CHAPTER8

Synaptic
Transmission and
the Neuromuscular
f unction
Edward G. Moczydlowski

a.,:t:..

'.::__.rifi'.1,

The ionic gradientsthat cells maintatl lcLossrhrn mcmhranesprovide a

lbrm of stored electrochemicalenergy cells can use lor electrical signalling.
The combination of a resting membrane porential of -60 to g0 mV and
a diverse array of voltagegated ion channelsallorvs excitablecells to gener
ate action potentials that propagate over long clistancesalong the surlace
membrane o[ a singlenerve axon or musc]e fiber. However, another classol
mechanismsis necessaryto transmit such electricalinfomation from cell to
cell throughout the myriad of neuronal netu'orks that link the brain lrrrh
sensoryand effector organs. Electrical signals must pass acrossthe special
.\..^ ,h1^5:*p
b ..1
"l l

'.

,:..-- ):.'t
t
-';;t't'.

s3@

16n6112re, r . r r- , ,llc.l a

sy-napse.The process underifing this cell to-cell transfer of electr.icalsignals is tenned synaptic transmission. Communication betr,veencells at a
synapse can be erLher elecuical or chemical. Electrical synapscspror.icle
direct electrical continuity beti,r,eencells by means of gap lunctions,
u,hereaschemicaLslnapses link two cells iogether by a chemical neurotransmitter thaLis releasedtrom or.receLland diffusesto another.
ln this chapter tve discr-rss
the generalpropertjes ol synaptic transnissioli
and Lhen focus mainly on s1'napttctransmissionbenveen a notor neuron
and a skeletal muscle fiber. This interface betg'een the motor neuron ancl
the muscLecell is called Lhe neuromuscularjuncljon. In Chaprer 12, Lhe
locus is on synaplic transmission between neulons in the central nen'ous
s)'stem(CNS).

MEC}IANISMSOF SYNAPTICTRANSMISSION
ElectricalContinuity Between Cells ls EstablishedEither by
Direct FIowof CurrentThrough Gap lunction Channelsat
an ElectricalSynapseor by Diffusion of a Neurotransmitter
acrossa ChemicalSynapse
Once the cor.rceptol bioelectrlcitl' hacl taken l.iold among physiologistsof
l h e J I L . e - r . r r r . b e , a m c ,c , r ' h . r r| e q u c < l . o n h o u c l . " r r ' . a-l i g r a .
"l
lorv between ce1lsposed a lundamental biologic problem. lmagine rhar rn'o
cells lie side by sicle lvrthout any specializeddevice for commr,rnicrting
betweenthem. Funhermore,imaginethat a l1at,20-pcm2
membraneareaoI
the first, or presynaptic, cell is separated-by 15 nm-from a stu.iilar
area of the seconcl,or posts)'naptic, cell. In his classic book on electro
physiology, Katz caLculatedthat a voltage signal aL the presynapricmem
brane would suffer ] 0.000-fold atLenuarionin the postsl-napricmembranc.

- _--:'

-l
and the NeuromuscLrlar
SvnapticTransmission
lunction / 8

TABLEA-I

CHEMI(AL
ELICTRICAL

lonotropi(

Metabotropic

Agonist

None

e.9.,ACh

e.9.,ACh

Membrane
proteln

Connexon

Receptor/
channel

Receptor/C
pfotein

speed

lnstantaneous I msec

secto min

Effect
ACh, acetylcholine;v-, membranepotential.

A simiLar calculation based on the geometry and cable

propeflies of a typical nenre-muscles).napsesuggeststhat
an action polential arriving al a nerve terminal could
depolarizethe posts)'napticmembrane by only 1 pV after
crossing the s).napticgap-an attenuation of 105. ClearLy,
Lhe evolution of complex multicellular organismsrequired
the development of special splaptic mechanismsfor electrical signalling to seNe as a workable means of interceLLularcommunication.
l w o . o m p e l i n gh y o o t h e s eesn e r g e d - t h e l o h c e n tury to explain how closely apposed cells could communicate electrically. One schooLof thought proposed that
ce 15 are dirpcll) linled b; micro,coDic cornecLn8
bridges thar enable electrical signals to flow directly.
Other pioneering physiologistsused pharmacologicobservations to infer that cell-lo-ceLltransmissionwas chemicaL
in nature. Ultimate resolution of this question awaited
both the development of electron microscopic techniques,
which permitted yisualization of the intimate contact re
gion between cells, and further studies in neurochemistry,
which identifred the small, organic molecules that are
responsible for neurotransmission.By 1960, accumulated
evidence led to the general recognition that cells use borh
direct electrical and indirect chemical modes of transmission lo communicatewith one another.
commuThe essentialstructural element o[ interceiLuLar
nicarion, the slrrapse, is a speciaiized point of contact
between the membranes of two different, but connected,
cells. Electrical and chemical rynapseshave unique morphologies, distinguishable by electron microscopy. One
major distinction is the dismnce of separationbetween the
two apposing cell membranes.At electrical s)mapses,the
acljacent cell membranes are separaled by about 3 nm
and appear to be nearly sealed together by a plate-1ike
structure lhat is a lraction of a micrometer in diameter.
Freeze-fractureimages of the intramembraneplane in this
region reveal a cLusterof closely packed intramembranous
particles that represent a gap junction. As described in
Chapter 6, a gap junction correspondsto planar arrays o[
connexons, each of which is made up of six connexin
monomers (see Fig. 6-18). The multiple connexonsfrom
apposing cells physically connect the two cells together
via multipLeaqueouschannels.
In contrast to the gap junction, the apposing cell membranes of rhe chemical s).napse are separatedby a much
larger gap. Approximately 30 nrn separatesthe two ce1l
membranes at a neuronal chemical synapse,and the gap

is as large as 50 nm at lhe vertebratenewe-muscle synapse.An additional characteristicof a chemtcal $mapse is

the presenceof numerous s)'napticvesicleson the side of
the slnapse that initiates the signal transmission,termed
the pres).napticside. These vesicles are sealed, sphedcal
membranebound structures that mnge in diameter tiom
40 to 200 nm and contain a high concentrationof chemical lreurotransmitter.
The contrastingmorphologies of electricaland chemical
s1-napses
underline the contrasting mechanisrnsby which
they function (Table 8 l). Electrical slnapses pass volt
age changesdirectly from one cell to another across the
low-resistance continuity that is provided by the con
nexon channels. On the other hand, chemical syrapses
link two ceLlsby the diffusion of a chemical transmitter
acrossthe large gap separatinglhem. The neurotransmlt
ter rhat is stored in the ry.napticvesiclesis releasedinto
the synaptlc space,diffuses acrossthe cleft of the slrrapse,
and activatesthe posts)'napticcell by blnding to a specifrc
receptor protein on the posts).napticcell membrane.
Direct evidence for the existenceof chemical transrnission predated the experimental confirmalion of electrical
slnapses. The foundations of s),TrapticphysioLogycan be
traced back io early studies of the aulonomic newous
system. Early in the 1900s, researchersnoted that adrenal
gland extracts,which contain epinephrine, elicited physio
Logicaleffects (e.g., an increase in heart rate) that were
similar to those elicired by stimulation of sl.mpathetic
nerve hbers. ln 1904, ElLiot proposed that sympathelic
nenes might release a subsmnce thal is analogous to
epinephrine that would functlon in chemicaLtransmission
b e L u e e na n e r r ea n d i r s r , i r g eot " g a r .) ' r ' a r < l L d . e s u g gested that the vagus nerve, which is parasyrpathetic,
p r o d u c e sa r e l d t e ds u b 5 l a n \ er h a t L r e ' o o n s i b ' eo r d e n r p < < in n

nf rl'"

L""rhpqr

A . l a , ' t c e x p e r . m e npt e r l o r r e d b v l o e w r n l 0 2 l r s
widely cited as the first definitive evidence for chemical
neurotransmission.Loewi used an ingenious bioassay to
rest [or the releaseof a chemical substanceby the vagus
n e r v e . H e r e p e a t e d l ys r ' m u r a t e dr h e r a g u s n e r v e o f a
cannulated frog heart and observed a slowing of the
heartbeat. At the same time, he collected the a ificial
saline thar emerged from the ventricle of this overslimulated heart. When he later perfused the same heart with
the fluid he had previously collectedwhile stimulaling the
vagus, he observed that this perfusate itself slowed the
heart rate in a manner that was identical io direct vagal
stimulation. He also later identified the acLivecompound
as acein the perfusion fluid, originally called Vagusstorf,
tylcholine (ACh).
Efforts by DaLeand coworkers to undefsland the basis
of neurotransmissionbe[ween motor nefl/es and skeletal
muscle culminated in the identification of ACh as the
endogenousexcitatory neurotransmitter. Thus, the inherent complexity of chemical slnaptic transmissionwas evident ftom these eariiest investigations,which indicated
rhat the same neurolransmitter (ACh) could have an inhibirory acdon at one qnapse (vagus nerve-heart) and
an excitatory acdon at another s)'napse(motor nerveskeletal muscle). For their work on nerve tmnsmission
acrosschemical s)'napses,Otto Loewi and Sir Henry Dale
@
receivedthe Nobel Prize in Medicine in 1936.

and the Neuromuscular

8 / SynapticTransmission
lunction
/

I (eleclrotoniccurrent)

Linearll,rvith the translunctional voltage (i.e., the y,,, difIerencebetween the tu'o cells). However, the crayfish syn
apse described b1. Furshpan and Potter allorvs depolariz
. l E . L r n l t o . r , , r e a dI ' o n l ) l - o n , d r r r t o f f o | . h e
'

..,-

-i.

r I

h.

n^-r..

