BT_GS 1.35 - Phys NMJ

Question 1

What is the NMJ? (Define it)

Answer 1

The neuromuscular junction is a specialised synapse for the transmission of a signal from the motor nerve terminal to a postsynaptic region on the muscle fibre.

Question 2

Give an overview of the structure of how skeletal muscle fibers are innervated by nerves

Answer 2

Skeletal muscle fibers are innervated by large myelinated nerve fibers that originate from large motoneurons in the anterior horns of the spinal cord.
Each nerve fiber, after entering the muscle belly, normally branches and stimulates from three to several, hundred skeletal muscle fibers.
Each nerve ending makes a junction, called the neuromuscular junction, with the muscle fiber near its midpoint.

Question 3

Describe the structure of the presynaptic membrane of the NMJ

Answer 3

Neuronal membrane featuring voltage-gated calcium channels and docked acetylcholine-filled vesicles that are ready for immediate exocytosis
The nerve fiber forms a complex of branching nerve terminals that invaginate into the surface of the muscle fiber but lie outside the muscle fiber plasma membrane.
is covered by one or more Schwann cells that insulate it from the surrounding fluids
Axonal terminal contains many mitochondria needed for synthesis of ACh
Once ACh synthesized (in the axoplasm) is absorbed rapidly into many small synaptic vesicles, about 300,000 of which are normally in the terminals of a single end plate.
presynaptic membrane has dense bars opposite the nicotinic receptors on the crests of the postsynaptic folds.
Some acetylcholine vesicles line up on either side of the dense bars to form active zones.
vesicles in these active zones are immediately available for release; the others form a reserve of acetylcholine.

Question 4

Describe the structure of the synaptic gap

Answer 4

Narrow (values range from 500 Å - 70nm) space filled with basal lamina proteins
large quantities of AChesterase which destroys acetylcholine a few milliseconds after it has been released from the synaptic vesicles.

Question 5

Describe the structure of the postsynaptic membrane (motor end plate)

Answer 5

Deeply furrowed/ invaginated membrane with synaptic gutters / troughs, 8-10x the surface area of the presynaptic membrane
At the bottom of the gutter are numerous smaller folds of the muscle membrane called subneural clefts, greatly ↑ the surface area at which the synaptic transmitter can act.
The acetylcholine-­ gated ion channels are located almost entirely near the mouths of the subneural clefts lying immediately below the dense bar areas, where the acetylcholine is emptied into the synaptic space.
The voltage-­ gated sodium channels also line the subneural clefts.
Covered in nicotinic acetylcholine receptors (10,000 per μm2).

Question 6

Draw and label a diagram of the NMJ

Answer 6

Question 7

Describe action potential transmission at the NMJ

Answer 7

An action potential arrives via the motor axon (a 100-120 m/s) wave of depolarisation spread by voltage-gated sodium channels in the cell membrane

At the presynaptic membrane, voltage-gated calcium channels open in response to membrane depolarisation

The intracellular calcium activates SNARE proteins which open acetylcholine vesicles via exocytosis

Acetylcholine spills into the synapse (abundantly, with a safety factor of 3-5 times the minimum amount required to achieve endplate depolarisation)

vesicular exocytosis hypothesis proposes that a motor nerve action potential evokes the exocytosis of about 60 vesicles each containing about 10,000 molecules of ACh., which produce the muscle end-plate potential (EPP).

5x in the amount of transmitter released at the neuromuscular junction

During its synaptic dwell time, ACh diffuses across synaptic cleft and binds to nAChRs on the myocyte membrane.

The nACh receptor is a pentameric ligand-gated cation channel, and when 2 ACh molecules bind simultaneously to the 2 binding sites on the α subunits → opens channel → allows influx of Na+ (and Ca2+and efflux of K+) → depolarises the motor end plate

The depolarisation associated with the spontaneous, random release of a single vesicle of ACh is called a miniature end plate potential (MEPP) → depolarisation spike of 0.5mV amplitude

In contrast, when a nerve impulse evokes release of a large quanta of ACh → a much larger summative end plate potential (EPP)

EPP depolarises the resting membrane potential (–70mV) past the threshold (–50mV) for activation of the surrounding voltage-gated Na+channels→ action potential along the muscle fibre cell membrane

AP is propagated down the t-tubule system → release of Ca2+ from sarcoplasmic reticulum

↑Intracellular [Ca2+] → excitation-contraction coupling

Whole sequence of events occurs within 5-10ms

Question 8

Is there a positive feedback loop during action potential transmission?

