6. Excitable Cells: Neural Communication

Question 1

Define the membrane potential.

Answer 1

The electrical potential difference that exists between the inside of a cell and its surrounfdings.

Question 2

How can membrane potentials be studied?

Answer 2

Using glass microelectrodes.

Question 3

Describe which cations and anions have higher concentration intracellularly and extracellularly.

Answer 3

Higher intracellularly:

• Potassium
• Phosphate
• Protein
• Magnesium
• Hydrogen

Higher extracellularly:

• Sodium
• Chloride (NOTE: Varies a lot intracellularly)
• Calcium
• Hydrogencarbonate

Question 4

What are the two types of gradient involved in membrane potentials?

Answer 4

• Chemical gradients
• Electrical gradients

Question 5

What is the equilibrium potential?

Answer 5

For each ion, it is the potential at which the chemical gradient balances the electrical gradient so that there is no movement of ions through the respective channel.

Question 6

What is another name for the equilibrium potential?

Answer 6

The reversal potential.

Question 7

In a neurone, what is the equilibrium potential for potassium, sodium and chloride ions?

Answer 7

• Potassium = -90mV
• Sodium = +58mV
• Chloride = -60mV

Question 8

What is the symbol for the membrane potential?

Answer 8

Em

Question 9

What is the symbol for the equilibrium potential of potassium and sodium?

Answer 9

• Potassium = E_K
• Sodium = E_Na

Question 10

Describe briefly how the membrane potential relates to the equilibrium potential of the different ions.

Answer 10

• The membrane potential is a balance between the different equilibrium potentials
• The membrane potential will be closer to the equilibrium potential of the ions with higher permeability

Question 11

Describe why the resting membrane potential is closer to the potassium equilibrium potential than the sodium equilibrium potential.

Answer 11

The permeability of the membrane to potassium is much higher than to sodium.

Question 12

What happens to the membrane potential when the permeability to a given ion is increased?

Answer 12

The membrane potential tends towards the equilibrium potential of that ion.

Question 13

What equation can be used to calculate the membrane potential at any time?

Answer 13

Constant Field Equation (a.k.a. Goldman, Hodgkins & Katz equation)

NOTE: This is not core material.

Question 14

State the Constant Field Equation (GHK equation).

Answer 14

Question 15

What equation can be used to calculate the equilibrium potential for an ion?

Answer 15

Nernst equation

Question 16

State the Nernst equation.

Answer 16

Question 17

In the Nernst equation, when are the concentrations taken?

Answer 17

At equilibrium

Question 18

When drawing models of cell membranes, what is it important to remember about the ions?

Answer 18

The ions of each side should usually ensure osmotic equilibrium.

Question 19

What is the effect of increasing extracellular potassium concentration (K⁺_o) on the membrane potential? Describe an experiment that showed this.

Answer 19

• As the extracellular potassium concentration increases, the membrane potential increases
• In Hodgkin and Horowicz’s 1959 experiment, this is a linear increase (according to the Nerst equation), but below 10mM potassium concentration, the line slopes off to the right -> This is due to some slight permeability of the membrane to sodium ions

Question 20

State how the Nernst equation simplifies at RTP.

Answer 20

Question 21

How can the effect of changing the extracellular concentration of an ion on the membrane potential be predicted?

Answer 21

By looking at the Constant Field equation.

Question 22

What is the Gibbs-Donnan effect?

Answer 22

The movement olf charged particles near a membrane due to charged particles that are impermeable to the membrane, but can still exert an electric force.

Question 23

Describe the double-Donnan distribution of cell membranes at rest.

Answer 23

• Intracellularly, there are negatively-charged proteins.
• Extracellularly, there is an excess of sodium ions.
• Both of these contribute to electric forces, but also ensure an osmotic equilibrium since the total number of ions intra and extracellularly should be roughly equal.

https://derangedphysiology.com/main/cicm-primary-exam/required-reading/cellular-physiology/Chapter%20121/gibbs-donnan-effect

Question 24

Describe the pump-leak model of ion homeostasis.

Answer 24

• An Na⁺/K⁺-ATPase is used to pump 3 sodium ions out of the cell for every 2 potassium ions being pumped in
• This counteracts the leakage of sodium and potassium through leak channels and help to maintain constant concentrations at rest

Question 25

In terms of homeostasis, what is the importance of the Na⁺/K⁺-ATPase moving sodium out of the cell?

Answer 25

Maintaining the extracellular sodium concentration is important because it helps to maintain osmotic equilibrium.

Question 26

Describe how the action of the Na⁺/K⁺-ATPase is adapted to different conditions.

Answer 26

The graph for the rate of sodium efflux against the intracellular sodiu concentration is a sigmoidal curve, showing how the pump is smart and responds to the concentration of sodium.

Question 27

What word describes the Na⁺/K⁺-ATPase?

Answer 27

Electrogenic

Question 28

What is Ouabain and what is its effect on resting membrane potential?

Answer 28

Ouabain is an inhibitor of Na⁺/K⁺-ATPases, so it causes slow depolarisation of the membrane.

Question 29

How does the Na⁺/K⁺-ATPase respond to an injection of sodium into a cell?

Answer 29

• The injection causes hyperpolarisation (since E_Na is reduced)
• The Na⁺/K⁺-ATPase increases this hyperpolarisation by increasing its action so that the membrane potential is hyperpolarisaed further
• If ouabain is injected prior to the sodium, the downwards dip is much smaller, showing that the Na⁺/K⁺-ATPase increases this hyperpolarisation

Question 30

What is the effect of these on cell membrane potential:

• Increase in extracellular potassium
• Increase in intracellular sodium

Answer 30

• Increase in extracellular potassium -> More positive Em
• Increase in intracellular sodium -> More negative Em

(In some ways this is not intuitive, but think about it in terms of the equilibrium potential of each ion changing according to the Nernst equation)

Question 31

Describe how the resting membrane potential arises in cell membranes.

Answer 31

• The outwards potassium current (through potassium leak channels) combines with the outwards current from Na⁺/K⁺-ATPase to balance the inward sodium leak currents.
• The Na+/K+-ATPase works by moving three sodium ions out of the cell for every two potassium ions moved into the cell (using energy from the hydrolysis of ATP), so it is electrogenic, but it also helps maintain the sodium and potassium gradients that are being run down by action potentials.
• There may also be some contribution from inwards chloride fluxes (contributing to the potassium and ATPase), but this is a small effect.

Question 32

What is the best way to visualise the effect of changing intracellular or extracellular ion concentrations?

Answer 32

I think of it this way:

• If the concentration on one side is increased, then there will be more flow to the other side, causing the potential to change in the way that is intuitive from there

Question 33

State Ohm’s Law in terms of conductances.

Answer 33

I = Vg

(where g = conductance)

This is because g = 1/R.

Question 34

Describe how the size of the current of a single ion across a membrane can be determined.

Answer 34

For potassium (for example):

I_K = (E_m - E_K) x g_k

Question 35

Draw an electrical analogue of a cell membrane.

Answer 35

Question 36

State the conductance equation.

(Note: Not core material)

Answer 36

Question 37

State the equation for the charge stored on a membrane and how this is used.

Answer 37

Q = VC

Where:

• Capacitance of biological membranes = 1μ/cm

This means that the charge stored on a membrane is very very small per unit surface area.

Question 38

Draw the shape of an action potential.

Answer 38

Question 39

What peak does an action potential typically reach?

Answer 39

30-40mV

Question 40

What is a typical resting membrane potential?

Answer 40

About -70mV

Question 41

What is a typical membrane potential during the hyperpolarisation of an action potential?

Answer 41

-90mV

Question 42

What is a typical threshold for an action potential?

Answer 42

-55mV

Question 43

Describe the events underlying an action potential.

Answer 43

• Rapid voltage-dependent activation of voltage-gated Na⁺-channels
• Influx of sodium ions leads to membrane depolarisation
• Rapid depolarisation due to positive feedback opening more voltage-gated Na⁺-channels
• Inactivation of voltage-gated Na⁺-channels
• Depolarisation also triggered voltage-gated K⁺-channels (delayed rectifier), which open more slowly
• Efflux of potassium leads to membrane repolarisation
• Hyperpolarisation occurs due to the delayed closing of potassium channels
• Repolarisation causes the sodium channels to go from inactivated to closed state

Question 44

Draw the conduction curves for sodium and potassium during an action potential.

Answer 44

These occur due to the opening of voltage-gated sodium and potassium channels.

Question 45

What type of potassium channels are involved in an activation potential?

Answer 45

Delayed rectifier

Question 46

State some evidence for the ionic mechanism of action potentials.

Answer 46

• Sodium-free solution -> No AP possible (graded reduction in sodium produces reduction in overshoot)
• Radiotracers -> Show entry of sodium and potassium
• Voltage-clamp and patch-clamp techniques
• Genes and proteins identified for sodium and potassium channels (+ X-ray crystallography shows structure and function)

Question 47

What is the differences between a voltage-clamp and patch-clamp?

Answer 47

A voltage-clamp is concerned with an entire membrane, while a patch-clamp aims to study a single channel.