-i.

cel. )L.n.eLl

-dl

synapsesare called rectifying synapses to inclicate thar

the underl,vir'rgjur.ictionalconducLanceis voltage depend . r r - r , - ^ . 1 , . . r ' , 1 " " r r r q , . q 1c l. T n . r r n . l - a v e
shown that the voltagedependenceof electricalsynapses
arisesfrom uniqrie gating properties o[ dillerenLconnexjn
e e p e r r J r n rv . , r ,
) u o r r s . o n . . - o l or ' , r - r t r l L a g d
others are loltage independent. lntdr-rsicr-ectilicaLioncan
also be altereclbl Lhe lbrmation of a gap juncLion Lhat is
c o n 1 ' o . e6d 1 1 1 n" p i . l ; - - c l - . . l c h m a l e u 1 'o t r . l e r
''

l, L L_id c"rnertns

a1L -J -Lo

heterotyprcchannels.
CHEMICALSYNAPSES.
By thejr \,ery nalure, chemical
synapsesare inherently rectif,vingor polarized. They propl r e f - c . ) n , r l r l i(." l l
J3,e ,-l enl in onr d r" lton
" r ' posrs).napticcell that
Lhat releasesthe transnirter ro the

C e l - c e l lg a p j u n c l i o n
F I C U R E8 1 . A n e l e c l r i c a lr y r p s e

A l l e l e c r r i c . lsl \ n i p s c c o n s i n s o l

cules.

Electricaland ChemicalSynapsesBoth
ConveySignalsFrom One Cell to Another,
but Differ Greatly in the Particulars
SYNAPSES.
ELECTRI(AI"
Whereas overwhelnT
ing sullporl
J n . n s r t o t r a c \ u nu l ae J i n h \
lo" .lr,-n..ll .yr. l..
lirst hall ol the 20th century. Lhe first direct evidencefor
p l c . l . . , . r ' : ] nm r - - . o r d n r u . h l : r . r f r o r c l . . r r o . l l ) ' l
of a crayfishnervepreparation.ln 1959,
ologicrecorclings
t u , - h 1 " r r d P ot e r u . e J r r r . ' D ' r i . o ' . t n . r r g : n C
recordingelectrodeslo shor,vthat depolarization
of a pre- ) n J p , r .n r , e f b e r ' . t l t ' . l ) r - , o J o r ' r l - . - \ r r r nerve ce]] (the mosulted in excjtationol a posts),naptic
tor nene to fie tail nruscle) wlth virtually no time clelay.
In contrast, chemrcals1'napsesexhibiL a characLerjsticder r - r L l r . - . r , - 1 n " r u' r . o l r : g ,
J\ o dffro\ lndlrl.
g n . r l; [ t e " e r , t t - . r o l 1 r . p r e , n . r p t r '. " L T l r ec l e . r o r
stration of an electrical s1-napse
belrveen L\\,onervc membranes highlighted an Lmportantfunctional differetlcebe
\l!(lr rrl r d.\er'.11 yr -c r r c ' r J . -s r g . .
r\^ccn
versusbrieilv clelayecl
propagation(electricaL)
communlca
tion (chemrcal)Lhroughthe lunction.
An electrical synapse rs a true strucLrLralconnecLion
ltel$'eentu'o cells,mecliatecl
by the connexonchannelsof
gap jlmctions thaL link the c,vtoplasmof the trvo cells
(Fig. 8-L). Thesechannelsthus provide a loi,v-resistance
path for electrotonic currellt lloi,v, and alioil' to]tage sig
n d - t o l o \ \ \ , t - l t r ( d L c rt - j o n . r J
J.la) bct'rcer
"
hvo or more coupled cells. lvlanytypes of gap junctions
-o. c.r..1 . . - ( e . r . J . u r r . n l \ l \ c . u r l e f t r . rn ' \
(reciprocal
synapses). ln other u,orcls,Lhe current
lions
passingthrough Lhe gap iLrnctionis "el'rnjc", jt varies

. ^r 'in. l- r- .rr^., h r' r. .o-i e oTJ .tn. t . Ld15

_lr''
For'r. r hr r'--e ' a \ \. iot ,,r 'atll-! tll ch.m-v
,
f
r n - r r ' . " n h p l, . t h p p o - - b i l r 1 r l - a . h e
i.
."1
p . - t : 1 n L pi , , . l l , . r n r r r f r e r . e r ) n , p . . . . , rr J o n o r
transmittel release b1' the preslnaptrc cell. Studies ol s1n
apse clevelopment ancl regulation hale sholltr that postsy
naptic ceLls also p1a1 an actrve role in s,r'napseformation
n r h r f N S n o < t * n r n n r r.c. l l < m ,l \ ' l l S O p r o c l U ( e r r ' o
grade signalling molecules, such as nitdc oxide (NO), that
d1ffuse back into the presvnaptic Lerminal and modulate
l c ' , el o h, .)n.rt.! co -ecrio rf. 122. F rrf,cr-,.'"

il.

h a n. h . . - ,

"

,-,,. .rl )nme

)ndl

.OT

rains receptor-sthat lnay eiLher inhibiL or facilltate the

l e [ - : e o l r n , r r i . i e b ] b . o ,l - e m i-. n p . h . r r> m ' . T u , .
. h ' c ; l - r ; - e , " u l , l h e , ^ , 1 '. , J , u n r d . r e . L i u r r a l
pathu.a).for signal propagation that can be modulated by
bidirectional chemical commur-ricationbetween two inleractingcells.
The process o[ chemical transmissic]ncan be summarizeclby the l,rllowingseriesof steps(Frg.8 2):
\(e ,
\)

. \cr.totrdn,n le
dp t. \(.t

h-

'lr,(i5.

- r o l e c Je - . _ ' r ' e
p c L . r g c dr r t o
-d

lofl

lf,.ein.

In Inc r\<t-

cle membrane use the energl of an H gradient Lo

energizeuptake o[ lhe neurotransniLLerrn the vesicle.
\ l c p 2 . A r , . o n L r J r (t
r r l r i . h i n r o r c , r o l t " g eB . r rJ'
"
N r . r d ( c h , - n e l ,l . ) 1 8 2 1r.r r , . . - , r r h c p - c s y - a p
Ltc ne nre Lermtnal.

Step 3r Depoladzation opens voltage-gatedCa2t channels,

* h . . l - - l l o ' r. \ J ' l o c l e . t . e p r P ) y r l . , \r pl Le r m i n l l .
SLep4; The increasein intracellularCart concentration
([Ca2-1,)triggersthe fusion of s]'napricvesicleswith Lhe
D - e . r\ e p t . I l e n b r ,r e A \ J t e 5 t t l p r , l . . r, ' . . g a n r . ro\ f
transmiLtermoleculesare releasedinto the synaptic
cleft.
.Lel ) lhL trn,nll'
n r ^ e . u l e 'd l I u , er , r o . . h c s y n 1 1 1 . . - e ' . n J ' J n , l ! c f r r r e ( e po r . o - . h e n e l n h' r.,

'r, p n

,rt rhn

nn*<mrnr,e

rpll

l h e b r J ' g o f l r r n J rl | l t c I J . L i \ d L c >h c r e ! c n . o r .
vr,hich in turn activates the posts).naptic cell.
.tro /. llrc p.u.c>- \ trlt'.la,- bt l',rz)-nat c ce-

SynapticTransmission
and the Neuromuscular
lunction / 8

Extfacellular
space

.,^2+
vorragegareoLa
channels oDen.