Answer 8

Presynaptic Positive Feedback Loop
ACh released into the synaptic cleft also binds to pre-junctional nACh receptors → accelerates the resynthesis of ACh and packaging into vesicles.

Question 9

Describe how the NMJ system is reset following an action potential

Answer 9

ACh in the synaptic cleft is rapidly broken down by acetylcholine esterase (AChE) — an enzyme which is tethered to the post-junctional membrane and also free floating in the synaptic cleft — to choline and acetate, which are taken up by the nerve terminal and resynthesised into ACh

In fact, a large proportion of the released ACh is broken down before it even reaches the post-junctional ACh receptors

The cation channel of the nACh receptor closes

The Na/K ATPase pumps out the excess Na+

K+ leaks out of the cell via K2p leak channels

unlike in the neuron, for myocytes the chloride concentration gradient plays a larger role.

Due to the need to maintain polarity in the face of repeated depolarisations.

The myocyte leaks potassium each time the nicotinic channels open, and with enough of these events the potassium gradient would diminish, reducing the potential difference across the membrane and fixing it in a depolarised state (similar to the effect of suxamethonium).

the membrane potential returns back to baseline

Repolarisation cycles the surrounding Vg Na+ channels from the inactivated back to the resting state

The NMJ is now ready to respond to the next nerve stimulus

Question 10

How is ACh synthesised?

Answer 10

synthesized in nerve axoplasm from
choline
- Diet
- liver synthesis
- Reuptake from synaptic cleft after breakdown of ACh
- and is taken up by membrane carrier mechanisms, which are blocked by several quaternary ammonium compounds including hemicholinium and triethylcholine.
acetyl coenzyme A
- produced in the mitochondria of axon terminals from pyruvate and coenzyme A
- Process is catalysed by pyruvate dehydrogenase.
reaction is catalysed by choline O-acetyltransferase
- is produced in the nerve cell body and transported to the axon.
- activity is increased by steroid administration
Nerve stimulation augments acetylcholine synthesis by increasing intracellular sodium concentration.

Question 11

How is ACh stored?

Answer 11

Mainly stored in vesicles in the nerve terminal.

The active transport of acetylcholine into vesicles involves an ATPase and can be inhibited by vesamicol, tetraphenylboron and quinacrine.

~ 80% of the acetylcholine in the nerve can be released by action potentials, and this represents the amount in vesicles.

~10,000 molecules of ACh in each vesicle

The other 20% cannot be released and forms a stationary store.

Another surplus store can be detected only when intracellular cholinesterase is inhibited by physostigmine.

The stationary and surplus stores consist of acetylcholine dissolved in the cytoplasm.

Vesicles contain: acetylcholine, ATP, vesiculin, cholesterol, phospholipids and calcium.

Question 12

ACh is used as a neurotransmitter in what locations?

Answer 12

NMJ
Preganglionic parasympathetic and sympathetic fibres
Post ganglionic parasympathetic and sympathetic fibres (sweat glands)
CNS

Question 13

What is the quantal theory of ACh release?

Answer 13

one vesicle empties, releases 1500 acetylcholine molecules and produces one MEPP 0.5–1 mV, are spontaneous, random and of constant amplitude

Question 14

Factors that ↑ MEPP frequency

Answer 14

AChesterase inhibitors (also ↑ amplitude)
↑ [Ca2+]ECF
↓ [Mg2+]ECF
Theophylline
Catecholamines
cardiac glycosides
The venoms of the black widow (α-latrotoxin) and the Australian redback spiders increase MEPP frequency greatly by enhancing vesicle discharge and emptying the nerve of acetylcholine

Question 15

Factors that ↓ MEPP frequency

Answer 15

Curare (abolishes)
Botulinum toxin from Clostridium botulinum
inhibits acetylcholine release from cholinergic nerves, through the proteolytic cleavage of SNAP-25, a protein essential for acetylcholine vesicle fusion and release
Aminopyridines may be used to treat the paralysis
β-bungarotoxin,
Australian tiger snake venom (notexin),
Adenosine
GABA

Question 16

How is ACh inactivated?

