repolarises without a second AP in the duration of the same stimulus. In a myotonic muscle the stimulus fires and the muscle undergoes excitation via depolarisation however there is a long after discharge witnessed and a continued firing of depolarisation is witnessed. The after discharge is the basis for the prolonged contraction.
What are the symptoms of Cl- channel myotonia?
Muscle stiffness, delayed relaxation or cramping (generally painless) upon initial activity or unusual exertion with associated muscle hypertrophy.
What part of ClC-1 channel is affected by the mutations causing dominant myotonia congenita (Thomsen disease)?
Mutations affect the common gating on the channel. Dominant negative mutations are concentrated on the interface between two subunits.
F307S is a dominant mutation and I556N is a recessive mutation. The Po of both these mutants is shifted by about 50 mV to more positive potentials when they are expressed separately. Explain what happens with Po curves when these mutants are expressed in equal amounts with WT ClC-1. Use diagrams of Po curves to demonstrate the difference.
The Po curves indicate that the recessive mutation 1556N actually makes no difference in terms of voltage dependence of the current. The WT and WT + 1556N act similarly in current indicating the recessive has no direct effect on the cell ClC-1 channels. The opposite is seen in F307S mutations. The WT and WT + F307S showed the same +50mV difference as it did when potentials were measured alone. The WT + F307S showed similar changes in voltage dependant current to the pure F307S current indicating that it does effect ClC-1 channels.
