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The neuromuscular junction physiology

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Radmilo J Jankovic Aleksandar N Nikolić

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THE NEUROMUSCULAR JUNCTION PHYSIOLOGY

Radmilo J. Janković, Aleksandar N. Nikolić; Anaesthesia, Reanimation & Intensive care Center;
General Surgery Clinic, Clinical Center Nis

The mammalian neuromuscular junction is one of the most studied and best explained of all the
synapses. It can be most conveniently divided into presynaptic, synaptic, and postsynaptic phase. The
presynaptic component of neuromuscular junction is a position where the synthesis, storage, cycling
and release of acetylcholine are taking place. The synaptic cleft is the area between the nerve and the
muscle cell with the strategic localization of acetyl cholin esterase, which, with its fast catalytic activity
and rapid hydrolysis of released acetylcholine, thereby controls the duration of receptor activation.
Specific receptors appear on the postjunction membrane which in fact represent the specific cationic
channel where instant movement of ions appears after the linking of acetylcholine. Ionic gradient
spreads formed action potential downstream through the entire sarcolemma causing the muscle to
contract.

Mammalian neuromuscular junction (NMJ) is one of the most studied and best explained of all the
synapses. NMJ represents a bond between myelinated motor nerves and skeletal muscles. This
structure is integral part impressive biological amplification system which turns action potential of
nerves into muscular contraction (1). Healthy NMJ enables efficient functioning of an important link of
the 'basic triad' of the general anaesthesia which is immobility or surgical relaxation. Muscle relaxants
affect directly to the NMJ, and they are nowadays widely used in many balanced techniques of general
anaesthesia, NMJ can most conveniently be divided into presynaptic, synaptic and postsynaptic part,
with respect to the structure and function.

PRESYNAPTIC PART

Every myelinated motor axon, by reaching its target muscle, branches out to the great number of
terminal fibers (20-100). Each of these fibers innervates individual muscular fiber. Combination of the
motor axon`s terminal fibers and belonging muscular fibers is called motor unit. Terminal fibers
contain potassium (K+) and sodium (Na+) channels which control the duration and amplitude of the
action potential.

As opposed, nerve terminal contains a small number of Na+ channels, hence action potential continues
passively at this point. Synaptic vesicles (SV) are located in the nerve terminal. Each SV has 5000-
10000 molecules of neurotransmitter acetylcholine (ACh) deposited. Neurotransmitter is packed
together with the molecules of ATP (adenozine triphosphate) and proteoglycan in the vesicles, added to
ions H+, Mg2+ and Ca2+. Molar ratio in the vesicles between ACh and ATP is in the range from 10:1
to 1:1 (2, 3). It is generally accepted opinion that synthesis and release of ACh is an entire set of events.
ACh is synthetised from the Acetyl coenzyme A (Acetyl CoA) and cholin in the nerve terminals
cytosol, in a enzyme acetylcholine transferaze mediated reaction. ACh transfer into vesicles is mediated
by Mg2+ dependable proton pump which is used to link the ACh molecules to the transportational
protein which diffuses through vesicles` membrane in both directions exchanging each ACh molecule
with hydrogen ion. Content of each SV is marked as neurtransmitter quantum. In absence of action
potential SV`s are trapped in the filamentous network containing of actin, synapsin and spectrine (4.5).
Modern findings point us to conclude that there are different types of ACh deposited neurotransmitting
vesicles.
Approximately 1% of vesicles forms immediately available ACh reserve within the active pool, which
is responsible for permanent discharge of neurotransmitters during low nerve activity. On the other
hand, around 80% of SV is a reserve pool which releases ACh as a response to nerve stimulus. The rest
of the SV is stationary reserve. Ca2+ channels open when nerve impulse reaches terminal axon,
Calcium ions enter terminal nerve ending and Ca2+ dependable discharge of ACh from approximately
50 to 100 vesicles occurs. It is necessary that ACh vesicles are anchored into special spots of the
terminal axon called active zones in order for the process to take place smoothly. These zones are
located opposed to post synaptic ACh receptors. These vesicles make immediately available reserves of
ACh. As soon as content of these vesicles is discharged, new amounts of neurotransmitters are
compensated from reserve pool. Vesicles of the reserve pool are tied in the cytoskeleton for vesicular
protein synapsine by the actin fibriles. A part of Ca2+ ions, which enter terminal nerve ending as a
result of action potential appearance, link to the calmuduline, what activates protein kinase II. Protein
kinase phosphorilates synapsin which then dislocates from reserve vesicles enabling their movement
towards the place of discharge, active zones of the terminal nerve ending respectively. Several enzymes
are included in the vesicles docking process within the active zones, consequently discharging synaptic
content. Primarily, those are Ca2+ linking proteins synaptotagmin and synaptobrevin, followed by a
25kDa synaptol protein named SNAP-25 and syntaxin, intergral protein of the terminal axon
membrane, which is dominantly present in the active zones. For a successful anchoring process of
synaptic vesicles, interaction between synaptobrevin, syntaxin and SANP-25 is necessary. This
formation is also known as SNARE complex (6). Synaptotagmin functions as a vesicular ramp in
absence of Ca2+ ions holding vesicles in pre expulsion tension and preventing discharge of vesicles`
content. Family of low molecule guanosine triphosphate linking proteins, called rabs, has an important
role in joining membranes and exocytosis of the SV in addition to synaptotagmin. Thus the specific
protein Rab3A is essential for quantitative sustainability of SV`s reserve pool, as well as accelerating
the exocytosis process in cases of repeated simulations (7). ATP synthasis is activated by including
Ca2+ and its linking to synaptotagmin, and it becomes a part of active zone complex. Hidrolysis of ATP
synthasis leads to discharging the content out of anchored vesicles, together with linking
synaptotagmin. SV`s proteins can be a target for toxins as in case of α-latrotoxin, which is a
neurotoxin of a black widow spider.