Question 48

Describe how a voltage-clamp can be used to study ionic currents in an action potential.

Answer 48

• The membrane potential is measured
• A required membrane voltage is input
• The two are compared and current is injected proportional to the difference, so that the membrane is maintained at a given potential
• The current injected is equal and opposite to the currents through channels in the membrane
• The currents through a single type of channel can be studied by using channel blockers of the other channels

Question 49

What are the blockers of voltage-gated sodium and potassium channels?

Answer 49

• Tetradotoxin (TTX) -> Sodium channel blocker
• Tetraethylammonium (TEA) -> Potassium channel blocker

Question 50

Draw the voltage-current graphs for sodium and potassium channels (involved in an action potential).

Answer 50

The x-intercept of sodium shows the reversing potential.

Question 51

Why is an action potential all or nothing?

Answer 51

The initial depolarisation must open enough voltage-gated sodium channels to trigger the rapid positive feedback explosion that leads to full depolarisation.

Question 52

Where is positive and negative feedback seen in an action potential?

Answer 52

• Positive feedback -> Rapid opening of sodium channels
• Negative feedback -> The opening of potassium channels leading to the repolarisation that causes them to close

Question 53

Describe why the conductance of the membrane to sodium decreases after some time in the action potential.

Answer 53

• The voltage-gated sodium channels inactivate after some time
• This means that they do not allow sodium to pass through them
• The channels can only recover from inactivation when the membrane repolarises

Question 54

What are the three states of voltage-gated sodium channels in an action potential?

Answer 54

Open, closed, inactivated

Question 55

When can recovery of sodium channels from inactivation occur?

Answer 55

When:

• The membrane repolarises (AND)
• Sufficient time has passed

Question 56

What are the two phases of the refractory period?

Answer 56

• Absolute refractory period
• Relative refractory period

Question 57

What is the absolute refractory period and what causes it?

Answer 57

The first part of the refractory period when not enough sodium channels have recovered from inactivation to allow for another action potential to be triggered. No AP is possible at all.

Question 58

What is the relative refractory period and what causes it?

Answer 58

The second part of the refractory period where sufficient sodium channels have recovered for an action potential to be triggered, but the threshold for the stimulus is higher than usual.

Question 59

Describe the patch-clamp technique used in action potential experiments.

Answer 59

• Glass pipette is pressed against membrane around a single channel and suction is applied
• Electrically tight seal is formed
• When pulled away, the patch of membrane along with the channel is excised and held in the pipette tip

Question 60

Describe a patch-clamp experiment to show the activity of sodium and potassium channels in an action potential.

Answer 60

When the membrane is suddenly depolarised:

• Sodium channels exhibit a single, short inwards current (before becoming inactivated), which is almost immediate
• Potassium channels open randomly and stay open, so the outwards current starts at any point and is prolonged until repolarisation occurs

Question 61

Describe the features of a voltage-gated sodium channel.

Answer 61

Transmembrane protein with 4 transmembrane domains:

• Pore through centre with a narrow region that acts as a selectivity filter
• Charged, moving, voltage-sensing region
• Activation gate coupled to the voltage-sensing region with inactivation particle

Question 62

Remember to read up on potassium channel structure.

Answer 62

Do it.

Question 63

Draw a graph to demonstrate the absolute and relative refractory period on an action potential.

Answer 63

Question 64

Explain this graph.

Answer 64

• Point A is the normal resting potenetial where there is no net flow of current.
• The arrows towards A show how the membrane will equilibrium to that potential below the threshold.
• Point B is the threshold, above which the membrane potential does not return to A, but instead there is a rapid net inward current that depolarises the membrane.

Question 65

Draw a diagram of a neuron.

Answer 65

Question 66

Describe how an action potential is propagated in an unmyelinated axon.

Answer 66

• Sodium influx in an active patch of membrane generates a strong local depolarisation
• Current flows from the active region along the axon, depolarising adjacent regions of the axon
• This depolarisation leads to the triggering of an AP in these parts of the membrane, which regenerates the strength of the signal

Question 67

In an unmyelinated axon, why is there a need for the constant regeneration of the action potential?

Answer 67

There is some outward leak from the axon, so the depolarisation is weakened and must be constantly regenerated by the opening of sodium channels (i.e. triggering new APs).

Question 68

Describe the local currents that exist during impulse propagation in an unmyelinated axon.

Answer 68

The outwards leak currents complete local currents that include flow in both forward (orthodromic) and backward (antidromic) directions.

Question 69

What is the clinical relevance of local currents in action potential propagation?

Answer 69

Local currents lead to changes in surface potential, which can be detected by ECGs. Extracellular electrodes can also be used in experiments to monitor the propagation of signals.

Question 70

What is some experimental evidence for the local currents that exist during action potential propagation?

Answer 70

• A nerve axon can be fixed with recording electrodes at different points along it
• A block is placed on the axon between the first two recording electrodes
• The size of the depolarisation is massively reduced between the first two recording electrodes, and degrades more at the subsequent ones

Question 71

What is the name for the flow of charge along an axon without propagation?

Answer 71

Electronic conduction

Question 72

What is the name for the way in which an axon can be modelled electrically?

Answer 72

Cable theory

Question 73

Describe cable theory of axons, with the help of a diagram.

Answer 73

The membrane can be split into a series of sections, each with:

• R_m = Membrane resistance
• R_l = Longitudinal resistance -> Due to resistance from the cytosol
• C_m = Membrane capacitance

Question 74

What are the two main passive properties of a membrane that affect conduction velocity?

Answer 74

• Time constant (τ)
• Length constant (λ) - The distance a potential can travel along a neurone before it degrades to 37% of the original amplitude

Question 75

For the length constant, state:

• Its symbol
• The definition
• What it is mathematically equal to

Answer 75

• λ
• The distance a potential can travel along a neurone before it degrades to 37% of the original amplitude.
• λ = √(R_m/R_l)

Question 76

For the time constant, state:

• Its symbol
• The definition
• What it is mathematically equal to

Answer 76

• τ
• A measure of how quickly the membrane potential is able to rise or fall (i.e. the time for the membrane potential to fall to 37% of its original)
• τ = R_m x C_m

Question 77

How can the length and time constant be represented on a graph?

Answer 77

• The graphs are voltage-distance and voltage-time respectively.
• Both show an exponential decrease, with the constant being the distance or time for the voltage to fall to 37%.

Question 78

Name the main factors that affect conduction velocity in nerves.

Answer 78

• Myelination
• Axon diameter
• Temperature
• Other factors (e.g. age)

Question 79

Describe the structure of a myelinated axon and how conduction along it works.

Answer 79

• The membrane of a Schwann cell wraps several times around an axon, creating a myelin sheath
• The gaps between the Schwann cells are nodes of Ranvier, in which voltage-gated channels are concentrated
• Between nodes of Ranvier, the conduction is purely electrotonic
• The nodes act as regenerating stations, so that the signal is reinforced

Question 80

Describe how myelination affects conduction velocity.

Answer 80

• Myelination increases conduction velocity
• This is because it increases the resistance of the membrane (R_m), so that the length constant is increased -> λ = √(Rm/Rl)
• Increasing the length constant increases the conduction velocity because more distance areas ahead of the impulse can be depolarised to threshold
• Although the C_m is decreased, the time constant is unaffected because the increase in R_m is roughly proportional to the decrease in C_m -> Otherwise, a decrease in time constant causes an increase in conduction velocity

Question 81

Describe how axon diameter affects conduction velocity.

Answer 81

• The larger the axon diameter, the faster the conduction
• This is because the longitudinal resistance (R_l) is decreased, so that the length constant is increased -> λ = √(R_m/R_l)
• Increasing the length constant increases the conduction velocity because more distance areas ahead of the impulse can be depolarised to threshold

Question 82

Derive the equation showing the effect of axon diameter on the length constant.

Answer 82

When x is the diameter of the axon:

• The longitudinal resistance is proportional to the area, so: R_l ∝ 1/x²
• The membrane resistance is proportional to the circumference, so: R_m ∝ 1/x
• These can be inserted into λ = √(R_m/R_l), which can be manipulated to give: λ ∝ √x

Question 83

Myelination or axon diameter - Which has a greater effect on conduction velocity in axons and why?

Answer 83

Myelination has a larger effect because it is roughly directly proportional to the conduction velocity, whereas the square root of the axon diameter is roughly directly proportional to the conduction velocity.

Question 84

Describe how the time constant affects the conduction velocity in an axon.

Answer 84

• The smaller the time constant, the faster the conduction velocity
• This is because depolarisation of the membrane ahead of the active patch can occur more rapidly, so the signal is transmitted more quickly

Question 85

State some experimental evidence for the structure of myelinated axons.

Answer 85

Fluorescently-labelled antibodies for sodium channels and potassium channels can be prepared and then observed under a microscope, showing the location of the channels.

Question 86

Describe the importance of having densely-packed channels in the nodes of Ranvier.

Answer 86

The large number of channels increases the magnitude of the inwards sodium current, thus making the local depolarisation more effective.

Question 87

What is the name for the process of conduction in myelinated axons?

Answer 87

Saltatory conduction

Question 88

Describe the effect of temperature on conduction velocity in axons.