Presynaptic
nerveterminal
of the nerve
\
cell
i(electrotonic
current)

Postsynaptic
cell

A neurotransmitterbreaksdown,
is takenup by the presynaptic
terminal or oiher cel1s,oi diffuses
away ftom the syr'upse.

n 1. r r h c m i c a ls t n a p s cc a n b e r h ) r g h t u [ . ] s o c c L r n n s r r s | r . . t . L r .
F I C U R E B 2 . A c h c m i c a ls ) n r p s c S l n a p L i cr m n s m i s s i o 1

str.ucrionof tl.retransmitler(e.g..hydro\,siso[ ACh b,v

acetyLcholinesterase
lAChEl), (2) uptake of transniuer
lnLo the presynapticnerve terminal or into other celLs
s)'stems,
or (3) diffusionoI
by Nr*-dependentiranspor-t
rl-,. r:n<m,rrnr m.l..rrl.<
t r \ \ , \ 1 rO l L n ( r \ n r P \
-fhe

molecular nalure oI chemical synapsespermrrs

enormous dilersrt,vin lunctional specializationancLrcgula
occrLrsat the leyel of the transrion. FunclionalcLlversillr
r' rPsPo-]c(
l"r5)-af
h, c .
u \ . q . r r l , , ( l r .a l " n d h
f o. ..e(
o
n
r
.
l
l.le.!.e. . e
c [, rcn
orn
u.,
scrve as neuroLransmitLers(p. 104). These
posed-to
molecuLcs lnclude botl.r small organic molecules such as
norepxrephrine, AC}r, serotonin (5 hyclroxyLryptamine [5,nrtt,r -rrl'.t

'nd
'1..

h .
t u.:.., . :r-rrd'dl rln^b l) | , .,' ,l i '.\B\1. ely
.
.dF. ..pr, - 'r.h.-.nJur'pl
a Jer \coh.'lrn,

The Transmitter at a ChemicalSynapseCan

Activate Either an lonotropic Receptorthat ls
Itself a Channel or a Metabotropic Receptor
that ls Linked to a C Protein
. . r . l r n ! rr | l . r . . P l r r - d r
.h, r'. br'..rrgoie.o
llgand gareclion cl.rannelproterns and G protein-linkecl
protejns(also calledscven- Lransmembrane-hcljx
receJrLor
recepLors,p. 92). SeIeraLneurotransmitteLmolecLLles.
- u . l r. g r
e , r . \ L * r < a . l i e " r ' ' l t- ' . s 1 1 ' 5 l , I
h o t 1 p , ' f r . . - r t o . - . n l . ' a l tc J . c . . . e c l u J

2Oa

SylaptiL

-tdn'Ti..'on

a1d tl-" \eL-omJ)culat ,LtL 01

IONOTROPIC
RECEPTOR

B METABOTROPICBECEPTOR

.,:

Axol
Electrical
srmulus

\,',,/

..

I
I

......

t\;
l' 1
: i,: l

l;:
|

\ .
.ll

N".l/
-'--"
@,

";
i l , ,' : . '

:,

. , , ,- |

..:..,.....,,. .:.
...,.:t..
' '. -ri ..._),i.,1t:1,.,,.,.
,/

.:.. ^
t

"

Acetylcholine
Atrialmuscle
cell membrane

aa

Skeletalmuscle
fibermembrane

Nico:inicACh receptor
channelactivatir
ln

lr

Il

-lr

[,4uscarinic
AChreceptor
acrvaron
]

R"b"r"
- P./
"l "TP G protein
fromthe helerotrimeric

IM".b"r*
lepolarization

F I C U R E8 3 . i d l o t r o p i c x n d n . r r
b o t r o f i c . r c . t \l ( l r { r l m r f e . e p r o r s A ,
T h i s e \ m p l c i l l u s r f : u r sI n i c o r i r l i c '
a c e l ) l c h o l i n cr c c c p t o r s h r c h i s r
tLg.Lml-gaLcd
rhannel on Lhe possr'naptic nembr.rnc In r sktleral mLrs
c l e , I h e f n d r e s u l t i s m r L s c l cr r r
LflcLion B, This eramptc illuslratcs
I r r u s c r f j r r i c ; l . e r ) l c h o l i n cr c r c p
1 o f .u h r r h i s c o u p l c d t o e L c r c r o u r
mcric a; trotern. ln I crr.lirc rrLrsc l c . L h cc m l L c s u l ti s . l e c r e i s . . ll r c a r t
rrte. Noie thrt rhc frcr\nairtrc r.,
l . 1 s eo l r \ ( l h i s \ . r \ s i r r i l r r l 1 e f .
. r n d L L rA - \ C h . a r c r \ l . h o h n r i ( , 1 P .
3 L L r n o n n 1e f p h o s p h x r e

- ]r

lr-

----r-

p"t""tb
,
"tb"
exctaiion
L

Activationoi lnward I
rectifierK- channelby py

**. l
t-r.,r"-.t
nyperporar
zaron

IVIusce
contraction

-t-

Decrease n
nean TaIe

mate. Ellutamete receptors thaL are ton chilt-tr-relsarc

knorvn as ionotropic receptors,and gluLanaterecepLors
to C proLcinsare calleclmetabotropic receptors.
cor,4t1ed
fhis nonenclature is berng increasingly used ro clescribe
thc Lwo malor l)rpes()1 rcccptols lof Lfansmittersother
llun llutamate.
lonotroprc ancl metabotropicreceptorsdetermine the
ultimatefunctionll responseto tftnsmiter re]ease.
Activaio

.rt u'n ror'.

.1.

lot .r..r',

,..^.',1,gnl

,n

chlnnels This channelaclit'aLjon]n ir,rrnresultstn depo

larizationor hl perpolerizatl,uof the postsFrapLic
membrane,tl.iechoicedepenc|ngon rhe ionic selecriljt)'01the
conclucLance
change.i\cLir.atlonof a mettllotroprcG protern lir.rkedreceptor resulls in the procLucuonof actir-ea
ancl By sr.rbunits,r'hich initiaLe a u,ide varieLl of cellular
responsesby clirect intelaction rvith either ion channel
prLrternsor oll'rersecond-messenger
eflectorproteins (lt.

95). B) lreir YeD nalure, ionotropic receptorsntecliaLe

fast ionic slnaptic responsesthat occur elt it nillisccond
t r c - . . ' 1 , r. . l t . r , . - , c . ' ' u - \ . r . . . p r , r - , . / r . . , \ \ .
medirted sy'napticresponses
biochenricalll'
in rhe rangeof
secondsLo minuteS.
Flgurc B-3 compares Lhe basic processesmecliare.lb)'
t\vo prototyPicACh receprors(AChRs):(1) rhe ACh acLi
vatecljon channcl.lt the neuromuscLLlar
lunction of skele
tal muscle, rn ionotroprc receptor also knou'l as the
nicotinic AChR (Fig. 8 3A). and (2) the c pfotein
LinkedAChR at Lhe.tnal parasynpaLheLic
synapseof the
heart. a metebotiopicreceptoralso knom as Lhemuscatp lh r'ol .
rinir \L"Rrl r;9
\ ' L r -t r J ' d i n i .
clistincfion r,vasa classicpharnttcologu.classificaLionbasecl
on rvheLherLheAChR is acLilatedbv nicotine or mlrscarine. The molecular basis for tl.iis disril.rcrionrvas discor,
ered oniv mucl hter. ln the caseof the ionorropic(nico-

SynapticTransmission
and the Neufomusculaf
lunction / 8

tinic) receptor, opening of the AChR channel results in a

transient increasein permeability to Na* and K*, which
directly produces a brief depolarization that activatesthe
muscle fiber. In the caseof the metabotropic (muscarinic)
receptor, activation of the G protein-coupLed recepior
opens an inward rectiFer K+ channel, or GIRK (p. I97),
via p7 subunits releasedfrom an activated heterotrimedc
G protein. Enhanced opening of these GIRKs produces
membrane hyperpoLarizationand leads to inhibiiion of
cardiac excitarion (p. 488). These two funcLionallydistinct
mechanisms are the molecular basis for the seemingly
conflictrng observations of early physiologisrs rhar ACh
(VagusstofJ)activates skeletal muscle bur inhibirs hearr
muscle.

TRANSMISSION
SYNAPTIC
AT
THE NEUROMUSCULAR
TUNCTTON
Neuromuscular
lunctionsare Specialized
with ActiveZonesof Synaptic
Synapses
(Neuronal)
Vesicles
on Presynaptic
Membranesand HighlyAmplifiedlunctional
(Muscle)Membrane
Foldson Postsynaptic
The chemical ry.napsebetween peripheral nerve terminals
and skeletal muscle fibers is the most intensely studied
synaptic connection in Lhe neryous system. Even rhough
the detailed morphology and the specific molecular components (e.g., neurotransmittersand receptors)differ considerably among different iypes of slnapses, ihe basic
electrophysiologicprinciples of the neuromuscular junction are applicable lo many other qpes of chemical slrrapses, including neuronal sl.naptic connections in the
brain, to which we wiLl retum in Chaprer 12. In this
chapter, we focus on the neuromuscularjunction in discu5sinS
l h e b a > r cp r i n . i p l e so f : l n a p L i cl r d n s m i 5 s , o n .
Motor neurons with cell bodies in rhe spinal cord have
long axons that branch extensivelynear the point of contact wirh the target muscle (Fig. B-4). These axon processeseach inneruate a separatefiber of skeletal muscle.
The whole assembly of muscle fibers innervated by the
axon from one motor neuron is called a motor unit.
T1pically, an axon makes a single point of synapdc
contact with a skeletal muscle hber, midway along the
lengt\ oi rhe muscle 6oer. thrs <pec.al.zed
s).napLiL
tegion is called the neuromuscular junction or the end
p l a t e ( F . 9 . 8 4 ) . A n . r d i v i d u a le n d p l a r ec o - ' s L ' o f a
smal1tree-like patch of unmyelinated nerve processesthat
are relerred to as terminal arboizations. The buLb-shaped
endings that finally contact the muscle fiber are called
boutons. Schwann cells are intimately associatedwith the
newe terminal and form a cap over the face of the nerve
membrane that is located away from the muscle membrane. The postslnaptic membrane of the skeletal muscle
fiber lJrng directly under the nerue terminal is characterized by extensiveinvaginarionsknown as postjunctional
folds. These membrane infoldings greatly increase the
surface area of the muscle plasma membrane in the posts)'naptic region. The interrening space of the s1'naptic
cleft, which is approximately 50 nm wide, is filled with a
meshwork of proteins and proteoglycansthat are part of

