Describe the physiological structure of the neuromuscular junction in skeletal muscle

1. Nerve terminal

2. Synaptic vesicles

3. Active zones

4. Synaptic cleft

5. Postsynaptic membrane folds

6. Postsynaptic membrane receptors

1.   Nerve terminal – at the termination of the nerve (motor neuron), unmyelinated banches of axon insert into grooves along the muscle surface. (Terminal bouton)

      2.   Synaptic vesicles – membrane vesicles inside the nerve terminal containing the neurotransmitter acetylcholine (“ACh”)

      3.   Active zones – ACh is released from synaptic vesicles at specialized regions of the presynaptic membrane called active zones located opposite the postsynaptic membrane folds.

      4.   Synaptic cleft – a space of 20-30 nanometers between the nerve and muscle membranes

      5.   Postsynaptic membrane folds – the membrane surface area on the muscle side of the cleft is increased by the presence of many folds

      6.   Postsynaptic membrane receptors – ACh receptors

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1.  Neurotransmitter molecules are synthesized and packed in vesicles

2.  An action potention arrives at the presynaptic terminal and drives membrane potential to +50mV

3.  Depolarization of the membrane opens voltage-gated Ca+2 channels.

4.  Calcium travels down concentration gradient (mM outside terminal and μM inside) and increased locally at the active zones.

5.  Calcium triggers fusion of synaptic vesicles with the presynaptic membrane.

6.Transmitter molecules (usually Acetylcholine) diffuse across the synaptic cleft.  ACh either diffuses away, is degraded by ACh Esterase, or binds to ACh receptor

7.  Bound receptors activate the postsynaptic cell and triggers and end-plate potential by increasing membrane permeability to sodium and postasium (opening of nicotinic ACh receptor).

8.  The vesicles are recycled at the presynaptic terminal.

In skeletal muscle, ACh binds to nicotinic ACh receptors leading to membrane depolarization and muscle contraction.

In cardiac muscle, ACh binds to muscarinic ACh receptors leading to membrane hyperpolarization and a decrease in muscle contraction (decrease heart rate).

Acetylcholine (ACh)

1. Where is it synthesized?

2.  Where does nerve get choline?

3. What makes up acetylcholine?  Which enzyme makes it?

4.  Where is ACh stored?

1.   ACh is synthesized in nerve terminals

      2.   Choline is taken up by nerve terminals

      3.   Choline and acetyl CoA à ACh (enzyme = choline acetyltransferase)

      4.   ACh is taken up by synaptic vesicles (~10,000 molecules per vesicle)

Presynaptic calcium channels

1.  Where are they located?

2.  When do they open?

3.  What does an influx of calcium do?

      1.   Voltage-gated calcium channels are localized near the active zone in the presynaptic membrane

            Presynaptic membrane particles which visualized by Em fall in rows on either side of the active zone are likely calcium channels

      2.   Calcium channels open to allow calcium influx into the active zone upon depolarization

      3.   Entry of calcium into the active zone causes the synaptic vesicles to fuse and release ACh

Synaptic delay

1.  How long is the synaptic delay

2.  What does this time relate to?

3.  What causes the delay?

      1.   Synaptic delay of 0.5-0.8 msec.

      2.   Time between the arrival of an action potential at the nerve terminal and the initiation of the muscle endplate potential (EPP).

      3.   Most of the synaptic delay is due to presynaptic events leading up to release of ACh, with an additional small delay due to diffusion of ACh across the synaptic cleft

            Much less delay (<.1 ms) occurs in the response to ACh applied close to the endplate through a micropipette

Quanta

1.  Definition

2.  What is the name for the depolarization from one quanta?

3.  How many quanta does a presynaptic action potential usually release?

1.   Quantity of ACh contained in a single vesicle is called a quantum

2.   Spontaneous release of quanta occurs at rest giving rise to small postjunctional depolarizations called miniature endplate potentials (MEPP)

3.   A presynaptic action potential normally causes simultaneous release of about 125 quanta, sufficient to depolarize the muscle membrane well beyond threshold (“One to One Transmission”).