Answer 88

• Positive (often linear) correlation observed between temperature and conduction speed.
• Because in cold conditions, there is a slowed opening of voltage-gated channels, so that the action potential is more slowly propagated.
• This is of particular importance in the population of elderly people and those with impaired blood circulation, who may struggle with responding to a cold external environment.

Question 89

Give some examples of other factors that affect conduction velocity in axons.

Answer 89

• BMI
• Age
• Height
• Sex

However, these are only weakly supported by experimental evidence. There are often confounding factors -> e.g. Sex is often correlated with height, so that sex differences might not be explained by the sex.

Question 90

Draw a graph of conduction velocity against axon diameter for myelinated and unmyelinated axons.

Answer 90

Question 91

Are myelinated neurons always faster than unmyelinated neurons?

Answer 91

No, below about 0.8 micrometer axon diameter, unmyelinated axons are faster (see graph).

Question 92

Remember to clarify evidence for saltatory conduction.

Answer 92

Do it.

Question 93

What are the different nerve fibre types in mammals? What are their diameters, conduction velocities and functions?

Answer 93

From fastest to slowest conduction velocity:

• A
  
  • α -> 12-20μm, 70-120m/s -> Proprioception, Somatic motor
  • β -> 5-12μm, 30-70m/s -> Touch, Pressure
  • γ -> 3-6μm, 15-30m/s -> Motor to muscle spindles
  • δ -> 2-5μm, 12-30m/s -> Pain, Cold, Touch
• B -> Less than 3μm, 3-15m/s -> Preganglionic autonomic
• C (unmyelinated)
  
  • Dorsal root -> 0.4-1.2μm, 0.5-2m/s -> Pain, Temperature, Mechanoreceptor
  • Sympathetic -> 0.3-1.3μm, 0.7-2.3m/s -> Postganglionic sympathetic

Question 94

For Aα fibres, what is the diameter, conduction velocity and function?

Answer 94

Aα:

• 12-20μm
• 70-120m/s
• Proprioception (position and movement of the body), Somatic motor

Question 95

For Aβ fibres, what is the diameter, conduction velocity and function?

Answer 95

Aβ:

• 5-12μm
• 30-70m/s
• Touch, Pressure

Question 96

For Aγ fibres, what is the diameter, conduction velocity and function?

Answer 96

Aγ:

• 3-6μm
• 15-30m/s
• Motor to muscle spindles (force generation)

Question 97

For Aδ fibres, what is the diameter, conduction velocity and function?

Answer 97

Aδ:

• 2-5μm
• 12-30m/s
• Pain, Cold, Touch

Question 98

For B fibres, what is the diameter, conduction velocity and function?

Answer 98

B:

• Less than 3μm
• 3-15m/s
• Preganglionic autonomic

Question 99

For C sympathetic fibres, what is the diameter, conduction velocity and function?

Answer 99

C sympathetic:

• 0.3-1.3μm
• 0.7-2.3m/s
• Postganglionic sympathetic

Question 100

For C dorsal root fibres, what is the diameter, conduction velocity and function?

Answer 100

C dorsal root:

• 0.4-1.2μm
• 0.5-2m/s
• Pain, Temperature, Mechanoreceptor

Question 101

What is a compound action potential (CAP) and where does it occur?

Answer 101

• A compound action potential is the series of action potentials in the different nerve fibres (A-alpha, A-beta, etc.) that are caused by a single stimulation of the nerve
• Due to the different conduction velocities, the CAP arrives as a series of peaks
• You need to appreciate that they ocur in peripheral nerves.

Question 102

What is the effect of TTX on the action potential?

Answer 102

• TTX is a sodium-channel blocker
• So the initial depolarisation phase is inhibited and action potentials cannot fire

TTX is found in pufferfish and poisoning is extremely dangerous.

Question 103

What is the effect of TEA on the action potential?

Answer 103

• TEA is a potassium-channel blocker
• So although the initial depolarisation is the same, repolarisation takes much longer (it is only due to sodium channel inactivation) and there is no hyperpolarisation overshoot
• The resting membrane potential is not affected because the potassium channels involved in that are different

The prolonged action potential can, for example, cause release of excess neurotransmitter into a synapse, so it can be used to to reverse the effects of non-competitive blockers, such as curare.

Question 104

What are some diseases related to the myelination of neurons?

Answer 104

• Guillaine-Barré syndrome -> Autoimmune condition characterised by a fast destruction of neurones (axonal type) or their Schwann cells (demyelinating type) in the peripheral nervous system. Often underlied by infection. The demyelinating variant (the most common type) has early symptoms such as tingling and weakness of the limbs, which can develop into severe mobility problems, pain, difficulty breathing and paralysis. Rarely, the syndrome may result in death. These symptoms are caused by improper signal conduction down the neurones, since the signal cannot be contained within the partially unmyelinated neurone and its strength dissipates rapidly between nodes of Ranvier.
• Multiple Sclerosis -> Demyelinating disease in which the CNS myelin sheaths are degraded.
• Diptheria (in about 10% of cases)

Question 105

Can a nerve innervate multiple muscle fibres?

Answer 105

Yes, but each muscle fibre can be only be innervated by one nerve.

Question 106

What is an endplate?

Answer 106

The location of the muscle fibre where each of the axon expansions terminate (i.e. the location of the NMJ).

Question 107

What is a synapse?

Answer 107

A specialised structure at which information transfer takes place without physical interactions between the two participating cells.

Question 108

Describe how a synpase works.

Answer 108

The electrical signal (action potential) is converted to a chemical signal (neurotransmitter) that diffuses between the presynaptic and postsynaptic cells.

Question 109

Describe an experiment that proved the chemical process of synaptic transmission.

Answer 109

• Otto Loewi placed two beating frog hearts, each in its own perfusion chamber – one preparation had the vagus nerve intact, while the other was denervated
• Next, he stimulated the vagus nerve supplying the first heart, causing it to beat more slowly
• When Loewi applied the perfusate of the first heart to the second heart, it too slowed down, as if its vagus nerve had been stimulated as well
• He named the inhibitory factor ‘vagusstoff’, which is known today as acetylcholine

Question 110

Draw a diagram of a NMJ.

Answer 110

Question 111

What is the neurotransmitter used at NMJ?

Answer 111

Acetylcholine (ACh)

Question 112

Describe the structure of acetylcholine.

Answer 112

It is an ester of acetic acid and choline.

Question 113

Describe the process of storage of acetylcholine at the NMJ.

Answer 113

• ACh is stored in vesicles in the presynaptic neuron
• Proton pump (V-ATPase) is used to move hydrogen ions in the vesicle, acidifying it
• The proton gradient created by this is exploited by antiporter called the vesicular acetylcholine transporter (VAChT), which moves ACh in using the outwards H⁺ gradient

Question 114

Name the proteins involved in the packing of ACh in vesicles.

Answer 114

• Proton pump (V-ATPase) -> Pumps H⁺ into vesicles
• Vesicular acetylcholine transporter (VAChT) -> Uses the H⁺ gradient to move ACh into cells

Question 115

Describe the process of release of ACh at the NMJ.

Answer 115

• Action potential causes depolarisation of presynaptic membrane
• This triggers the opening of voltage-gated calcium channels and causes an influx of calcium into the presynaptic neuron
• The Ca²⁺ ions bind to calcium-sensitive proteins on the vesicular surface, namely synaptotagmin.
• This leads to docking of the calcium to the membrane, by the joining of SNARE proteins.
• v-SNARE proteins on the vesicular surface attach to t-SNARE proteins on the presynaptic membrane, and a tight complex (a SNARE-pin) is formed. This is seen when synaptobrevin on a vesicle interacts with syntaxin and SNAP-25 on the presynaptic membrane surface.
• The complex enables calcium-dependent fusion of the vesicle with the membrane and exocytosis of the ACh can occur.

It is worth noting that the “kiss and run” mechanism of ACh release occurs occasionally, where the vesicle only partially fuses with the membrane before sealing shut again, but this is rare and does not allow for complete release of ACh into the synaptic cleft.

Question 116

Describe the proteins involved in the fusion of a vesicle with the presynaptic membrane at the NMJ.

Answer 116

• Synaptotagmin -> Vesicular protein that senses cytoplasmic Ca²⁺

SNARE proteins:

• v-SNARE -> Synaptobrevin -> Vesicular protein that binds to t-SNARE proteins
• t-SNARE -> SNAP-25 and syntaxin -> Protein on presynaptic membrane that binds to v-SNARE proteins

Question 117

How can you remember the different v-SNARE and t-SNARE proteins?

Answer 117

• v-SNARE proteins are on the Vesicles
• t-SNARE proteins are the Target
• Synaptobrevin is a v-SNARE protein
• Syntaxin and SNAP-25 are therefore the t-SNARES

Question 118

What drugs can influence the storage of ACh?

Answer 118

• Proton pump inhibitors
• VAChT inhibitors (e.g. vesamicol)

Question 119

What drugs can influence the release of ACh at the NMJ?