the extracellularmatrix. A particular region ol the muscle

basemenrmemb-anecalledrhe sl.nrpricbasal dmjna contains various proteins (e.g., collagen, laminin, agrin) that
mediate adhesion of the neuromuscularjunction and play
important roles in slnapse developmentand regeneration.
The s'-naptic basal lamina also contalns a high concentralion of the enzl-ne acetylcholinesterase (AChE), which
ultimately terminatess)'naptic transmissionby rapidly hy,
drolyzing free ACh to choline and acerate.
Electron micrographs of the bouton region demonstrate
the presenceof numerous sphericalslnaptic vesicLes,
each
with a diameter of 50 to 60 nm. The cell bodies of motor
neurons in the spinal cord produce thesevesicles,and rhe
microtubule-mediatedprocessof fast axonal transport (p.
26) translocatesthem to the nerve terminal. The quantal
nature of transmitter release(describedlater in more detail) reflects the fusion of individual stnaplic vesicleswith
t F e p l a : m am e m b r a n eo f r h e p r e s y - l p L i it e r m i n a l .F a c r
slnaptic vesicle contains 6000 to 10,000 molecules of
ACh. The ACh concentration in synaptic vesicles is approximately 150 mM. ACh is synthesized in rhe neNe
terminal-outside the vesicle- from choline and acetylcoenz).me A by the enz).me choline acetyltransferase.
t h e A C h m o v e st n t o 1 F q5 , ' n r t , 1 .\ e s r ! l c \ r a d 5 p e c i f i (
ACh-H exchanger,which coupLesthe inward transport of
ACh to the efflux o[ H*. Energetically, this process is
driven by the vesicular proton electrochemicalgradient
(positive voltage and low pH inside), which in rurn is
produced by a vacuolar-qrpeH* pump fueled by adenosine triphosphare (ATP) (p. 6a). The newe terminal also
contains numerous mitochondria that produce the ATP
required to fuel energymetabolism
The processof fusion of synaptic vesiclesand releaseof
ACh occurs at differentiated regions of the pres)'naptic
membrane called active zones. ln electron micrographs,
active zones appear as dense spots over which slnaptic
vesicles are closely clustered in apposition to the membrane. High resoiution images of active zones reveaLa
double, linear array of si.naptic vesicles and intramembranous particles. These zones are oriented directly over
secondaryposrs).napticclefts that lie between adjacenrpostjunctional folds. Molecular localization studies have
shown that the density of ionotroplc (nicotinic) AChRs is
very high at the crests of postjunctional folds. Examinaiion of the detailed microarchitectureof the neuromuscular sy.upse thus revealsa highly specializedstructure for
delivering neurotransmittermoleculesto a preciselocation
on lhe posrs)-napticmembrane.

Acetylcholine
Activatesthe lonotropic
(Nicotinic)Acetylcholine
Receptorto Produce
an End-Plate
Currentand Thusan Excitatory
Postsynaptic
Potential
ELectrophysioLogical
experiments on muscle fibers have
characterizedthe electrical nature of the DosLs\,'naDtic
re\nnn<p

,r

rne

mr .elp

enrl

nlrrp

liorrrp

R-5

, lr rrrurp.

results obtained lrom a classic expeiment performed by

Fatt and Katz in 1951. Their work is the frrst description
of how stimulation of the motor nerve affects rhe membrane potential (V-) at the posts)'napticregion (i.e., mus-

210

junction
and the Neuromuscular
8 / SynapticTransmission

Spina cord

Nervecellbody

Musclecell
or I ber
Postjunctional
folds

Axon/

Synaptic
vestcles

'o
Act ve zone
\@

Acetylcholine
(re easedfrom

oI

vesrcles
Presynaptic
memorane

,t

laT.rna

Acetylchollne
receprors

@k'

F I C U R E8 - 4 .

Postjunctional
folds
Postsynaptic
membrane

Ihc \efrcbr:rte

bou1ons'i1ss'cLllsthcspcciali:.rLiolso|l1rcposls,vnaplL
.onL:rLLrirgthe
tionrl loltls). Depol^iz.rLiorl
flclr Thc Aa:h nrolccrLlcsrnrLsLdiffusc
\ c s j c l e sb ) r c s \ ' n t h r s i 2 i n' g\ ( . h e n d l r i r n s p o r t r f gr b L sl c h i n t r t h c v c s r r l r \ i r r n A C h - H c x c l a n g c r .

junction / 8
SynapticTransmission
and the Neuromuscular

Voltagerecordiqg

\\

llJ-

211

MorotneNe

The muscleis treatedwith

curareto limit ACh
receptorachvahonto
subthresholdresponses.

Stimulus
of
motornerye

F I C U R I 8 - 5 . E n d p l a i e p o t e r h a l se l i c i r e da r r h c l r o g n u r o muscular lunclion by sLmulaturg the mot,:lr neuron. The magr i r ' ' " o f t l . .! . c r . ' rr \ I o . w
1i. r.l IDP . s,....
near thc end plxre and deca)s farther awry (De!e fonr Fati I,
Kelz B: An anal)sis of the end-p1atepotenLialrecorddwirh an
i n L r a c e l l u l aerl e c t r o d eJ. l , h y s i o ll l 5 : 1 2 0 3 7 0 , 1 9 5 i . )

The delay in
response is a
function of
acetylcholi11e
release,
diffusion, and
activation ol
Pos6ynaPtlc
aecepto$,

10
0
Excitatorv
1o postsynaptic
(orend plate)
0 potenlial
(mv)
10

1 . 0( m m )

The delay
in response
tlme
increasesas
a function
of the
distance
hom the
end plate.

0
10
0
10
0

cle cell) of lhe neuromuscularjunction. Normally, nen'e

stimulatron rvouid drir.e the V,,, of the muscle above
threshold and elicit an action potentlal (p. 172). However, Falt and Katz were interestecl not in seeing the
acrion polential, but in sLudyingthe sn.rall,graded electd
cal responsesthat are produceclas ACh binds to receplors
on the muscle cell membrane. Therefore. Fatt and Katz
g r e a \( r e J ., e d h c r e , p o n i eo l r h , c C h R i b 1 b . o . k r n g
mosl of them with a carefully selectedconcentrationof d. u b o . u r ai n u h r .h , r e d i r ru < - , l e r . T h c y - , e r e r : a
h C - f l l d m i , r o e l . r t r o d , r o r h e e n d p l . . l r er e g , o Fo . . 1
frog sartorius muscle hber. Tl.ris arrangemenr allowed

r h , m r n m .' ' '.' 'r' 'r '. , /r n v , . " h o p .

spoto[ the musclecell.

in

\ V h e n l J l " i r d h a t - e l e c t r L " l l re \ ( i r e d l h , ' m o r o r

neNe axon, they' obsen'ed a transient depolarizaLionin
the muscle membrane alter a deiay of a few milliseconds.
l L e J e l a ' r p r p q p t r h e - r p - e .u i e q l o r r h e r e l e a , eo l
\ ( h . l , d i l u , o n a r r o . . r h e . ) n a p s r . a n q , j c r i \ ' . j r i oonl
posls)rnaplicAChRs. The positive voltage changefollows a
biphaslc tln.recourse: V., rapidly rises to a peak and then
more sLowly relaxesback to the resting value, consistent
\\'iLh an exponential time course. This signal, known as
the end-plate potential (EPP),is an example of an excit-

212
A

and the Neuromusculaf

8 / SynapticTransmission
lunction
PREPARATION
EXPERII\,lENTAL

The muscle is treated with

curare to limit ACh
receptor actrvation to
subthresholdresponses.