      ACh is released from vesicles of nearly uniform size containing a fixed quantity of transmitter molecules

Acetylcholinesterase

1.  What is it's function?

2.  Where is it present?

3.  What does it's action cause?

1.   Acetylcholinesterase is an enzyme which hydrolyzes ACh

      2.   Present in the synaptic cleft and concentrated at external surface of postsynaptic membrane

      3.   Endplate Potential is terminated by the hydrolysis of ACh

Endplate Potential (EPP)

1. Definition

2. Characteristic shape

3. Size

4. Time

5. Caused by

6. Ended by

1.   Transient depolarization of postsynaptic membrane

      2.   Fast rise, exponential decay

      3.   Size 10-30 mV

      4.   Time 5-10 msec

      5.   Caused by activation of ACh receptors at endplate by the simultaneous release of ACh from about 125 vesicles

      6.   Ended by hydrolysis of ACh

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See a fast rise, slow decay.  As the EPP travels away from the end plate, the EPP amplitude drops and there is almost no change in membrane potential 2.5mm away.

Miniature endplate potential (MEPP)

1.  What is it?

2.  When does it form?

3.  How often do these occur?

4.  How much does it depolarize the membrane?

5. What causes it?

6.  Effect of drugs

1.   Small transient depolarization of postsynaptic membrane.  (Does not cause muscle action potential)

      2.   Absence of nerve stimulation; it's the result of vesicles fusing by chance and then releasing contents

      3.   Approximately 1 per second

      4.   0.4 mV depolarization

      5.   Caused by spontaneous release of a small number of vesicles of transmitter into synaptic cleft

      6.   Same response to drugs as EPP

Aceylcholine receptor

1.  What does ACh when bound?

2.  What does an open channel cause?

3.  If an AP caused release of ACh, what will be the result?

4.  What is the channel permeable to?

1.   Binding of ACh to individual receptors, causes individual ionic channels to open

      2.   Opening the ACh-receptor channel permits current flow, depolarizing the muscle membrane (EPP).  Sodium flows into the muscle cell and potassium flows out of the muscle cell

      3.   Sufficient ACh is released by a single presynaptic action potential to depolarize the muscle to threshold whereupon a muscle action potential is triggered

      4.   Ionic basis of the endplate potential.  In contrast to the ion channels giving rise to the action potentials, ACh-receptor channel is permeable to both Na⁺ and K⁺ ions.  Na⁺ conductance is 1.7 times K⁺ conductance

1. What is the structure of an nicotinic acetylcholine receptor?

2. How many membrane spanning segments?

3.  Which subunits bind ACh?

4. How many ACh must bind to open the channel?

1.  Heteropentameric receptor; alpha (x2), beta, gamma, and delta.  Embryologically, the receptor has two epsilon subunits which are later replaced by alpha subunits.

2.  Each subunit has four membrane-spanning segments.

3.  Alpha subunits

4.  Each alpha must have an ACh bound (so two)

Pharmacology of neuromuscular transmission

1.  What kind of agonists can bind ACh receptors?

2.  What blocks the ACh receptor?

3.  What is the function of anticholinesterases?

4.  What prevents re-uptake of choline?

5. What blocks the Calcium channel?

6.  What blocks ACh release?

      1.   Other cholinergic agonists are also effective at binding to the ACh receptor and leading to channel openings (i.e. carbachol, nicotine) – can cause muscle spasms.

      2.   Curare, d-tubocurarine, and α-bungarotoxin (this one binds irreversibly) blocks neuromuscular transmission by binding to the ACh receptor, preventing ACh binding, and thereby preventing channel opening.  Curare does not affect the Na⁺ or K⁺ channels involved in action potential propagation.

      3    A variety of agents act as anticholinesterases (for example, neostigmine) inhibiting the breakdown of ACh and thereby increasing and prolonging the depolarization following transmission.  Physostigmine and DFP also block acetylcholinesterase activity

      4.   Hemicholinium prevents the re-uptake by the presynaptic terminal of choline (produced by the hydrolysis of ACh and used for its resynthesis) thereby leading to ACh depletion and failure of transmission

5.  ω-Conotoxin (made by a snail to paralyze fish).  An AP may come down the axon, but if the calcium channels are blocked there will be no neuromuscular transmission