Answer 119

• Botulinum toxin -> Proteolytic so it can cleave the 3 main SNARE proteins at different sites
• Verapamil or Magnesium -> Block calcium channels (so release is indirectly prevented)
• Vesamicol -> Inhibition of VAChT so that vesicles are not filled
• Black widow spider venom -> Massive release and depletion of vesicles

Question 120

What are some clinical uses of botulinum toxin?

Answer 120

• Blepharospasm (uncontrolled contraction of eyelid)
• Salivary drooling
• Axillary hyperhidrosis
• Achalasia (oesophageal spasm)
• Cosmetic reasons

Question 121

How is a steep concentration gradient maintained across the NMJ?

Answer 121

• Quantal secretion of ACh
• Acetylcholinesterase is found in pits on the post-synaptic membrane, while the acetylcholine receptors are on the projections.

Question 122

What receptors are present on the postsynaptic membrane of the NMJ?

Answer 122

Nicotinic AChR (nAChR)

Question 123

Describe the diffusion and binding of ACh at the NMJ.

Answer 123

• Diffusion occurs due to Fick’s Law (J = D x A x ΔC/Δx)
• ACh binds to nAChR, a non-selective cation pore is formed, allowing Na⁺ to diffuse into the postsynaptic membrane and K⁺ to diffuse into it, depolarising the membrane
• This depolarisation of the membrane is called the end-plate potential (EPP)
• An EPP can trigger an action potential in the muscle due to the depolarisation of the membrane, which voltage-gated sodium and potassium channels in the membrane are sensitive to

Question 124

What is the potential at the postsynaptic membrane of the NMJ called?

Answer 124

End-plate potential

Question 125

Is the end-plate potential propagated?

Answer 125

No, but it is usually strong enough to trigger an action potential, which is propagated and causes muscle contraction

Question 126

What are miniature end-plate potentials (mEPPs)?

Answer 126

• Small depolarisation of the postsynaptic membrane which do not trigger an AP
• They are caused by the random exocytosis of a single vesicle containing ACh

Question 127

Describe the breakdown of ACh at the NMJ.

Answer 127

• Acetylcholine in the synaptic cleft is broken down by acetylcholinesterase (AChE), which occurs after it dissociates from nicotinic receptors -> This action is so fast that about half of the ACh is broken down before it even reaches the receptors
• Products of ACh hydrolysis are acetate and choline

Question 128

Where can AChE be found at the NMJ?

Answer 128

• In the pits of the postsynaptic membrane
• It is secreted by the muscle and is attached to the basal lamina by collagen connections

Question 129

What type of reaction is the breakdown of ACh?

Answer 129

Hydrolysis

Question 130

What are the products of the breakdown of ACh?

Answer 130

Acetate and choline

Question 131

What is the difference between acetate and acetic acid?

Answer 131

Acetate is the conjugate base of acetic acid (i.e. it doesn’t have the H).

Question 132

What are some chemicals that can influence the breakdown of AChE?

Answer 132

• Novichok
• Neostigmine (used to treat myasthenia gravis)

Question 133

Describe the mecahnism and effects of Novichok.

Answer 133

• Inhibits AChE action, which causes ACh build up in the synaptic cleft
• So ACh permanently occupies the nicotinic receptors
• The result of this is that the pores formed by the nicotinic receptors remain open, allowing free flow of sodium and potassium ions, which gradually depolarises the membrane above its threshold. After a brief period of involuntary muscle contraction (due to the rapid firing of action potentials), the voltage-gated sodium channels in the muscle are permanently inactivated and are forced into a permanent absolute refractory period, so that further action potentials in the muscle cannot be triggered. This is because the channels cannot exit their inactivated state until the membrane returns to its resting potential.
• There are many antidotes to Novichok, such as pralidoxin (which activates acetylcholinesterase), but without intervention death occurs due to cardiac arrest or suffocation within minutes.

Question 134

What type of drug is neostigmine?

Answer 134

It is a anti-cholinesterase.

Question 135

What is myasthenia gravis and how can it be treated?

Answer 135

• Autoimmune neuro-muscular condition caused by auto-antibodies against nAChR at the NMJ
• Antibodies act in three ways:
  
  • Steric block of ACh
  • nAChR internalisation and degradation
  • Encourage immune system attack on muscle cell membrane
• Symptoms: Tiredness, muscle weakness, eyelids drooping, double vision
• Treatments: NEOSTIGMINE (anti-cholinesterase), Surgical removal of thymus, Removal of antibodies from blood

Question 136

Describe how neostigmine works.

Answer 136

• Binds to the active site of AChE
• So more ACh are able to reach the small number of nAChR
• This helps to trigger an action potential

Question 137

Describe the process of recyling and synthesis of ACh.

Answer 137

• Choline is taken back into the presynaptic neurone via choline transporters, which is a sodium-dependent process
• Choline may also be obtained from the diet in the form of phosphotidylcholine (a phospholipid)
• Acetate is not recycled
• Instead glycolysis converts glucose to pyruvate (in the presynaptic neuron), which is then converted to acetyl-CoA (in mitochondria)
• Choline acetyl transferase (CAT) is the enzyme that transfers an acetyl group from acetyl-CoA to choline, forming ACh

Question 138

What drugs can be used to target the synthesis of ACh?

Answer 138

The CAT enzyme is a drug target that is currently being studied, because stimulation of its activity (or prevention of its inhibition) would lead to an increase in ACh production, allowing for an increased release of ACh from vesicles after an action potential. This offers the potential for alternative treatments for myasthenia gravis and Alzheimer’s disease, amongst others.

Question 139

Describe an experiment to show that the EPP is variable in size and is not propagated.

Answer 139

• Muscle fibre in a bath, with microelectrodes inserted at 1mm intervals along the fibre
• Stimulate using motor axon
• Record the shape of the end-plate potential at each microelectrode

Question 140

Describe an experiment to show that ACh release is calcium-dependent.

Answer 140

Flash photolysis in the Calyx of Held (a particularly large synapse in the mammalian CNS):

• Fill presynaptic terminal with caged calcium (calcium ion trapped within a ‘cage’)
• Flash light to release Ca²⁺ and use intensity to vary [Ca²⁺]
• Record the excitatory postsynaptic current

Question 141

Describe an experiment to show that the release of ACh is quantal.

Answer 141

• Record a series of EPPs
• Plot a bar chart of the frequency of each EPP amplitude value
• The peaks exist at 0.4mV intervals, showing that the release is quantal

Question 142

Describe an experiment to show the action of nAChRs.

Answer 142

Patch-clamping can be used to show the currents through the non-selective pore that forms in the nAChR.

Question 143

Describe the structure of an nAChR.

Answer 143

• 4 transmembrane regions
• M2 region is pore-forming
• Two alpha units when ACh binds -> 2 ACh molecules bind to each receptor

Question 144

Name all of the experiments involved in the NMJ.

Answer 144

• Otto Lowi’s frog hearts
• EPP microelectrodes
• Flash photolysis of caged Ca²⁺
• Patch clamping
• Plotting EPP sizes

Question 145

Name the main mechanisms of neuromuscular blocking drugs and give examples of each.

Answer 145

• Competitive non-depolarising -> Tubocurarine, Vecuronium
• Depolarising -> Suxomethonium
• Vesicular releasem-> Botulinum toxin

Question 146

Name the non-depolarising blockers at the NMJ and explain how they work. Also state the main clinical uses.

Answer 146

Examples: Tubocurarine, Vecuronium

• Antagonists that compete with ACh for AChR binding sites
• Therefore do not depolarise membrane but prevent transmission

Clinical uses: Muscle relaxation in anesthesia

Question 147

Name the depolarising blockers at the NMJ and explain how they work. Also state the main clinical uses.

Answer 147

Examples: Suxamethonium -> Structure is like 2 acetylcholine molecules joined by a methyl group

• Are similar to ACh so they can bind to nAChR and cause depolarisation of the postsynaptic membrane, just like ACh does
• However, they are much more resistant to breakdown by AChE, so they can keep binding to the AChR and causing depolarisation
• Phase 1 is characterised by muscle twitches, while phase 2 involves paralysis due to the sodium channels in the postsynaptic membrane being inactivated (they are unable to return to normal due to the membrane potential never returning to rest)

Clinical use: Anesthesia

Question 148

Describe how each type of neuromuscular block can be reversed.

Answer 148

• Non-depolarising block -> Reversed by anticholinesterases, such as NEOSTIGMINE, because they can increase the concentration of ACh so that is out-competes the blocker
• Depolarising block -> Harder to reverse

Question 149

Describe the structure of local anaesthetics.

Answer 149

Homologues of cocaine with:

• Hydrophobic group
• Ester or amide linkage
• Ionisable group (usually an amine)

Question 150

Name some widely-used local anaesthetics. Give their potency and duration.

Answer 150

• Procaine -> Potency: 1, Duration: Short
• Lidocaine -> Potency: 4, Duration: Medium
• Tetracaine -> Potency: 16, Duration: Long
• Bupivacaine -> Potency: 16, Duration: Long

Question 151

Describe the mechanism of action of local anaesthetics.

Answer 151

• Local anaesthetics block voltage-gated sodium channels
• So action potentials cannot be initiated
• So sensory and motor information cannot be conveyed

Question 152

Why is the effect of lidocaine on sodium-channels use dependent?