END-PLATECURRENTSOBTAINEDAI
HOLDING
POTENTIALS
VARIOUS
400
200
End-plate
0
current
(nA)
-200
-400
012345678
Time(msec)
FOR PEAKEND.PLATECURRENT
C I-V F]ELATIONSHIP
I(nA)

Clamped
membrane
potential

(mv)

FICURE 8 6. End-plate currents obtained at dillerenr membrane poten

tials in a vohage-clamp expenmen!. A, Two electrode vokage cLamp is
used to measurethe end-plate cunent in a frog muscle 6ber. The tips of
rhe rwo microelectrods are in the muscle fiber. B, The six records
represenr end-plaLecurrents that \'r'reobiaind while the motor nerve
rvas stimulated and the poslslnaptic membrane was clamped to V,,,
91, 68, 37, +24, +38 mV Notice that ihe peak
v a l u e so f - I 2 0 ,
cunem reveres from inward to out\rard as the holding polential shifts
from -37 to +14 mV. C, The rvelsaLpotential is near 0 mV because
rhe nicotinic ACh receptor has a poor selecnuty for Na+ versus K-.
(Data kom Magleby KL, Stevens CF: The effect of voltag on lhe lime
c o u r s eo f e n d - p l a t ec u r r e n t .J P h y s i o l2 2 3 1 5 1 - 1 7 1 , 1 9 7 2 . )

atory postslrnaptic potential. lt is produced by the trans i e n r o p e n i n Bo l A C h R t h a n n e l . . r n h t , h a r e > ee r L ' \ e l )

permeableto monovalentcalions such as Na*and K-.
The increase in Na* conductance drives V* to a more
e B o n .I n
p o c r \ e v d l L ei n l \ e \ r c r n I ) o ' t h e e - d - p J a Lr e
rhis expenment, curare blockade al1ows only a small
number o[ AChR channels lo open, so that the EPP does
nor reach the threshold to produce an action polential. If
the experiment is repeatedby inserting the microelectrode
at various distancesfrom the end plate, the amplitude of
the potential change is successivelydiminished and its
peak is increasingly deLayed.This decrement with disl a n c eo . . u r ( b e c a u s er h e E P Po r g r a t e . a t t h e o n d - p l " t e
region and spreads away from this site according to the
^ , . < ' . p . r h p n . o n er r e qr n ' C 0 r o [ L h e r . u ' . l e f b e r .
Thus, the EPP in Figure B 5 is an example o[ a propa. ouerer. wrhout tl'e curdre
g a r e d .g r a d e d r e s p o n c e H
blockade, more AChR channels would open and a larger
EPP rvould ensue, which would drive V,,,above threshold
and consequently trigger a regeneratingaction potentiai
(p. 172).
What ions pass through the AChR channels during
generation of rhe EPP?This question can be answeredby
using the same voltage-clamp technique that was also @
. s e d r o , t r d 1 t h e b a c i so f l h e d c l o ' p o l e r t, l r 5 e eF . 8 .
7 5B). Figure 8-64 illustrates the experimenlal prepara
tion for a two-electrode voltage-clamp expedment in
which the motor nerve is stimulated while the muscle
fiber in the region of i|s end plate is voltage-clampedto a
chosen V-. The recorded current, which is proportional
to the conductance change at the muscLeend plate, is
called the end-plate current (EPC). The EPC has a characrerisric rime course that rises to a peak within 2 ms
after stimulation of the motor newe and falls exponenL i a l l ;b a c r t o e r e q f ' g B - o B ' . l h e l i m e c o u - " eo f , h e
EPC correspondsto lhe opening and closing of a population of AChR channels, governed by the rapid binding
a n d d i , p p e a r a n , eo [ A C h a s r t d i l l u ' e . t o t h e p o . t s y n a p tic membrane and is hydrolyzed by AChE.
A < * h o r l m ' n F i o , n e8 - o B u h e r h e m u ' l " f b e r i .
120 mV, we ob
clamped to a "holding potentiaL" of
serve a large inward current (i.e., the EPC). This inward
r , r r e n l d e L r e a ) et 5n r a g n t t u d oa s V . i ' n a d e m o l e p o ' ',"
d i r e c t i o nt o b e c o r e a n
"nr l-.
at
positive
values
of V-. A plot of Lhe
outward current
peak current versus the clamped V- shows that the reversaLpotential for the EPC is close to 0 mV (Fig. B-6C).
B e . a , - c t h e E P r . . p e c i f c a 1 c o t e s p o n d <l o c u - r e - L
through AChR channeLs,this reversalpotential reflectsthe
Na*
ionic selectivity of these channels when extraceLLuLar
and Kt concentrations(lNa=]" and lK*1.) are normal.
By varpng the concentrationsof the extracellular ions
w r l e m o n i l o r i n tgh e , l ' i L ' - I n e r p \ e r - apl o r e n l i aolf t l ^ e
EPC, researchersfound that the AChR channel is permeabLeto Na*, K*, and Ca']*,but not to anionssuch as CL
concentration,lhe current
Becauseof its low extracelLuLar
attributable to Ca2* is smalL under physiological condluons and its conLribution can be ignored. By pLuggingthe
vaLuesfor the various cations into the Goldman-Hodgkin
Katz voltage equation (Equation 6-9), one can obtain the
permeabiLityof the AChR channel lo various alkah monor a l e n ti o " , . - l d u v el o \ a p e r m e a b i l L tTyh e r e , u - tt - t h e

SynapticTransmission
and the Neuromuscular
lunction/ 8

213

ACh RECEPTORVIEWEDFROI\IABOVE

O,SUBUNITOF ACh RECEPTOR

cBosssEcrloNoFAchRECFPTOR

f\\

t\\

Extracellular
space

P^rc

trw,r.^^,,,,,-,

Neurotransmitte
rpocKer

Cytoplasmic
veslibule
(ACh) receptor. The njcotinic ACh receplor is a h eteropenramer\llth ihe subunir composirronof
FICURE8-/. Structure ol lhe rlicotinic aceLyLcholine
drB76. Thes subunits are highly homologous to one another, and each hAs four nembrane spanning segmenrs(N11-M4).

following sequenceof relative permeabillty:0.87 (Li,),

1.00(Nat), 1.ll (K'), and 1.42(Cst).This weakionic
. e l e r t ' .L v - t : n d s
r arleJ ,o rrr-r ro LVpcdrollage
g " t e d\ " , h a r , < l - .
1 n d \ e P \ / P . I n l r o -o d p p - o \ r "hi,
mately 20, and voltage gated K channels, which have
P,/r" ratios greater than 100. On Lhis basis, the ionotropic (nicotinic) AChR channel at the muscle end plare is
ofien classiliedas a nonselective cation channel. Never
theiess, the weak ionic seLectivityof the AChR is well
suited to its basic lunction of raising V," above the Lhresh
o1d of about -50 mV, r.vhich is necessaryfor llring an
action potenlial. When the nicotinlc AChR channeLat the
muscle end plate opens, the normally high resting perme
ability of the muscLeplasma membrane for K+ relaiive to
Na* falls so that Nat and K+ become equally permeant
and V* shifts to a value between E" (approximately -80
+50 mV).
mV) and Er. (approximateLy
As lve shall seein Chapter 12, whrch focuseson synap' ' . t r a r - T s s i u ni n t h e r \ S , . m r l a rp n r r r p l e ,h o l o r o r
generating postsFlaptic currents by oiher types of agon i - Lg r r .J . h a n r r l - . [ o r e x a n p l . r h r e ,e p r o r - g a t e d
channels for serolonjn and glutamate are cation selective
and give rise to tlepolaizing excitatory postsynaptic potentials. In conlrast, the receptor-gatedchannels for gLyctne and GABA are anion selectii,eand dnve V,,, in the
h)?erpolarizing direction, toward rhe equilibrium potential for Cl . These hyperpolarizingpostsynaptic responses
are called inhibitory postsynaptic potentials.

The lonotropic (Nicotinic) Acetylcholine

Receptorls a Member of a Superfamilyof
PentamericLigand-Gatedlon Channels
The molecular nature o[ the nicotinic AChR channel was
rere:lerl hr

-t dre.

rlr:r

rn. lrrdef

nrn ern

n,rri6c;r '"

amino acicl sequencingof isolated subunits, and molecu

lar cloning. Purificatlon of the receptor was aided by the
recognirion that the elecuic organs of certain fish are a
n , r r ' ,u a - l r r , L r n . r . e o [ r l p - . c o r r n , A C h R . l - t h e
c l e c r r i .e e a n d t o r p e d o. a 1 . r h . e l e ,t r r c o r g a n -a r e , n bryologlcally derived from skeletal muscle. The Torpedo
ray can deliver largc electrical dischargesby summating
the simultaneousdepolarizationsof a stack o[ many disklike cells called electrocytes.These cells have the skeletal
muscle rsoform of the nicotlnic AChR, which is activated
by ACh releasedlrom presynapticterminals.
The purilied TorpedoAChR consists of four subunits
(a, p, 7, and 6) jn a pentamericstoichiomerryol 2a:lp:
17:16 (Fig. B-7). Each subunit has a molecularmass o[
approximateLy50 kDa and exhibits relatively high homology to lhe other sllbumts. The primary sequencesof nico
tLnic AChR subunits are also highly conservedin evoLu
r o . r \ ' , r r - h " - l c - e , : l r r r * l e t . o l o r m :o [ t h e a
subunit are approximately 907o identical among the Tor. e d nr a , ' - o o . h , ( " - r n . r r e ; , L n u m a n .
The a, B, 7, and 6 subumts each have four distinct
hydrophobic regions known as M1 through M4, which
. o r ' - e - p od r o r e m b - a n e- p a n i n g > e g n - 1 1 .l.h e , : g o n . ' L binding site 1or ACh is in the extracelLularN-terminal
domain o1 the a subunit. For each of the subunits, the
M2 transmembranesegmentlines the aqueouspore of the
. h:nnel nrnrpin lr i-

hrn,,oh r. r

^.rp

hr,

Na' and K+ cross the membrane.

