When dynamin is disrupted
vesicles do
not recycle and become
depleted
Wild-type
Dynamin mutant
Fly temperature-sensitive mutant
Endocytosis
Uncoating
Hsc70
Auxilin
ATP
ADP+Pi
Neurological conditions affecting the vesicle cycle
Congenital
myasthenic syndromes
(loss of
small vesicles)
Lambert-Eaton
myasthenic syndrome
(antibodies against Ca channels)
Clostridial toxins
(botulism-paralysis
tetanus-muscle spasms)
Signaling at the neuromuscular junction
(colored electron micrograph)
The Neuromuscular Junction
The motor neuron axon branches to form
multiple synapses (boutons) in the middle
of the muscle.
Each bouton is physically separated
from the muscle endplate by a
synaptic cleft 100 nm wide.
ACh filled vesicles and Ca2+ channels cluster
at active zones
A basement membrane lining the cleft contains
collagen and acetylcholinesterase an enzyme
which hyrolyzes ACh.
Each bouton lies above junctional folds
in the muscle membrane packed
with nicotinic acetylcholine receptors.
Depolarization of the muscle membrane
initiated by ACh receptor activation, then
opens voltage-gated Na+ channels
Autoradiograph of neuromuscular junction
Black grains
indicate binding
of radioactive
(-bungarotoxin) to ACh
receptors enriched at
top of junctional folds
(10,000 receptors/µm2).
-bungarotoxin extracted from the venom
of Braided Krait snakes, is a potent inhibitor
of some nicotinic ACh receptors
Reconstructed image of a nicotinic ACh receptor
8.5nm
Pore 0.8nm
Receptor is a complex of probably five subunits which together form a
channel
Toshima and Unwin, 1988
The muscle nicotinic ACh receptor is a typical ionotropic receptor
The receptor is made up
of five subunits; 2  and 3 non- 
Binding of 2 ACh molecules
opens the channel through a
conformational change
The inhibitory snake venom -bungarotoxin competes with ACh for binding sites
ACh receptor subunits have conserved domains
Subunits are 50% conserved
suggesting similar structure
M2 domains line the
channel pore
4 hydrophobic
transmembrane domains
of 20 amino acids form 4
alpha helixes
Functional model of the nicotinic ACh receptor
channel
Aligned negatively charged
amino acids (purple) flanking
M2 of each subunit form rings
that contribute to Ion selectivity
(repulsing anions)
Ring of
hyrdophobic
leucine residues
occlude the pore
where the M2 is
kinked inward
Conformational changes are
thought to reorientate the
residues within the pore, allowing
Ions to flow.
threonine/serine ring contribute ion
selectivity filter
Voltage-clamp experiments show the inward synaptic current
produced by motor axon stimulation
The endplate potential lags behind the ACh generated current
due to the charging of the membrane capacitance
The ACh receptor is permeable to Na+ and K +
At reversal
potential
the driving forces
for Na+ and K+ are
equal and opposite
so the net ion flow
is zero
At resting potential the
inward driving force for
Na+ is large and the
outward driving force
for K+ is small
Na+
K+
Na+
K+
IEPSP=gEPSP X (Vm - EEPSP)
Na+
K+
Na+
K+
ACh-gated channels conduct a square-shaped unitary current
On-cell patch clamp
Erwin
Neher
Burt
Sakmann
Unitary current at -90mV
is 2.7 pA(10-12A). This current
represents the passage of
~17,000 ions
The open time varies for
individual channels
Unitary current at -130mV
is 3.9 pA(10-12A).
Single open ACh receptors behave as simple resistors
The EPSC time course results from the summed contributions
of individual ACh-gated channels
ACh is released
in response to an action
potential
ACh is removed from cleft
rapidly(>1ms) by hydrolosis
(acetylcholine esterase) and
diffusion
Channels close randomly
in absence of ACh with
a mean open time of 1ms
At a typical endplate, the current is -500,000 pA and the single channel current is -2.7 pA
so ~ 200,000 individual ACh receptors open to make the smooth endplate current
Summary of single-channel currents, end-plate current and
end-plate potential
Summary of events underlying neurotransmission at the
neuromuscular junction