Answer 152

• The degree of blocking increases when some action potentials are sent through the nerves
• This is because the lidocaine is more effective at binding to the sodium channels when they are open

(Check this)

Question 153

Which nerve fibre types are involved in the sensing of noxious stimuli and how is this converted into an action potential?

Answer 153

• Aẟ and C fibres sense different modalities of noxious stimulus (e.g. pain, heat, etc.)
• This is converted into electrical activity by transient receptor potential channels (TRP channels) and purinergic channels
• This is amplified by sodium channels to elicit an action potential

Question 154

Describe the effect of neuron diameter on sensitivity to local anaesthetic.

Answer 154

• Narrower neurons are more sensitive to block
• So myelinated Aẟ and unmyelinated C fibres carrying pain information are the most sensitive

Question 155

Describe the equilibrium that exists within local anaesthetics and what the importance of this is.

Answer 155

BH⁺ -> B + H⁺

• Only the B can diffuse through the lipid bilayer to get into the cytoplasm
• This is required for the LA to work
• Therefore the effectiveness of the LA is dependent on the pKa and pH

Question 156

What are some factors that influence local anaesthetic activity and what effect do they have?

Answer 156

• pKa -> The pKa is the pH at which the number of ionised and unionised fractions of the drug is in equilibrium. The lower the pKa, the more the unionised fraction is present for any given pH and hence the faster the onset of action.
• pH -> The lower the pH, the lesser the potency because of the equilibrium (BH⁺ -> B + H⁺) so there is less of the unionised form to cross the membrane and block sodium channels
• Lipid solubility -> The more lipid soluble the LA, the higher the potency, the faster the onset of action and the longer the duration, since more of the drug can cross the lipid bilayer and form a depot of it in the cytoplasm
• Intermediate chain -> The longer the intermediate chain, the more potent the LA
• Protein-binding -> The higher the degree of protein-binding, the longer the duration of action

Question 157

Describe some experimental evidence for the model of local anaesthetic action.

Answer 157

• QX-314 and QX-222 are permanently charged LAs
• They only work when induced straight into the cytoplasm, not outside the neuron
• This shows that the active form of LAs (the charged form) can only enter and bind on the cytoplasmic side of the sodium channels
• They also bind cumulatively with each depolarising pulse and then don’t unbind at rest

Question 158

What are the two main pathways for local anaesthetics to access sodium channels?

Answer 158

• Hydrophilic pathway
• Hydrophobic pathway

Question 159

Draw and explain the hydrophilic pathway of local anaesthetics accessing sodium channels.

Answer 159

Question 160

Draw and explain the hydrophobic pathway of local anaesthetics accessing sodium channels.

Answer 160

Question 161

Describe the structure of a voltage-gated sodium channel.

[EXTRA]

Answer 161

• Made up of 3 subunits: α, β₁ and β₂.
• The alpha subunit consists of 4 transmembrane domains, each of which is made up of 6 membrane-crossing segments that are alpha helices
• The S5 and S6 segments from all 4 domains make up the pore of the sodium channel
• The S4 segments contain a sequence of positively charged residues that sense the voltage across the membrane and move in response to a change in membrane potential
• The inactivation gate is the loop between the III and IV domains

Question 162

Do local anaesthetics bind permenently or reversibly?

Answer 162

Reversibly

Question 163

Is the action of local anaesthetics confined to blocking sodium channels in nerves?

Answer 163

• No, they can also block other channels, such as nicotinic acetylcholine receptors, ryanodine receptors and K+ channels.
• No, they can also block channels in the heart.

Question 164

Give another clinical use of local anaesthetics.

Answer 164

They can be used as anti-arrhythmic drugs (e.g. lidocaine).

Question 165

Name a more potent sodium channel blocker that local anaesthetics and describe its action.

Answer 165

• TTX
• It is a more specific and potent blocker than LAs.
• It can bind sodium channels extracellularly.

Question 166

Describe the differences between the metabolism of different local anaesthetics.

Answer 166

• Esters (e.g. procaine, tetracaine) -> Metabolised in the blood by plasma cholinesterases or liver esterases. These have a short half-life.
• Amides (e.g. lidocaine, bupivacaine) -> Amides are widely distributed via the circulation and are metabolised exclusively by enzymes in the liver. Half-life of these compounds is therefore longer.

Question 167

What are some of the factors affecting local anaesthetic absorption?

Answer 167

• Dosage
• Injection site
• Vasoconstricting agents
• Drug-tissue binding

Question 168

What is the effect of vasocontricting agents on local anaesthetic action?

Answer 168

• There is loss of LA from the site of local application into the circulation.
• Vasoconstricting agents can cause the local anaesthetic effect to be prolonged and circulating anaesthetic levels to be reduced.

Question 169

Describe some of the toxic effects of local anaesthetics.

Answer 169

If the LA gets into the circulation:

• CNS toxicity (initial light-headedness, but may lead to convulsions and eventually respiratory depression and coma).
• Cardiovascular effects (myocardial depression with reduced heart rate and stroke volume, vasodilatation, reduced blood pressure).

Question 170

What are some of the methods of administrating anaesthetics?

Answer 170

• Surface
• Direct infiltration into tissues
• Injection close to nerve trunk
• Regional anaesthesia
• Spinal anaesthesia
• Epidural anaesthesia

Question 171

Draw a diagram of how regional anaesthesia may be administrated and state what this is called.

Answer 171

Bier’s block

Question 172

What is a more specific method of anaesthesia and how may it be achieved?

Answer 172

• Nociceptor-specific anaesthesia may sometimes be preferrable
• It could be achieved by targetting TRVP1 channels on small unmyelinated C-fibres that selectively express this channel

Question 173

Describe the sequence of local anaesthetic blockade.

Answer 173

• Pain first
• Then general sensory
• Motor last

Question 174

In spinal and epidural anaesthesia, where is the injection site? Draw a diagram of this.

Answer 174

• Spinal -> In the L2-L5 region, Lumbar CSF which bathes the cauda equina
• Epidural -> In the lumbar epidural space (but can also be in the thoracic and sacral areas)

Question 175

In spinal anaesthesia, what tissues are transversed by the needle?

Answer 175

• Skin
• Fat
• Interspinous ligament
• Ligamentum flavum
• Epidural space
• Dura

Question 176

Describe the patient positioning for spinal anaesthesia injection.

Answer 176

The patient should be sitting down, curled up so that the area between the vertebrae is opened up on the posterior side.

Question 177

How is spinal anaesthesia used to reach different dermatomes?

Answer 177

Gravity is used to reach different dermatomes. The curvature of the spine is useful for this.

Question 178

What are the uses of spinal and epidural anaesthesia?

Answer 178

• Spinal -> Pain relief and muscle paralysis during surgery
• Epidural -> Analgesia in labour, Pain relief and muscle paralysis during surgery

Question 179

Briefly summarise the differences between spinal and epidural anaesthesia.

Question 180

What type of molecule is acetylcholine and how can you remember this?

Answer 180

It is an ester. You can remember this because it is broken down by acetylcholinESTERase.

Question 181

Where in the body may acetylcholine be found?

Answer 181

• All autonomic ganglia
• All NMJs
• At many autonomically innervated organs
• At many sites in the CNS

Question 182

Describe the divisions of the nervous system.

Answer 182

Note: The enteric nervous system may also be listed alongside the sympathetic and parasympathetic divisions of the autonomic nervous system.

Question 183

Can the CNS function on its own?

Answer 183

No, it requires the sympathetic and parasympathetic systems to provide a link between the CNS and peripheral organs.

Question 184

Describe the structure of the autonomic efferent pathway.

Answer 184

There are two neurons arranged in series. These are the pre-ganglionic and post-ganglionic and these are joined at an autonomic ganglion.

Question 185

Where is the autonomic nervous system is acetylcholine released?

Answer 185

• By all pre-ganglionic and post-ganglionic parasympathetic neurons
• By all pre-ganglionic sympathetic neurons (and in the post-ganglionic too with sweat glands ONLY)

Question 186

Draw a diagram illustrating the different sites of ACh release in the peripheral nervous system.

Answer 186

Question 187

Describe the difference between the two types of ACh receptor.

Answer 187

• Nicotinic -> Ligand-gated ion channel permeable to monovalent and divalent cations, but not anions
• Muscarinic -> G-protein coupled receptors

Question 188

Are acetylcholine receptors only found on the postsynaptic membrane?

Answer 188

No, there can also be autoreceptors on the presynaptic neuron, which serve in negative feedback in signal transduction.

Question 189

Describe the different locations of acetylcholine receptors in the body, acounting for nAChR and mAChR.

Answer 189

• CNS -> nAChR and mAChR
• Autonomic -> nAChR and mAChR -> After all pre-ganglionic and post-ganglionic parasympathetic neurons, and after all pre-ganglionic sympathetic neurons (and post-ganglionic too in sweat glands ONLY)
• Somatic (NMJ) -> nAChR

Note that mAChRs are not always quoted as being in autonomic ganglia because transmission is mostly due to nAChRs (see flashcard about this).