Answer 16

rapidly destroyed by the enzyme acetylcholinesterase
Hydrolyses acetylcholine to choline and acetate
Anionic site
A negatively charged site that attracts the positively charged quaternary amide head of the ACh molecule
Esteratic site
The acetylcholine molecule is broken at the ester bond to form choline, while the acetyl group is transferred to the esteratic site of the enzyme
The short time that the acetylcholine remains in the synaptic space—a few milliseconds at most—normally is sufficient to excite the muscle fiber. Then the rapid removal of the acetylcholine prevents continued muscle re-­excitation after the muscle fiber has recovered from its initial action potential
The choline is recycled: it is taken up into the nerve terminal and reused in synthesis of new ACh

Question 17

Postjunctional nACh receptor structure

Answer 17

a pentagonal array of 5 subunits (labelled ⍺, ß, δ, 𝛄 and 𝜀). 2x identical ⍺ subunits and these each contain a binding site for acetylcholine.
α-subunits have adjacent cysteines essential for ACh binding (α-δ and α-ε/γ interfaces).
17 nicotinic subunits have been cloned.
Simultaneous binding of 2ACh molecules needed (but not sux).
Highly concentrated, and in close proximity to ACh-releasing sites (~10,000/μm2)
The clinically used neuromuscular blocking drugs also bind to this site on the ⍺ subunits.
Protein complex with MW 275,000
Fetal AChR composed of 5 subunit proteins: 2x ⍺, 1x β, δ, 𝛄
Adult AChR 𝜺 (E for elderly) replaces 𝛄 (y for young)
Subunits penetrate both sides of plasma membrane
lying side by side in a circle to form a tubular channel

Question 18

Difference between mature and foetal nAChR

Answer 18

Mature / Adult receptors
Net driving force for outward movement of K+ is near zero due to electrical gradient.
For Na+, both the concentration and the voltage gradients act in the same direction → large intracellular movement of Na+
For Ca2+, directional gradients are similar to that of Na+ but extracellular concentration is much lower → significantly less movement into cell.
stable metabolically, with a half-life ~ 2 weeks

Immature/foetal receptors
Proliferation caused by: when there is deprivation of neural influence or activity, as in the fetus or after denervation
Denervation injury (e.g. burns/trauma, spinal cord injury, UMN/LMN lesions,).
no longer localized to the endplate region but are inserted throughout the muscle membrane into the junctional, as well as extrajunctional, area, ∴ also referred to as extrajunctional receptors
Prolonged block of mature receptors at NMJ.
Compared with mature receptors:
↑ sensitivity to ACh and suxamethonium.
1/10 -1/100 of dose can effect depolarisation
↓ sensitivity to non-depolarising muscle relaxants.
half-life ~24 hours
smaller single-channel conductance, but a 2- to 10-fold longer mean channel open time.

Question 19

Postjunctional nACh receptor function

Answer 19

channel remains closed until 2 ACh molecules attach to the two ⍺ subunits, causes a conformational change that opens the channel,

ACh gated channel diameter of 0.65nm. Large enough to allow the important cations—sodium (Na+), potassium (K+), and calcium (Ca2+)—to move easily through the opening. Thousands transfer per millisecond

Anions e.g. Cl- do not pass through because of strong negative charges in the mouth of the channel that repel these negative ions.

Question 20

What ion flows through the nAChR channel in the largest concentration

Answer 20

far more Na+ ions flow through the ACh-­ gated channels than any other ions for two reasons:
there are only 2 positive ions present in large concentrations—Na+ in the ECF and K+ in the ICF.
the negative potential on the inside of the muscle membrane, −80 to −90 millivolts, pulls the positively charged sodium ions to the inside of the fiber while simultaneously preventing efflux of the positively charged potassium ions when they attempt to pass outward.

Question 21

Describe the NMJ end plate potential, thresholds and draw a diagram

Answer 21

Sudden influx of sodium ions into the muscle fiber when the ACh-­ gated channels open causes the electrical potential inside the fiber at the local area of the end plate to ↑ in the positive direction as much as 50 - 75 millivolts, creating a local potential called the end plate potential.
a sudden increase in nerve membrane potential of more than 20 to 30 millivolts is normally sufficient to initiate more and more sodium channel opening, thus initiating an action potential at the muscle fiber membrane.