\ , h R r o l n o r m d l" r d t r r , , r l e f i b e , ,a r e p r e s , n r r h r g h
de-,t1
L L e i u n , t . o n a lf o . d r o f t e f o r , s v n d p r l L
ne-n
brane. However, in developlng muscle fibers of the mammalian embryo and rn denervatedfibers of aduLt skeletal
- < , p A , h a . , , . , 1 , . " l p r ) : r " i br r . d r t - e m e m brane outside the end-plate region. The tu'o types of
A r \ R s . , , 1 e d . u n c r i oa a n d n o n j u n . r r o
al re.epror-.

214
A

and the Neuromuscu

ar lunction
8 / SynapticTransmission

CURRENTS
SINGLE-CHANNEL
Embryonic
ozJltS

Single-channel
currents(PA)

0
Single-channel^
-z
currents(pA)

20 40 60 B0 100 120 140

Time(msec)

B t-v RELATTONSHIPS
2

-100
Embryonic
crz0t6

FICURE 8 8. Properties ol embrlonic and adrlt acetylcholine (ACh)

receptors fron skeletal muscle A, The resulLsol pelch cLamp expe rnenrs, sith lhe p:rlch pipeues in th outside out configutation end the
parch exposed io 0.5 pNl ACh, are sumnarizcd ln thc rpper pdncl, rhe
ir estigatoE expresscciLhc emb$onic AChR. $'hich has the subuni!
compositjon d:87.5, in Xdrril]rrlsooc]tes. ln thc ldv.f ldnul, thc invesli
garors expresscd lhc .ldrlt,A.Chlt, $,hich has the subunii co]nposlion
arB5. No(ice thlr rhe meen open times ar great,rr furr the errbfyonic
forn, s'hereas Lhc unitary currens are greater lor the adult lornl. B,
Ihe lrfo lires summarize d:ia that are simihr to those obtarned rn A.
The single channel conductance of the aduLt lorm (i9 pS) is hiilher
rhan rhaL ol Lhe eLnbry'tnlictorm (40 pS) (Data {rori \,lishina M, Takai
T, Imoro K. ei a]: lvloleclrlar disrincrion between feLaland aduLt forns
o l m u s c l ea c e L l l c h o l i nree c c p L o fN a L u r e1 2 1 : 4 0 6 - 4 1 1 , 1 9 8 6 . )

\naut,
0rP.6

have clilferent functional properties. The uniLary conduct

ence of nonjunctionaL receptors 1s approximately 50%
larger and the single channel liletime is longer in duraLion
rhan thar of junciionaL receptors. The basis for this phenomenon is a differencein subunit composilion. The nony t r ' rt ,: . r r t cl o
i c n b r r o n r c l e c P F l o 1a5c r D e n l . r r - r c ,r o' .l n
plex with a subunit composition of arp76 1n mammals,
just as in rhe eLectricorgan of the TolTedoray. For the
.luncriondlAChR in adult skeletal muscle, subsiitution ol
an e subunit for the leta] 7 subunit results in a compLex
ivlth tl.recomposition arPe6.
T e l u n . ' r o n "fl r o p e r t e so l t . e t u o t ; p . o ' e e p t o - s
l.ravebeen studied by coexpressrngthe cloned subunits ir.t
) , , ' . , p u ,o o c v r e , t r g L r cB - 8 4 - L o s p a c h c l a m o r e c o . d
ings of single ACh-activatedchannels in oocytes ihat had
been injected u'ith nRNA encoding either a, B. 7, 6 or
a, B, e, 6. Measurementsof currents at different vohages
yreLded single-cl.rannelI-V cun'es (Fig. 8-BB) showing
r h a tr r . . t a n n e lf o r . n . dr r i h t \ e e ' u b u n . L] ^ a d" . r r r l
c o d u c L d n . eo I s o p S $ h e . p n . r h " r b r n c , l v r h r h e y
subunit had a conducranceof 40 pS. The mean lifetime
ol single-channelopenLngsat 0 mV was 1.6 msec for etype and 4.4 msec for y-type receptors, closely corre
sponding to values founcl in native IetaLand adult muscle,
respectively. The different functional properties o[ iuncr r o n a ; n d r o n l t r n t . o n a ln r . o t l n . A a h r e ,e n l o r . D - e c - r

r ,liDir .nD.irli?a;
r^1.< rr
' ) ' l * P L( L r d n r m- > r o l r
'9r1n"11"n
ier> cle\eofrl.c.li a d ,1nrp-e
MolecuLarcloning of genes that encode AChR subuniLs
of the TorTedoray electric organ and mammalian skeletal
r ..1,r lcd ro rh" rd rt.f' ot.on o[ ..r rrge -umbcr o[
relaLedgenes for AChR channel proteins. For example,
mammals have a family ol at least eight genesthat encode
homologous a subunits of nicotrnic ACh activateclrecepr f t h , - L r l . t " n u r c e e c e p l o - , -h p
L o( . , l | e o s , b r , n . o
procluct of a gene called al. Seven additional genes desiElnatecla2 through aB encode a subur-rilsLl-ratare expressedin neuronal Lissues.Only the proLein producLsol
genes n1, a7, and aB bind the snake venom protein
called a bungarotoxin. In addiLion, at least lour B subunits exist. Besidesthe B subunit of the skeletal muscle
AChR which is calLedB]-there are three neuronaL
homologs (82. 83. P+). Heteromeric associatronof differ
ent combinalions oI these subunrts could potentially pro
J u . , , l a r g e- u - r b o o l l u r . t i o n . , r e . e p t o t r o o r n ' . A l though the exacl physiological role of nicotinic AChR
channels in various neuronalpathways remains lo be established, it is likely that AChRs in the brain play a roLe
in addictron to the nicotine contarnedin tobacco.
Besidesnicotinic AChRs, Lhree other related classesof
agonist-activatedchannels are recognized,including iono
tropic receptor channels that are activated by serotonin
"hl"

..flD

SynapticTransmission
and the NeuaomLrscular
lunction / 8
A

215

GLYCINE

2
Macroscopic 0
current
_,1
(nA)

0
S ngle- -'
channel"
current -4
(pA) -6
-8
23456
Tinre (sec)

B GABA
5 ngre- channel u
curreni -2
(pA)
4

Macro- 0
scopic
cutrent_,1
(nA)

0
1
2
3

1000
1500
Tme (msec)

23456
Tirne (sec)

FICURE B 9. (:ltfrcnrs acttrrred by glycrne and 1aminobut).ric Acid (cAB,\) ,A These e\pernrcnts Nere performed on culLurcd mousc splnaLcord
neurofs usjng palch clamp lechniques The lcli pnncl sho*,s rhe macroscopic Cl clrrrcnl, *hich s rneasuredin the rvhole ce11con[iguration and
carricdb1'gl1cLne|eccPlo](G\'R)channclstvhene\posed1og]lcil1'Thellghtpdn.lshori'sslglc.channcl
Lhe hol.ting porenlial \\'rs -70 m\r n, The L/t pancl shoNs rhe mrcros.opic Cl currenr ihai is carried bv
ouL patch conngufallof In both scenariLrs,
a;ABA, re.eptor channcls when exposed to CABA. The lighl pdncl shors single-chamreLcurrents (Det:r lroln Bofmann.l, Hamill OP. Salinann B:
\'lechxnjsnofaniLrnpernreetiLlL,rLhloughchanrre

(5-HT- receptor),glycine (GlyR),and GABA (GABA, re, e f . u i . A q m n t . nc d p c \ o u r ) . \ r h l r n d 5 H l r e

ceptor channels are boLh permeable to cations and thus
produce exciLaLorycurrents, rl''hereasglycine acti\,atedancl
GAB{, chrnnels are permeableto anions such as Cl and
produce rnhibitory crirrenls. Fillure 8-9 shorvs exanples
. ' r c n L - n e d a L e qh _
o - r r J \ r o r . n p c . r r d L r r r i t a rr l
- e r . r v r t e da d u { B A . . h . r . l - C l " n e d
51,
te,c>en' o d i - g . u l u n t - o h c - . a t t o r ' r p l u r . r n n ,\ r r . o J e
t ) t r ' . c i r -t h . r ' . 1 , o n o l o o . u . . q r h R > L r b - t' . f h . r
primary amino acid sequencessharc a common arrangcment of ML, N{2. M3, and M4 transmembrane
segmenLs,
as describedearlierlor the mcotinicACl.rR(seeFjg. 8-7).
T Lc - e p r o L e - L h . sa l b . l o n g r o - J E . d c r e r . r r y r L J l
is knoi,vn as the ligand-gated ion channel superfamily
f p L 0 \ . . , o r . , . e r l r l , . i . o l l - c . cI n c - . u t L , . r - . L J l
'hp. '^'"
., In,a ,rce-lo h, b -j. ror
. r . L r T \ c - . r r r i , , . ' e . r ' \ l \ a D D c ,r o c - i d e ' l ' 1
rvjthin the tr42 segmenr.\4utatlon of only three resLdues
ri.ithin Lhe M2 segment of a cation-selectivea subunit of
a neuronal nicotinic AChR is sufFcLentto conven it lo an
anion-selectrvechannel actlr.atedby ACl.r.