Question 190

Where does cholinergic transmission stimulate mAChRs and mAChRs exactly?

Answer 190

mAChRs:

• Autonomic ganglia
• Organs innervated by parasympathetic nervous system
• CNS

nAChRs:

• Autonomic ganglia (+ adrenal medulla)
• NMJ
• CNS

Question 191

What is the main neurotransmitter released at organs innervated by the sympathetic nervous system and what is the main exception to this?

Answer 191

Norepinephrine (a.k.a noradrenaline), except at sweat glands, where ACh is released instead.

The way to remember this is Akerman going “NAAAAAW” to symbolise being sympathetic.

Question 192

What acetylcholine receptors are found at autonomic ganglia?

Answer 192

Off Wikipedia, so double-check this:

• ACh is always used as the transmitter within the autonomic ganglion. Nicotinic receptors on the postganglionic neuron are responsible for the initial fast depolarization of that neuron. As a consequence of this, nicotinic receptors are often cited as the receptor on the postganglionic neurons at the ganglion.
• However, the subsequent hyperpolarization and slow depolarization that represent the recovery of the postganglionic neuron from stimulation are actually mediated by muscarinic receptors, types M2 and M1 respectively.

Question 193

Describe the structure of mAChRs.

Answer 193

Single polypeptide containing 7 transmembrane regions that are alpha helices.

Question 194

Are mAChRs ever found presynaptically? What is the purpose of this?

Answer 194

In the CNS, they are found both presynaptically and postsynaptically. The ones of the presynaptic membrane are used to modulate the release of ACh.

Question 195

Describe the different types of muscarinic receptors and their functions.

Answer 195

• M1 -> Excitatory -> CNS responses such as memory, arousal, attention and anaesthesia + exocrine glands
• M2 -> Inhibitory -> Lowers heart rate
• M3 -> Excitatory -> Smooth muscle
• M4 -> Inhibitory -> CNS
• M5 -> Excitatory -> CNS

Question 196

Which mAChRs are excitatory and what type of GPCR are they? How do these work?

Answer 196

• M1, M3 and M5
• They couple to Gq proteins
• Which activate the inositol phosphate pathway

Question 197

Which mAChRs are inhibitory and what type of GPCR are they? How do these work?

Answer 197

• M2 and M4
• They couple to Gi/Go proteins
• Which inhibit adenylate cyclase and so reduce intracellular cAMP

Question 198

Describe the different types of nictonic ACh receptors and their locations and functions.

Answer 198

• N1 (or Nm) -> At NMJ
• N2 (or Nn) -> Autonomic ganglia, CNS and adrenal medulla

Question 199

Compare the structures of N1 and N2 nAChRs.

Answer 199

• N1 (at NMJ) -> 2 alpha, 1 beta, 1 gamma and 1 delta subunit -> Each subunit has 4 transmembrane regions
• N2 (neuronal and ganglionic) -> Only made of 2 subtypes: alpha and beta

Question 200

Describe the agonists and antagonists of nAChRs and mAChRs.

Answer 200

nAChRs:

• Agonists -> ACh and nicotine
• Antagonists -> Tubocurarine

mAChRs:

• Agonists -> ACh and muscarine
• Antagonists -> Atropine

Question 201

What is the name for the depolarisation of the postsynaptic cell due to opening of channels in AChRs?

Answer 201

• At the NMJ -> EPP (End-plate potential)
• In nerves -> EPSP (Excitatory postsynaptic potential)

Question 202

Compare the onset and duration of nAChR and mAChR action.

Answer 202

• nAChR -> Rapid and short-lasting
• mAChR -> Slow onset and long-lasting

Question 203

What is the name for the change in membrane potential of the postsynaptic cell due to the action of mAChRs?

Answer 203

• ESPS (Excitatory postsynaptic potential) -> When the membrane is slowly depolarised
• IPSP (Inhibitory postsynaptic potential) -> When the membrane is slowly hyperpolarised

Question 204

Summarise simply everything about nAChRs and mAChRs.

Answer 204

• The nervous system is split into the CNS and PNS
• The CNS uses both nAChR and mAChR which bind ACh, but it also makes use of many other neurotransmitters -> The excitatory receptors are N2 and M1 and M5, while the inhibitory ones are M4.
• The PNS is split into the somatic and autonomic nervous system
• The somatic nervous system only features one efferent neuron from the CNS to the effector (i.e. no ganglia), so the only neurotransmitter released is at the NMJ where ACh binds to nAChR
• The autonomic nervous system is split into the sympathetic and parasympathetic (and also the enteric, but this is sort of like the parasympathetic)
• The autonomic nervous system features two efferent neurons from the CNS to the effector (joined by an autonomic ganglion)
• At the ganglion, there are nAChRs (N2) which are predominantly used to allow transmission here, although there are also mAChR which play a role in repolarisation (check this)
• The sympathetic autonomic nervous system uses mostly norepinephrine (noradrenaline) at the effector organ (this is adrenergic transmission), and the only major exception to this is sweat glands, at which mAChR receptors are used (M2)
• The autonomic nervous system uses mAChRs at the effector organs, with M2 receptors being inhibitory for the heart muscle, while M1 receptors are excitatory for exocrine glands and M3 are excitatory for smooth muscle
• mAChRs that are excitatory are Gq-coupled, which work by activating the inositol phosphate pathway
• mAChRs that are inhibitory are Gi/Go-coupled, which work by inhibiting adenylate cyclase and so reduce intracellular cAMP

Question 205

What are the physiological effects of ACh at low and high concentrations? Describe an experiment to show this.

Answer 205

Question 206

What is Endothelial Derived Relaxing Factor (EDRF)?

Answer 206

Nitric oxide (NO) - it causes smooth muscle relaxation.

Question 207

Describe the importance of snake toxins in physiology and medicine.

Answer 207

Bind very tightly and selectively to nAChRs at the NMJ, so:

• Useful for identification and purification of nAChR protein
• Useful for studies of NMJ transmission and ion channel function
• Potential treatments for MS, infections caused by herpes viruses, and retroviruses

Question 208

What are the different types of drugs that affect cholinergic transmission?

Answer 208

• Muscarinic agonists/antagonists
• Ganglion-stimulated drugs
• Ganglion-inhibiting drugs
• Neuromuscular-blocking drugs
• Acetylcholinesterases and other drugs that enhance cholinergic transmission

Question 209

What is the effect of muscarinic agonists and what is another name for these drugs?

Answer 209

• They closely mimic the effects of parasympathetic nerve stimulation (decreased heart rate, increased smooth muscle contraction, increased exocrine secretions)
• They may be called parasympathomimetics because of this

Question 210

Give some clinical uses of parasympathomimetics (muscarinic agonists).

Answer 210

• Glaucoma -> Pilocrapine constricts the pupil and improves drainage, reducing the pressure that is characteristic of glaucoma
• Suppression of atrial tachycardia (rarely)

Question 211

What is another name for muscarinic antagonists?

Answer 211

Antimuscarinic drugs

Question 212

What are some effects of muscarinic antagonists?

Answer 212

• Inhbition of exocrine secretions
• Tachycardia (very fast heart rate)
• Pupillary dilation
• Relaxation of smooth muscle

Question 213

Give some clinical uses of antimuscarinic drugs.

Answer 213

• Cardiovascular -> Treatment of bradycardia (slow heart rate)
• Ophtalmic -> Pupil dilation
• Respiratory -> Asthma (due to smooth muscle relaxation)
• Gastrointestinal -> Decrease secretions
• Smooth muscle -> Urinary incontinence

Question 214

What types of drug are ganglion stimulants?

Answer 214

nAChR agonists

Question 215

Do all nAChR agonists act on both neuronal and NMJ nAChRs?

Answer 215

No, because there are different types of nAChR at each (N1 and N2). For example:

• Nicotine -> Autonomic ganglia + CNS
• Lobeline -> Autonomic ganglia
• Suxamethonium -> NMJ

Question 216

What is the only depolarising blocking drug currently in use?

Answer 216

Suxamethonium

Question 217

What is the location and duration of action of suxamethonium?

Answer 217

• Only lasts a few minutes (before it is metabolised by plasma esterase)
• Acts at NMJ because it is not broken down by AChE at the NMJ

Question 218

What are the non-depolarising blockers of nAChR at the NMJ and at ganglia?

Answer 218

• NMJ -> Tubocurarine + Vecuronium
• Ganglia -> Mecamylamine

Question 219

Both depolarising and non-depolarising blockers may be used in surgery. Compare how easily each is reversed.

Answer 219

• Non-depolarising blockers (e.g. tubocurarine and vecuronium) are easily reversed by neostigmine (AChE inhibitor) since they are competitive blockers and the increase in ACh can reduce their effect
• Depolarising blockers (e.g. suxamethonium) are not easily reversed

Question 220

What is the most common cholinesterase inhibitor?

Answer 220

Neostigmine

Question 221

What is an important condition for when cholinesterase inhibitors will work?

Answer 221

They only work when there is pre-existing ACh release.

Question 222

Give some examples of clinical uses of anticholinesterases.