Clostridium neurotoxins lead to splitting SNAP 25, syntaxin and synaptobrevin, what results in SV`s
exocytosis inhibition (8). SVs are, after the process of exocytosis, renewed in the process of
endocytosis. Since all SVs contain unique proteins, the process of SV recycling must result in renewing
these proteins. Modern physiology suggests 3 possible explanations of SV recycling, with kiss-and-run
theory having the greatest foothold. Betsy and Wu announced a theory, wanting to explain instant
renewing of SV after the exocytosis, which states that discharge of vesicle`s contents through the
fusional cleft runs really fast, in milliseconds. After the cleft closes, SV takes cytosol`s ACh using
active transport, and it is ready for a new round of events. This theory implies that SVs do not lose their
structural integrity during the exocytosis. New SVs are set in immediately available reserves after the
recycling, and they can be part of expulsion of neurotransmitters the same way as SVs received from
reserve pool (9). The entire cycle lasts approximately 1 minute (10).
Discharging ACh takes place either spontaneously or as a response to a nerve stimulus. There are 4
different modalities of ACh release: quantal, subquantal, comprehensive and a molecular leaking
mechanism. In absence of action potential, miniature potentials of final motor plate appear, 0,5 – 1 m in
size. It is considered that these miniature action potentials appear as direct consequence of quantal
release of the ACh. Quantal release of the ACh occurs after exocytosis of synaptic vesicle, while
subquantal occurs after exocytosis of insufficiently fulfilled vesicle. Comprehensive release occurs
when a big part of axoplasm is extruded from motor neuron. Molecular leaking occurs by low intensity
diffusion of ACh over neural membrane.
SYNAPTIC PART

Synaptic cleft is almost 50nm wide space which is located between nerve terminal and postsynaptic
membrane. ACh diffuses through synaptic cleft in milliseconds after release from main nerve terminal.
However, almost a half of total released ACh never reaches its goal on a postsynaptic membrane
because it quickly decomposes to choline and acetate under the influence of acetylcholinesterase
(ACHE), enzyme which is strategically located inside the synaptic cleft. Specific transport system
repeats taking the choline in the cytosol terminals, making it available for ACh resynthesis. ACHE
belongs to B subtype of carboxylesterase (enzyme classification 3.1.1.7) and represents one of the most
efficient catalytic systems in nature. ACHE hidrolisys capacity is massive, almost diffusion limited, and
it implies hydrolisis of aproximately 4000 ACh molecules per active zone in a second (11). ACHE is
coded by a gene located on a 7q22 chromosome at people. This enzyme is located in its assymetric A12
form in a synaptic cleft, which consists of 3 tetrameric subunits assimetrically conneted with junctional
basal lamina (12). high concetration of this enzyme limits the activity of ACh, preventing excessive
activation bith presynaptic and postsynaptic nicotine cholinergic receptors (PNHR).