Equation 8- I

ln the case of an cgoni-st

activateclchannel, such as the
AChR channel, ar least one additional state must be
p r e e n l b ( . l J - \ t h , . o ' c d . r ; n n . c : r ' e i t h e rI ' r n d ' l B
nisl or not:
a

Closedchannel
No aElonist

la

Equation 8-2
r.-]

Closedchannel
Agonist llound

Open channel
Aplomstbound

I t h i . t r ' . . p - h , ' c L h c. l o : c d , L , . L, c, I o t . ' ec . r o

nel must brnd one molecule of the agonist ACh Lo lorm
' - 1 e. ;
J r d s ^ r i - lb " u n C . \ n n " l L . r r' - . l o , " d l r
r r, .,.--

, l _ r, n " , I r l - ,

(AO). Llon'evcr. even this scheme is oi'er1ysimplistic because u.e knolr, that each of the two cv subunits of the
AChR channel must bir.rd ACh srmultaneously for the
cl-rannelLo open:
Equation 8-3

AcetylcholineReceptorChannelsCannot
Open Until Two Acetylcholine
MoleculesBind
I c P ( r . . - t - . r , o l n . r r . - . n g .- . h ; p . r . r f c n l .
each representrngthe opening of a slngle AChR chalLnel
aL the neuromuscular lunction. Earlier rve descnbed the
random openingand closingof an idealizedchannelin a
nvo-state model ln which the channel coulcL be eirhet
closedor open (p. 1BL):

2o

/
I agonist

2 agonists

Open

Unclerstanclingthe kinetics oI channeLopening can be

veD' impoltant Ior clarifl.lng the mechanism by which
c c L d n ( r n n e i r r l r i \ t t o r -u o " l . F o f , \ a r p l e . " . 0 1 f . '
tive inhibitor could preventbinding of the agonisrACh.
Ho\'\'ever.many noncompetitive antagonistsof the AChR

216

Transmission
8 / Synaptic
andthe Neuromoscular
lunction

charLnel,including some local anesthetics,act by enteing

rhe lumen of rhe channel and blocking the flow of ionic
current. Figure B-l0A shorvsthe results of a patch-clamp
experiment in which a single AChR channel opened and
closed in responseto its agonist, ACh. After adding QX222, an analog of the loca1anestheticagent lidocaine (p.
189), to the extracellular slde, the channel exhibits a
rapidly flickering behavior. This flickering represents a
series of brief interuptions of the open state by numerous closures(Fig. B- 10B). This tlpe of flickering biock is
causedby rapid binding and unbinding of the anesthetic
drug to a site in the mouth of the open channel. When
the drug binds, it block the channel to the flow of ioru
(ArB). Conversely,when the drug dissociates,the channeL
becomesunblocked (ArO):

E q u a iro n 8 - 4
d/O-ArB
Blocked

Channel blockers have proved to be effective tools to

study the mechanism of ion pemeation. For example,
QX-222 helped ln locating amino acid residues on the
M2 transmembranesegmentthat form part of the blocker
binding site, thus identifiring residues that line the aqueous pore.

PotentialsRevealthe
MiniatureEnd-Plate
Nature
of
Transmitter
Release
from
Quantal
Terminals
the Presynaptic
Under physiological conditions, an action potential in a
presynapticmotor newe axon produces a depolarizing
pastsynapticEPP rhat peaks at approximately40 mV more
positive than the resting V.. This large signal results from
the releaseof ACh from only about 200 sp.Lapticvesicles,
each containing 6000 to 10,000 molecules of ACh. The
neuromuscularjunction is clearly designed for excesscapacity inasmuch as a single end plate is composed of
numerous s)'napric contacts (-1000 at the frog muscle
end plate), each with an active zone that is lined with
dozenr oI malure Synr'ptrcvesicles.lhus, a large -rer
tory of ready vesicles()l0a), together with the ability to
s)'nthesizeACh and package it into new vesicles,allows
the neuromuscular junction to maintain a high rate of
successfultransmlssionwithout significant loss of function
as a result of presy.rapticdepletion of vesiclesor ACh.
The originaLnotion of a vesicular mode of transmitter
delivery is based on classic observationsof EPPs under
conditions of reduced ACh release. 1n 1950, Fait and
Katz observed an interesling kind of electrophysioLogic
"noise" in their continuous, high-resolution recordings of
V- with a microelectrodeinserted at the end-plate region
of a frog muscle frber. Their recordings from resting muscle fibers that werc not subjected to nene stimulation
revealedthe occurence of tiny depolarizationsof approximately 0.4 mV that appeared at random intervaLs.These
small depolarizationswere blocked by curare, an antagonist of AChR channels, and they increased in size and
duration with the application of neostlgmine,an inhibitor
the spontaneous\- fl-ctuaLlon' also
ol AChf. Becar-rse
exhibited a time course that is similar to that of rhe

normal EPP, they were named miniature end-plate potentials (also known as "MEPPs" or "minis"). These observations suggestedthat even in the absence of nerve
stimulation, there is a certain low probability of transmitter release at the presl'naptic terminal, resulting in rhe
opening ol a small number of AChRs in the postslnaptic
membrane. An examination of the size of individual
MEPPs suggestedthat they occur in discrete muLtiplesof
a unitary amplitude. This findlng led to the notion that
ACh releaseis quantized,with the quantum event corresponding to ACh releasefrom one slnaptic vesicle.
Another way of studying the quantal releaseof ACh is
lo stimulate the preslnaptlc motor neuron and monitor
V- at the end plate under conditions when the probability of ACh releaseis greatly decreased.How can we decrea5pthe probabilityo' ACh release?
t he a-nplitudeol
the EPP that is evoked in responseto nerve stimulation is
de.reasedb1 lowering [Ca2 1. ard -creasinS[tt4g' . A
low [,-;2 l. decreasesCa ' errD nro Lhe pre'ynapLic
rerr al rFig. B 2. stry 3). A high IVg, ]. panially
blocks the pres)-napticCa'z* channels and thus also decreasesCa2+ entry. Therefore, the consequenceof either
decreasedlCa'z*1"or increased lMgz*J. is a falLin [Ca'zt],
in the presFaptic terminal, which reduces transmitter
releaseand thus the amplirude of the EPP (Fig. I 11).
Del e"-trllo and Kat- explortedthrs suppressiorof trans
miter reLeaseunder conditiors of 1ow [Ca,*]. and high
fMg'?*1"to obsewe the V- changescausedby the quantal
releaseof transmitter. Figure B-12A shows seven superimposed records of MEPPsthat were recorded from a frog
muscle hber during seven repetitive trials of newe stirnulation under conditions of reduced [Ca'z*]. and elevated
l\,4or I

The

rc, ard< zrc rlionprl rr rhp nn<rinn

nf rhe

nerye stimulus artifact. The amplitudes of the peak responses occur in dlscrete multiples of approximately
0.4 mV. Among the seyen records were one "nonfesponse," two responsesof approximately 0.4 mV, three
response)ol approximatell0.8 rV. ard o-e re5ponseo[
a p p r o \ l m a l e l )1 . 2 m V . O n e o [ t h e r e c o r d i n g <
al.o -evealed a spontaneousMEPP with a quantal amplitude of
approxlmately 0.4 mV that appeared later in the trace.
Del Castillo and Katz proposed that the macroscopicEPP
is the sum of many unitary events,each having a magnitude of approximately 0.4 mV. Microscopic observationo[
numerous vesicles in the synaplic terminal naturally led
to the supposition that a single vesiclereleasesa relarively
fixed amount of ACh and therebv Droduces a unitary
M F D P .A c . o r d i n gt o t n j s v i e w .t h e q u i n r i z e dV E P P <t n u s
cor espond lo lhe f l..or of d'screterurrbers of svnapti. *
v e s i L . e0
>, l , 2 . l , a n d s o o n .
@
For elucidating the mechanismof spraptic transmission
at the neuromuscular junction, Bemard Kaiz shared the .
1970 Nobel Prize in Physiologyor Medicine.
@

Direct Measurementsof Extracellular

Transmitter LevelsAlso Show That
Transmitter Releaseis Quantal
lrstead of using the postqmaptic AChR as a biologic
detector of quantum release,one can use a microscopic
electrochemicalsensor to measureneurotransmitter Levels
directly. Figure 8-13 shows results from an expedment
in which a fine carbon fiber electrode was placed very

SynapticTransmission
and the Neuromusculaf
Junction/ 8
A

CONTROL

Single-channel
current (pA)

F I G U R E8 - 1 0 . T h e e f f e c to f a l o c a l a n e s t h e r i co n
receplor channeL(AChR) A, Sin
rhe acerylcholLLle
gle-charnel recording ol nicotinic ACh receplor ex
pressd in a .Xenopftsoocyre The patch rvas in rh
ou[side out conllguralion, and the holding potentia]
rvas -150 mV. The continuous presence of L pM
A r '.u*d b " \,r re op r g B. ll-, e'l i
ment is similar to lhai in A, excepr ihat in addition
Lo rhe ACh, the lidocaine analog QX 222 (20 /rM)
was preseni at the exrracellularsur{acof th recp-,
'rr
e tr" r r"l op n.rg .- d,
| ,cl \o p
1..^rp,nr o b. r.T,. li r"r "g a. :d L,r nan o rcf
channel closures The dme scale of th lower panel
is expanded 10 fold. (Data lrom Leonard RJ, r-abarca
CG. Charnet P, et al: Il,rdence that lhe M2 mem
brane'spanning region lines rLle ion channel pore ol
rhe nicotinic recepror. Science 242:1578-1581,
l9BB.)