Answer 222

• Skeletal muscle -> Reversing NMJ block + Treatment of myaesthenia gravis
• CNS -> Treatment of Alzheimer’s disease (questionable benefits)
• Eye -> Treatment of glaucoma

Question 223

Give a short summary of the drugs influencing cholinergic transmission.

Answer 223

Question 224

Give some experimental evidence to demonstrate that the nAChRs in ganglia are different from those at the NMJ.

Answer 224

• Hexamethonium is a non-depolarising blocker that acts at autonomic ganglia
• Decamethonium is a non-depolarising blocker that acts at NMJ

Their structures are similar.

Question 225

Draw a nice diagram showing the organisation of the different divisions of the nervous system.

Answer 225

Note that the sensory division is not divided into sympathetic or parasympathetic -> Check

Question 226

Draw a summary of the efferent pathways of the different nervous sytem divisions.

Answer 226

Question 227

Compare briefly the somatic and autonomic nervous system transmission methods.

Answer 227

Question 228

Aside from cholinergic and catecholaminergic transmission, what are some other types of signalling? Define each and give an example of each.

Answer 228

• Purinergic -> Extracellular signalling mediated by purine nucleotides and nucleosides (e.g. adenosine and ATP)
• Gaseous -> Signalling mediated by gases (e.g. nitric oxide)
• Neuropeptide -> Signalling mediated by small protein-like molecules (peptides) used by neurons to communicate with each other (e.g. Neuropeptide Y and vasoactive intestinal peptide)

Question 229

What is the concept of co-transmission?

Answer 229

• The control of a single target cell by two or more substances released from one neuron in response to the same neuronal event, does occur in experimental situations.
• It has not been shown to occur in the normal operation of an animal, but the likelihood that it does is great.

Question 230

Remember to reivse muscle structure in OB deck 6.

Answer 230

Do it.

Question 231

What intracellular change is responsible for muscle contraction?

Answer 231

Increase of cytosolic Ca²⁺.

Question 232

Draw an EM image of a sarcomere, showing the different bands and lines.

Answer 232

Question 233

Draw a diagram to show how sarcomeres change in appearance upon contraction.

Answer 233

Question 234

What is excitation-contraction coupling?

Answer 234

The physiological process of converting an electrical stimulus to a mechanical response. It is the link (transduction) between the action potential generated in the sarcolemma and the start of a muscle contraction.

Question 235

Describe excitation-contraction coupling in skeletal muscle. [IMPORTANT]

Answer 235

• An action potential causes release of ACh at an NMJ
• This ACh binds to AChR on the muscle fibre, leading to depolarisation of the sarcolemma, which travels along the membrane and into the T-tubule
• Each T-tubule is flanked by 2 cisternae of the sarcoplasmic reticulum (called a triad)
• Sarcolemma depolarisation causes opening of L-type Ca²⁺ channel (LTCC) in the sarcolemma
• Each LTCC is mechanically coupled to a ryanodine receptor (RyR) on the sarcoplasmic reticulum
• Calcium exits via the RyR into the cytosol
• Some of the Ca²⁺ entering via the LTCC can also activate the RyR to open (calcium induced calcium release), but this pathway is not essential in skeletal muscle
• Calcium activates troponin C and triggers contraction

Question 236

Label this. What muscle type is it?

Answer 236

It is a triad in skeletal muscle.

Question 237

What proteins are involved in release of calcium in skeletal muscle once depolarisation reaches T-tubules?

Answer 237

On the T-tubule:

• L-type Ca²⁺ channel (LTCC) -> a.k.a. DHP receptor

On the sarcoplasmic reticulum:

• Ryanodine receptor (RyR) -> a.k.a. Ca²⁺ release channel

Question 238

What is the form of coupling in skeletal muscle between the L-type calcium channel and ryanodine receptor?

Answer 238

• Mechanical coupling

There is also calcium-induced calcium release -> But this is not required for contraction like in cardiac muscle.

Question 239

Describe the arrangement of the contractile filaments in skeletal muscle at rest.

Answer 239

• There are thin actin filaments that have tropomyosin wrapped around it in a helical fashion
• This tropomyosin blocks the heads of thick myosin filaments from binding to the actin (at myosin binding sites)
• There is a troponin complex that regulates the position of troponin

Question 240

What are the three components of the troponin complex?

Answer 240

• TnT -> Binds to Tropomyosin
• TnI -> Inhibits the myosin binding site
• TnC -> Binds to Calcium

Question 241

Describe how an increase in cytosolic calcium leads to contraction of skeletal muscle.

Answer 241

Calcium binds to troponin C, which causes tropomyosin to undergo a conformational change so that troponin I no longer blocks the myosin binding site and cross-bridge cycling can occur.

Question 242

Draw the process of cross-bridge cycling in skeletal muscle.

Answer 242

Question 243

Describe the different points at which ATP is required during cross-bridge cycling.

Answer 243

• Binding of ATP to myosin head causes release of the head from the binding site
• ATP hydrolysis causes the head to return to its relaxed state (but the free phosphate remains bound)
• Once a new cross-bridge is formed, release of the phosphate causes the power stroke to occur

Question 244

What change must occur in order for skeletal muscle contraction to end?

Answer 244

The cytosolic calcium levels must drop.

Question 245

Describe how skeletal muscle contraction is terminated and what proteins are involved.

Answer 245

Most important:

• Ca²⁺-ATPase on sarcoplasmic reticulum -> SERCA pump

Less important:

• Ca²⁺pump on plasma membrane -> PMCA
• Na⁺/Ca²⁺-exchanger on plasma membrane -> NCX

These lower the levels of cytosolic calcium by pumping it out of the cell or into the SR.

Question 246

What does SERCA stand for and why is the most important mechanism for terminating contraction of skeletal muscle?

Answer 246

• Sarco-endoplasmic reticulum calcium ATPase
• It is the most important mechanism because it is on the SR and intracellular calcium must be conserved

Question 247

What are the 3 ways in which the tension produced by skeletal muscle can be regulated?

Answer 247

• Selective recruitment of a greater or smaller number of motor units
• Changes in the frequency of stimulation
• Changes in the starting length of the relaxed muscle

Question 248

Draw how motor unit recruitment can be used to vary the tension produced by skeletal muscle.

Answer 248

The more motor units are recruited, the greater the tension in the muscle will be.

Question 249

What is the name for increasing the number of motor units recruited in a skeletal muscle in order to produce a greater tension?

Answer 249

Spatial summation

Question 250

Describe how modulation of stimulation frequency can be used to vary the tension in skeletal muscle.

Answer 250

Since muscle contraction lasts longer than an action potential, multiple action potentials in sequence can cause the tension to be increased.

Question 251

What is the name for increasing the frequency of stimulation of skeletal muscle in order to produce a greater tension?

Answer 251

Frequency summation (or temporal stimulation)

Question 252

What is tetany and what are the two types?

Answer 252

• It is the prolonged, constant contraction of skeletal muscle resulting from a very frequency of action potentials, so that the muscle contractions fuse
• The two types are fused tetanus and unfused tetanus

Question 253

Describe how modulation of muscle length can be used to vary tension.

Answer 253

In general, the more you stretch a muscle, the less force it can exert when contracted.

Question 254

What are the two types of muscle contraction?

Answer 254

• Isometric
  
  • Length is constant
  • But tension is generated
• Isotonic
  
  • Length changed
  • Tension is constant

Question 255

Draw the relationship between the length of muscle and the tension that can be generated. [EXTRA?]

Answer 255

Question 256

What parts of cardiac muscle allow it to act as a electrical and mechanical syncytium?

Answer 256

• Gap junctions -> Electrical coupling
• Desmosomes -> Mechanical coupling

These are two components of intercalated discs along with adherens junctions.

Question 257

Summarise briefly the idea of the Frank-Starling mechanism.

Answer 257

The greater the filling of a cardiac chamber, the greater individual myocardial fibres are stretched, and hence the greater the subsequent force of contraction .

Question 258

Draw the shape of an SAN action potential and explain the currents that are part of it. [IMPORTANT]

Answer 258

• Phase 0: Depolarisation due to inward calcium current
• Phase 3: Repolarisation dependent on outward potassium current
• Phase 4: Pacemaker potential due to funny current and also changes in potassium and calcium currents

Question 259

What is the symbol for the funny current?

Answer 259

I_f

Question 260

What channel carries the funny current?

Answer 260

HCN -> Hyperpolarisation-activated cyclic nucleotide channel

Question 261

How does the funny current work?

Answer 261

It is an inward current that slowly depolarises SAN cells in response to hyperpolarisation.

Question 262

What are the different currents that contribute to the pacemaker current?

Answer 262

• Inward Ca²⁺ current
• De-activation of outward K⁺ current
• Funny current (Inward cation current)
• NCX current
• Background inward Na⁺ leak

Question 263

Draw the shape of a ventricular action potential and explain the currents that are part of it. [IMPORTANT]

Answer 263

• Phase 0: Fast upstroke. Due to inward calcium and sodium currents.
• Phase 1: Rapid repolarization. Almost total inactivation of sodium and calcium currents.
• Phase 2: Prolonged plateau. Continued entry of Ca²⁺ or Na⁺ ions through their major channels and via NCX1 exchanger.
• Phase 3: Repolarization due to outward potassium currents.
• Phase 4: Electrical diastolic phase

Question 264

Show the different refractory periods in the ventricular action potential.