6.  Tetanus toxin and botulinium toxin

Biochemistry of AChR

1.  What is the density at the endplate?

2.  Are the subunits homologous?

3.  What is the property of the N-terminus?

4.  What type of channel (cation or anion selective)

5.  Which subunits are required for function?

1.  Density of AChR at endplate 20,000/um² (Limit 50,000/um²)

2.  Yes, there is a high degree of homology (sequence) among subunits

3.  N-terminal portions are hydrophilic, glycosylated and exposed to extracellular space

4.  Cation-selective channel activated by ACh

5.  All subunits, α₂βγδ, are required for function

Neuromuscular junction

1.  Which three cells are at the NMJ?

2.  Describe the synaptic portions

3.  Where are the AChR genes expressed?

4.  What three signals affect AChR distribution?

1.   The NMJ comprises portions of three cells – motor neuron, muscle fiber, and Schwann cell

      2.   Synaptic portions of all three cells are highly specialized, containing high concentrations of molecules found at low concentrations extrasynaptically

      3.   Nuclei throughout newly formed myotubes express AChRs subunit genes, and AChRs are diffusely distributed on the myotube surface

      4.   The nerve then sends three signals to the muscle that affect AChR distribution

            a.  Agrin, which interacts with transmembrane protein tyrosine kinase (MuSK) to organize AChR clustering

            b.  Neuregulin, which interacts with erbB kinases to induce selective expression of AChR subunit genes by synaptic nuclei

            c.  Acetylcholine, which activates AChRs to generate a voltage- and calcium-dependent signal that represses AChR subunit gene expression in extrasynaptic nuclei

            d.  Together, these signals lead to selective synthesis of AChRs in synaptic areas and precise accumulation of AChRs in the postsynaptic membrane

Myasthenia gravis

1.  What is it?

2. Key characteristics (# of cases, sexes affected, muscles affected)

3.  Physiological studies (MEPP and ACh)

4.  Treatments

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1.  Autoimmune syndrome that results in paralysis because inability of the neuromuscular junctions to transmit signals - antibodies produced againts the nicotinic ACh receptor.

2.  5 cases per 100,000, generally affects women in their 20s and men in their 60s.  Likely affects cranial muscles like eyelids/eye muscles and limb muscles.  Severity varies within the course of a day, but there is no sign of denervation.

3.  MEPP are greatly reduced, but normal amounts of ACh.  Antibodies reduce the number of functional ACh receptors or impede ACh interaction.  Junctional folds are also reduced.

4.  Inhibitors of acetylcholinesterase (like neostigmine), steroids and immunosuppressive drugs (block autoimmune process), removal of thymus (might remove autoimmune initiation), and plasmapheresis (to decrease the patient's antibodies)

1.  There is an impaired release of ACh (so not an autoimmune antibody to AChR like in classic Myasthenia gravis)

2.  Anti-presynaptic calcium channel antibodies present.  This decreases the amount of calcium channels and decreases the amount of ACh released. 

3.  Similar symptoms because there is a decrease in neuromuscular transmission.

Describe Congenital Myasthenic syndromes

(several rare diseases)

1. Autosomal recessive

2.  Loss or functional mutation in the epsilon subunit of AChR, acetylcholinesterase, or collagen (ColQ)

3.  ColQ helps bind AChE to the site; a mutation causes less AChE at the site and less ACh recycled because it diffuses away and is not hydrolyzed to be recycled.

4.  If it is an epsilon mutation, the first couple years of life have epsilon as the subunit in AChR and then alpha is turned on.  If a doctor can manage the patient, the patient will cure themselves as there is a change in AChR subunit expression.

A patient with Eaton-Lambert myasthenia gravis was found to have antibodies to the   presynaptic calcium channel.  Pharmacological inhibition of __________ would be useful in treating this patient.

a. Acetylcholinesterase

b. Acetylcholine receptor

c. Voltage-gated calcium channel

d. Voltage-gated sodium channel

e. Calcium release channel

Activation of the acetylcholine receptor results in the depolarization of the post-synaptic membrane.  The activated acetylcholine receptor allows:

      a.   Sodium and potassium to flow into the muscle cell

      b.   Sodium and potassium to flow out of the muscle cell

      c.   Sodium to flow out of the muscle cell and potassium to flow into the muscle cell

d.   Sodium to flow into the muscle cell and potassium to flow out of the muscle cell     

      e.   Calcium to flow into the muscle cell

In order to increase the speed of synaptic transmission one needs to decrease the time for which step in neuromuscular transmission?

      a.   Opening of the acetylcholine receptor ion channel

      b.   Binding of acetylcholine to the acetylcholine receptor

      c.   Diffusion of acetylcholine across the synaptic cleft

     d.   Presynaptic events leading to the release of acetylcholine

      e.   Synthesis of acetylcholine