ACHE activity is partly regulated by muscular activity. Active, fast muscles express several times
bigger ACHE levels in comparison to slow and inactive muscles. This phenomenon correlates with
relative abundance of specific mRNA in those muscles. Excitable membrane depolarization blocking
drugs, e.g. Tetrodoxin, at the same time reduce ACHE accumulation in the synaptic cleft (13). On the
other hand, Na+ channel agonists, e.g. Veratridine, dramatically increase collection of ACHE (14).
Apart from ACh hydrolisis, ACHE also has different and very significant role in sustaining integrity of
the NMJ such as nerve growth promotion, activity modulation of activity and PNHR. Except ACHE,
significant number of other komplex proteins is present inside the synaptic cleft, which affect forming,
grouping and integrity of the PNHR (15). ATP is released together with ACh, and it is subject to instant
hydrolisis into adenosine which links to the prejunctional P1 purinoceptors in the synaptic cleft.
Activation of the P1 receptors depresses neuromuscular transmission across the G protein mediated
block of the Ca2+ channel (16).

POSTSYNAPTIC PART

Postsynaptic membrane pulls itself in the secondary synaptic folds on the top of which are located
grouped together PNHRs in the concentration of ≈ 20000 receptors per µm. PNHR concentration is a
thousand times smaller outside the motor end plate. ACh receptors are postjunctionally placed in the
frame of ending plateau and they are nicotine type dominantly. It is known today that mammalian
PNHR consists of 5 protein subunits, joined together so they create a cylinder which is prominent on
the both sides of the postsynaptic membrane. Each protein subunit is coded by a particular gene and
each of them has different physical and chemical properties, including different molecular weight.

Different PNHR subunits are marked with the Greek alphabet letters: α, β, γ, δ and ε. α subunit is the
first isolated protein molecule of the postsynaptic membrane. Further analysis of the amino acid
sequences and PNHR ligand affinities revealed that N and C amino acid endings go in front of the
receptor structure in the synaptic cleft, while the rest of the repeated hydrophobic amino acid sequences
form 4 transmembrane helices marked M1-M4 (17) (fib.1a). 9 different types of the α subunits (α1-α9),
4 different types of β subunits, and one subunit of γ, δ and ε each were revealed until today.
However, every mature adult PNHR consists of the exact order of protein subunits: double α, and one
β, γ, δ and ε subunit each ( 2α1β1δε). Presynaptic α3β2 nicotine cholinergic receptors function as
autoreceptors, securing sustainable release of the neurotransmitters on the NMJ level, in the high
demand conditions for ACh. Classical TOF fade phenomenon occurs just as a result of this receptors`
inhibition by nondepolarizing muscular relaxants. On the other hand, well known depolarizing
neuromuscular transmission blockator succinylcholine does not indicate any affinity towards these
receptors (18). In the fetal stage, γ subunits are present istead of ε subunits (2α1β1δγ)(fig. 1b).

Figure 1: Postsynaptic nicotine cholinergic receptor (PNHR)

figure 1a: Order of amino acid remainings, building specific AH linking space, which juts out of the
receptor surface into synaptic cleft, while subunits M2 helices build cationic channel
figure 1b: Order of the protein subunits at fetal PNHR (above), and mature, adult PNHR (below)

Expression regulation of humane γ and ε genes together with these receptor subunits` way exchanging
is insufficiently explained today. Today, we know for sure that mRNA levels coded by γ and ε subunits
exchange reciprocally immediately after birth. Complete exchange of γ PNHR gradually completes
within the first 3 weeks of life, during the dynamic phase of synaptogenesis, respectively (19, 20).

Activation of the ε PNHR increases the Ca2+ concentration in the subsynaptic cytoplasm. As Ca2+
represents a very important secondary messenger, it is possible that exchanging γ into ε subunit and
consequent activation of the ε PNHR, which brings a high influx of Ca2+, represents another local
regulatory mechanism determining the architecture and NMJ functionality (21). On the other hand,
overactivation of the ε PNHR can overwhelm ending plateau with Calcium ions and initiate
degenerative processes by multiplying Ca2+ activated phospholopasis, DNase and calpain.