Answer 264

Question 265

Compare the action potential in sub-epicardial cells vs sub-endocardial cells of the ventricles.

Answer 265

The action potential is longer in sub-endocardial myocytes.

Question 266

What are some differences between the SAN action potential and ventricular action potential?

Answer 266

• Ventricular action potential features a steeper upstroke -> For speed of transmission
• Ventricular action potential features a prolonged plateau -> Enables plentiful entry of calcium into the cell

Question 267

Describe the process of excitation-contraction coupling in cardiac muscle.

Answer 267

It is essentially the same as in skeletal muscle, except the LTCC and RyR are not mechanically coupled.

Question 268

What makes excitation contraction coupling different in cardiac muscle compared to skeletal muscle?

Answer 268

Calcium-induced calcium release is required, since the L-type calcium channel and ryanodine receptors are not mechanically coupled.

Question 269

What is different about the T-tubules in cardiac muscle compared to skeletal muscle?

Answer 269

The T-tubules only have one sarcoplasmic reticulum cisternae next to them, rather than two.

Question 270

Explain the concept of CICR.

Answer 270

Calcium induced calcium release:

• When depolarisation travels into a T-tubule, it causes L-type calcium channels in the membrane to open
• In cardiac muscle, the LTCC are not coupled to ryanodine receptors (RyR)
• Instead, the calcium that enters the cytosol via the LTCC causes release of calcium from the SR via RyR
• Therefore, calcium flowing in from the outside is NECESSARY for contraction to occur, unlike in skeletal muscle

Question 271

Describe the feedback regulation of calcium levels in the cytosol of cardiac myocytes.

Answer 271

• Increased cytosolic Ca²⁺ inhibits LTCC so that there is less influx of calcium
• It also increases NCX activity

Question 272

What are the 2 main ways in which cardiac output can be modulated?

Answer 272

• Inotropy -> Change in force of contraction
• Chronotropy -> Change in frequency of contraction

Question 273

What are the main ways in which inotropy and chronotropy can be regulated?

Answer 273

Inotropy:

1. Starling’s Law -> Change in cell length
2. Cardiac nerves and circulating hormones

Chronotropy:

• Nerves and hormones

Question 274

Draw a graph to show Frank-Starling and the mechanism that explains it.

Answer 274

It happens in two ways:

• Stretch leads to changes in double actin overlap
• Stretch leads to changes in troponin complex affinity for calcium

Question 275

Draw a graph of tension in heart muscle against intracellular calcium.

Answer 275

Question 276

Describe which parts of the heart can be regulated in terms of inotropy and chronotropy. Which type of innervation enables this?

Answer 276

• Inotropy
  
  • Ventricles
  • Sympathetic innervation only
• Chronotropy
  
  • SAN/AVN
  • Sympathetic and parasympathetic innervation

Question 277

What is phospholamban and what does it do?

Answer 277

Protein that inhibits the SERCA pump.

Question 278

Draw the mechanism for the sympathetic regulation of inotropy.

Answer 278

This occurs in ventricular myocytes.

Question 279

Draw the mechanism for the sympathetic regulation of chronotropy.

Answer 279

This occurs at SAN and AVN cells.

Question 280

Draw the mechanism for the parasympathetic regulation of chronotropy.

Answer 280

• It is the opposite process to symnpathetic regulation, since it involves inhibition of cAMP synthesis rather than driving of cAMP synthesis.
• ALSO, the βγ subunit of the G-protein activates K⁺-channels, leading to hyperpolarisation of the cell

Question 281

Describe the effects of cardiac glycosides. Give an example. [IMPORTANT]

Answer 281

Digoxin:

• Inhibits sodium-potassium ATPase pump in cardiac muscle cells to alter their function.
• So intracellular sodium concentration therefore increases.
• Raised intracellular sodium levels inhibit the function the NCX (sodium-calcium exchanger), which is responsible for pumping calcium ions out of the cell and sodium ions in.
• Thus, calcium ions are also not extruded and will begin to build up inside the cell as well -> Leads to increased calcium uptake into the sarcoplasmic reticulum (SR) via the SERCA transporter.
• Raised calcium stores in the SR allow for greater calcium release on stimulation, so inotropy is increased.
• The refractory period of the AV node is increased, so cardiac glycosides also function to decrease heart rate.
• For example, the ingestion of digoxin leads to increased cardiac output and decreased heart rate without significant changes in blood pressure; this quality allows it to be widely used medicinally in the treatment of cardiac arrhythmias.

Question 282

Is calcium required for smooth muscle contraction?

Answer 282

Yes, just like with all types of muscle.

Question 283

What are the different things that can regulate smooth muscle contraction?

Answer 283

• Myogenic activity
• Sympathetic / Parasympathetic innervation
• Hormones and local compounds

Question 284

Describe how innervation, myogenic activity and hormones can evoke an increase in calcium in smooth muscle cells.

Answer 284

Myogenic activity:

• Voltage-gated Ca²⁺ channels (VGCCs) can open upon membrane depolarisation
• This increases intracellular calcium

Sympathetic innervation and hormones:

• Bind to ligand gated Ca²⁺ channels

OR

• Bind to G_q-coupled GPCRs
• This leads to an increase in IP₃ (second messenger)
• IP₃ binds to IP₃ receptors on the sarcoplasmic reticulum
• This leads to calcium release

NOTE: Don’t worry too much about parasympathetic innervation for now.

Question 285

What types of calcium channels are found on smooth muscle cells?

Answer 285

L-type calcium channels

Question 286

How does increased cytosolic calcium lead to contraction of smooth muscle?

Answer 286

Relies on a change in myosin structure:

• Calcium binds to calmodulin (CaM) to give a Ca²⁺-CaM complex
• This in turn activates myosin light chain kinase (MLCK) to phosphorylate myosin on serine 19 on the light (regulatory) chain.
• This exposes the site that binds to actin, preparing the myosin for cross-bridge formation.
• The Ca²⁺-CaM complex also stimulates ATPase, which is necessary for the cross-bridge cycle.

Question 287

What are two important enzymes involved in control of contraction of smooth muscle? How do they do this?

Answer 287

• Myosin light chain kinase (MLCK) -> Stimulates contraction
• Myosin light chain phosphatase (MLCPh) -> Inhibits contraction

MLCK and MLCPh work by phosphorylating and dephosphorylating myosin. When it is phosphorylated, the site that binds to actin is exposed, so cross-bridge cycling is enabled.

Question 288

What does myosin light chain kinase do and what things can regulate its activity? [IMPORTANT]

Answer 288

• Drives smooth muscle contraction by phosphorylating myosin so that it cross-bridge cycling can occur
• Activated by: Calcium (in Ca²⁺-calmodulin complex)
• Inhibited by: PKA and PKG

Question 289

What does myosin light chain phosphatase do and what things can regulate its activity? [IMPORTANT]

Answer 289

• Terminates smooth muscle contracting by dephosphorylating myosin so that the sites that bind to actin are not exposed and therefore cross-bridge cycling cannot occur
• Inhibited by: PKC

Question 290

What are the effects of PKA, PKC and PKG on smooth muscle contraction?

Answer 290

• PKA and PKG inhibit MLCK -> Therefore lead to relaxation
• PKC inhibits MLCPh -> Therefore lead to contraction

Question 291

How can agonists (e.g. hormones) cause sensitisation of smooth muscle to calcium?

Answer 291

• Bind to GPCR that activates an enzyme called Rho-kinase
• Rho kinase inhibits MLCPh (which usually drives relaxation of smooth muscle)
• Therefore, the phosphorylation of myosin is prolonged and therefore the muscle remains more contracted

Question 292

How does cAMP affect smooth muscle and why? [IMPORTANT]

Answer 292

• Inhibits contraction
• By inhibiting the actions of MLCK

Question 293

How does cGMP affect smooth muscle and why? [IMPORTANT]

Answer 293

• Inhibits contraction
• By stimulating the actions of MLCPh

Question 294

What is meant by L-type calcium channels?

Answer 294

Long-lasting -> They stay open for as long as the membrane is depolarised.

Question 295

Describe how the endothelium of arteries allows for vascular smooth muscle relaxation. [IMPORTANT]

Answer 295

Question 296

Draw a summary of the different things that can cause smooth muscle contraction or relaxation.

Answer 296

Question 297

Draw how sympathetic innervation can affect smooth muscle contraction.

Answer 297

This shows that the receptors that are present on the muscle determine what response the muscle has to sympathetic stimulation.

Question 298

How does viagra work?

Answer 298

• It is a phosphodiesterase 5 inhibitor, which means it blocks the breakdown of cGMP to GMP
• Therefore, the cGMP can continue to cause smooth muscle relaxation

Question 299

What two things can lead to smooth muscle relaxation?

Answer 299

• Drop in intracellular calcium
• MLCPh activity

Question 300

What are the 3 classes of calcium-blocking drugs that act on smooth muscle? [EXTRA]

Answer 300