Question 22

Compare the Muscle EPP to the action potential current flow

Answer 22

Question 23

Structure and function of presynaptic nAChR

Answer 23

α3β2
ACh released into the synaptic cleft also binds to pre-junctional nACh receptors → accelerates the resynthesis of ACh and packaging into vesicles
Responsible for ↑ ACh release into synaptic cleft during high frequency stimulation of the presynaptic nerve terminal (positive feedback)
This contributes to the high margin of safety
Inhibition by non-depolarising NMBDs during high frequency repetitive (i.e. tetanic) stimulation causes typical tetanic fade phenomenon.
Also causes fade with TOF.
Not inhibited by suxamethonium (rather, is activated by it → muscle fasciculations)

Question 24

Structure and function of other nAChRs

Answer 24

Ganglionic type
α3β4 i.e. (α4)3(β2)2
Brain type: α4β2
Important in ascending reticular activating system (ARAS)
Drugs must cross blood-brain barrier (lipid soluble)
Agonist: nicotine -> stimulation (note addictive)
Antagonist: volatile anaesthetic/ketamine -> sedation, hypnosis

Question 25

Describe the muscle action potential

Answer 25

In striated muscle, the action potential propagates over the cell surface in a similar manner to nerves. The same as transmission of APs in nerve except for a few quantitative differences: The resting membrane potential is about −80 to −90 millivolts in skeletal fibers, about 10 to 20 millivolts more negative than in neurons. The duration of the action potential is 1 to 5 milliseconds in skeletal muscle, about five times as long as in large myelinated nerves. The velocity of conduction is 3 to 5 m/sec, about 1/13 the velocity of conduction in the large myelinated nerve fibers that excite skeletal muscle. Action potentials spread to the Interior of the Muscle Fiber by Way of Transverse Tubules skeletal muscle fiber is so large that action potentials spreading along its surface membrane cause almost no current flow deep within the fiber. Maximum muscle contraction, however, requires the current to penetrate deeply into the muscle fiber to the vicinity of the separate myofibrils. This penetration is achieved by transmission of action potentials along transverse tubules (T tubules) that penetrate all the way through the muscle fiber, from one side of the fiber to the other, The T tubule action potentials cause release of calcium ions inside the muscle fiber in the immediate vicinity of the myofibrils, and these calcium ions then cause contraction.

Question 26

What features contribute to a 'high margin of safety' at the NMJ

Answer 26

Excess ACh released Far in excess of the amount required to depolarise the motor end plate 50% metabolised while diffusing across synaptic cleft - hydrolysis occurs nearly at the rate of diffusion Excess number of ACh receptors The number of nACh receptors at the motor end plate greatly exceeds the minimum number needed to be able to depolarise to threshold only 10% of the nACh receptor channels need to be open for the influx of cations to be enough to depolarise the membrane to threshold Up to 70% of nACh receptors can be blocked by antagonists without impairing transmission Need 75% of receptors blocked for reduction in twitch height After blocking the initial 75% of the receptors, the effect (twitch depression) with continued relaxant administration is relatively more rapid. The “iceberg phenomenon.” At least 95% receptor occupancy is necessary for complete suppression of twitch more than 95% of receptors must be blocked to facilitate intubation NAChR has a half life of 14 days With repeated nerve stimulation, for each consecutive nerve impulse, slightly less ACh is released pre-synaptically as there is insufficient time to resynthesise and repackage the ACh into the vesicles transmission of the signal is not hampered because of the aforementioned reasons

Question 27

What is the spare receptor concept and how does it affect dosing of NMBDs

Answer 27

only 10% of the nACh receptor channels need to be open for the influx of cations to be enough to depolarise the membrane to threshold, Need 75% of receptors blocked for reduction in twitch height, with > 95% of receptors must be blocked to facilitate intubation. Subsequent dosing should be reduced to 1/4 or 1/5 of intubating dose as a large proportion of receptors will still be occupied despite return of neuromuscular activity Priming involves a small dose prior to the intubating dose to occupy spare receptors and increase speed of onset

Question 28

Drugs that modify ACh release

Answer 28

Calcium entry modifiers Drugs that prevent Ca2+ entry into motor nerve terminal can impair neuromuscular transmission and prolong non-depolarizing neuromuscular blockade. Mainly reported with the aminoglycosides and polymyxins (both are antibiotic classes), but may also occur with various other antibiotics (e.g. colistin, tetracyclines, lincomycin). Patients with myasthenia gravis especially at risk. Calcium salts can reverse antibiotic-induced neuromuscular blockade. Local Anaesthetics ↓ Na+ entry into motor nerve fibres and prevent conduction of the nervous impulse → ↓ Ca2+ entry into motor nerve terminal → ↓ ACh release. After systemic administration or absorption, they affect conduction in unmyelinated nerve endings Conduction blockade of major or minor nerve trunks may prevent transmission of nerve impulses from anterior horn cells. Inhalational Anaesthetics ↓ release of ACh Profoundly affects degree of neuromuscular blockade. CNS depression → ↓ generation of nerve impulses. Direct effects on the muscle cell membrane. CVS effects may modify pharmacokinetics of NMBDs.