Membrane lipids and cholesterole have extremely powerful influence on the functionality and the
integrity of PNHR, in addition to Ca2+. A series of experimental studies point at neccessity of
cholesterole rich postsynaptic membrane for PNHR`s full activity (22). It seems that aforementioned
influence of the cholesterole is not carried out over the main lipid mass of the postjunction membrane,
but over specific cholesterole linking spots outside the lipid-protein matrix. Those specific spots are in
the frame of M1 and M4 helices of γ subunit. The Organichlorid insecticides can block PNHR by
incompetitive linking to these specific spots (23). PNHRs are synthetised inside the muscular cells by
means of petal circular allocation of protein subunits, creating a specific cylindrical shape.
Extracellular linking space used for linking of the ACh and similar agonists is formed by means of this
grooping, as well as nicotine receptors. Each receptor spreads transmembrane, on the both sides of
postjunctional cellular membrane. A central tunnel, which represents specific cationic channel, is
formed by specific linking of protein subunits. M2 helice of each subunit borders the inside of the
cationic channel (23). Ionic channel is 4 nm in diameter at its widest spot at the enterance of the
channel itself. After that, diameter gradually reduces below 0.7 nm around cellular membrane.
Receptor`s complex is 11 nm long, half of which juts out of the cellular membrane towards the synaptic
cleft. Only 2 nm of transmembrane nicotine AChR widens inside cytosol of the muscular cell.
Several specific proteins seem to have an important role in linking receptors themselves to the
cytoskeleton, with rapsyn and agrin having the most important role.
When 2 ACh molecules link to pentameric complex, it causes conformation changes inside the subunits
themselves, primarily in the α subunit, what causes the channel opening. On its widest part, which is
inside the synaptic cleft, channel is wide enough so it enables smooth ion movement in both directions.
Potassioum ions leave the muscular cell towards the synaptic cleft, but their intensity and speed are
smaller in comparison to the movement of Na+ ions, which quickly enter the muscular cell in a process
mediated by an open ionic channel. The consequence of potassium`s leaving the cell is
electronegativity inside the cell itself and membrane inactivity potential of 80 mV. Entering of Na+ into
the cell causes reduction of electronegativity. When inaction potential correction reaches value of 50
mV, voltage dependent Na channels inside the sarcolemma open, what causes even higher Na+ influx
into the muscle. This phenomennon increases the depolarisation intensity leading to creation of the
action potential which spreads over the muscular fiber surface towards transverse tubules, particularly
rich with Ca2+ channels. Dihydropyridine receptors (DHPR), acting as voltage sensors, are placed in
the transverse tubule system. They detect potential difference and open adjacent type 1 ryanodine
receptor Ca2+ channels (Ry-R1).

Linking DHPR and RyR1 causes release of great Ca2+ amount from the sarcoplasmic reticulum,
resulting in muscular contraction (24, 25). Transforming the electric signal on the muscle surface into
release of intracellular Ca2+ of the sarcoplasmic reticulum is also known as excitation-contraction
coupling. Linking Ca2+ ions to the troponin complex changes interaction between tropomyosin and
contraction mechanism, enabling correct link between actine molecules and myosin heads. That way
muscular contraction occurs through fine slide of the myofilaments. As Na+ channel activation
weakens, Cl- enters the cell through slower, voltage dependent chloride channels. This phenomenon
causes reversing of the postsynaptic membrane potential to the previous inaction potential of
approximately -70 mV to -90 mV. ACh amount released under influence of nerve action potential far
exceeds the one needed to cause excitation of ending motor plateau. Only 5-26% of secreted ACh is
enough to excitate the opening of ionic channels.

Activated PNHR remains opened barely for one month, and more than 105 ions of Na+ enter the cell in
that period. Nicotine receptor and its channel represent powerful amplificator which turns the initial
stimulus produced by 2 ACh molecules into directed movement of thousands of ions of Na+, K+ and
Ca2+. On the other hand, these specific receptors act as a kind of physiological ramp to ion movement.
Channel is opened until ACh links itself to specific places on the receptor, after which channel opens in
order to pass through the stimulus. However, channel closes again as soon as ACh leaves the receptor
(26).
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