Closed

open

ANALOG
LIDOCAINE
0

Single-channel
-3
current (pA)
-6

End-plaie4
potentral

(mv)
3

0.2

750
500
Time(msec)

217

0.4
0.6
lca'zl"(mM)

0.8

FiCURE 8 11. The effect of exrracellular Car* and Mg'z* on end plare
potentials. The dara, obtained by stimulating th motor neuron and
moiitoring the evoked subthreshold EPP, show that the EPP is slimu,
laLed by increasing levels of Ca'7. but inhibite.L by increasing levels of
Mgz' (Data from Dodge fA Jr, Rahaminoft R: Co operative action of
calcrum ions in iransmiuer release aL Lhe neuronuscular junciron. J
P h y s i o l1 9 3 : ' 1 1 9 - ' 1 3 21, S 6 7 . )

close to the pres).naptic terminal membmne o[ a leech

neuron lhat uses serolonin as its only neurotransmitler.
The carbon liber is an electrochemica]detector of seroronin (see Flg. B-13A), the currenl measured by this
electrodecorrespondsto four eLectronsper serotonin mol
ecule oxidized at the tip. Stimulating the leech neuron to
produce an action potential aLsoelicits an oxidation current, as measuredby the carbon hber, that correspondsto
rhe releaseo[ serotonln. At a [Ca'?*]. of 5 mM, rhe cure n r i J l a r g ea n d r o m p o s e do ' m a n , ' m a l l < p i l e <f s e eF r g .
8-138, top). On the other hand, reducing [Ca':*]" to 1
mN4-presumably reducing Ca']* influx into the ner-ve
terminal and thus reducing the number of quanra released-reveals individual spikes of serotonin release.The
releasespikes come in two sizes,small and large (see Fig.
B-I38, bottom), correspondingto two separateclassesof
s).napticvesiclesthat are evident on electron micrographs.
lnjecting the cell with tetanus toxin, which blocks the
releaseof slnaptic vesicles,aboLishesthe serotonin release
spres. TLu'. .tsesp kes rep-e>enr
ge-ujne eventsof s;'naprlc exocyrosls.
The nearLyimmediate appearanceof the small release
spikes alter electrical stimulation of the cell shows that
this t1.peof vesicular releaseis extremeiy rapid. From the
height and duration o[ the sma]l and large spikes in
F o -p c-

lB

nnF .rn

e.l mrtc lhe a rourl

of ejecl-t.a

charge and thus the number of serotonin molecules oxidized at the carbon fiber per spike. A unitary small event
corresponds to the release of approximately 4700 serotonin molecules, whereas a unitary large event corresponds to the releaseoI 15,000 to 300,000 serolonin

218

Transmission
8 / Synaptic
and the NeLrromuscular
Junction
A

(MEPPS)
MINIATUREEND-PLATEPOTENTIALS

0.9

l.JLrfiber of
q',jania

(rnv) 0.3

-0.3

10
1t
Time(msec)

DISTRIBUTION
AN4PLITUDES
OFI\,1EPP
22
20
18
14

Numberof
12
observations

10
B
Gaussian
curve
for 5 quanta

6
4
2
0

0.8

1.2 1.6 2.0 2.4

Amplitude
of EPP(mV)

2.4

3.2

N u m b e r o f q u a n t a r e l e a s e d( x )
FICURE 8-12. Evoked and sponlaneousminiature end-plate potentiaLs(MEPPS) A, The rnvstrgatorsrecorded y,,, in trog skeleral Inuscle Iibcrs LhaL
rvere exposed to extracellularsolulions having a tca'z'l of 0.5 mM and a IMg,'l of 5 mN4.These lalues mininrize lransrnifter release,and (herefore it
rvas possible !o resolve ihe srnallesrpossible MEPP, whrch corresponds Lo the reLeaseof a single synaptic vesicle (i e , 1 quantum) The invenjgators
stimulatd Lhe molor neuron seven consecutivetimes and recordd the evohed MEPPS ln one irial, ihe stimulus eloked no response(0 quanta). In
rwo lrials, the peak MEPP was ebout 0.4 mV (1 quantum). In LhreeoLhers,the peek responsewas abour 0.8 mV (2 quanla) Finally, in one, rhe peak
was about l.2mV (l quanta) ln once case,e MEPP ol Lhe smallest magnirude appeard spontaneously.B, The histogram summarizesclaLalrom 198
trials on a ca! neuromuscularjunctioLl in Lhe presenccof 12 5-mM extracellularMgz . The data are in bins $'r!h a width of 0.1 mV The disiribution
has ight peaks The first represenlsslimuli that evoked no responses.The olher seven representslimuli that eYokecL
MEPPSLhaLwere rcLlghly Lntcgral
muhiples of the smallesr MEPP Thc cur.,'eoverlying each clusrer ol bins rs a gaussianor'normal" luncuon and facjliralescalculaiion of the average
MEPP for each cluster of bins The peak r,rlues of these gaussianstollow a PojssoLldistribution. (Data trom Magleby KL: Neuromuscular lransmission.
In Engel AG, Franzini-Armsuong C (eds): Myolo$/, Basicand Clinical, 2nd ed r.welvYorL. Mccraw-Hil1, pp 442 ,163, 1994.)

molecules.Thus, the amount of serotonin releasedby the

small slnaptic vesicles of the leech neuron is about half
the number of ACh molecules contained in a q.naptic
v p rc c a t t , e l r o g r e u r o ' r u ' t u l a r j t n . i o n . I h e . e : n d
other obseNationsoI the s).naptrcfunction of nerve mus
cle and ner-ve-nerves)mapseshave led to the conclusion
Lhat chelnical neurotrdnsmissionoperaLesby a lundamentally
similar mechdnismat many t1pes o[ s]Trapsesin different
animal species(Chapter 12).
( L n - - t e m o r l o n o - r . r m , a n o e si n L h e r e - a t r v ee l f i .
ciency of neurolransmitter releasecan increaseor decrease

the str.englhof a particular slnapse and thereby give rise

r o d n l l t c r a r . o nr b c h a u o r .T h r e er y p e -o 5 ) r r , : r (p rm o o
u l : r t i o no ! L u - a ' l h e n e L r o r r u <url a r * t , t o n . e n d t L c y
differ 1n how they affect the quanial releaseof neurotrans
mitter:
@
Facilitation is a shor.flived enhancen.ientof Lhe endplate pofential in response to a brLeLincrease in the
l r e q l e n . i o f e a , / e) L . n J d l . o n O e w a y t n o r [ " r i l i t a t r o n
may occur rs by a transient increasein the mean number
^f

n,,'n,r

npr

npn,a

cnm,,l,,.

Potentiation (or posLtetanic potentiation) ls a long-

SynapticTransmission
and the Neuromuscu
ar Junction/ 8
A

219

SEROTONINRELEASE

EXPERIMENTALPREPARATION

80

(mv)

Current
or catoon
fiber(pA)

./Serotonin
Stimulus
artilact

--_1uoo

I
It'luoo
Li .

Postsynaptic
membrane

10
1
0msec
msec
"
""*ne.h!ra,+'v,'*.y-.*.-^
.a'n,
+

Sn-a. clea., a.,

vesces
\
Curreri

'\

l r.^,,*"*-.-**"*****"*.4

o'carbon
\
fioer{pAr [*.'t

/
****,

",*"*,,."*.

/I
/{ywdl"airp.n****r,r,,r+,,r,.r*r.y,
l-*'
'/

: : i : " " " 1 " , J : :1 . ' ' * * , ' t * u ' t , i . . , t t t - ; , , , - . n ^ * t /

F|cURE8]3'Dctec1io]r0[scroLonlndraLLslclcascd|ronsYnt1pti.vesicles1,.Ihcscro(DLn
ncurn.lnbcdetectedelectrochenlicell1'usLngacltbon|rbernrlcrocLccLroclcIhecu[en|clrr]edb1lhe
rccordccl front
(blghle\'e]o|5crotoninreLeasc)anda[(-^:-].,'o|
th.rt the Ielease
rd illLlsrLiLes
\'esiC1cslndlaLgcdcnsccoleRsicIes'botho|whichc1nll.ll1)ser\cd
.-ensmilrcf fele.rsefrom single s)'napriclcsiclcs N.{urc 377:62 65. l9g5 )

/iled and pronour]ced increasein transmitter releasethal

, . . - r ' : ! I . t r i ' , , - 1 - r ' d o l r i g l rl f c . . u c r r c e
) \'!. ir-r-ularion. This ellect can lasLlbr minuLcsafter the conclition
mav be causeclby' a ll.rtoc, ol
lng stimulus.Petentiatior-l
in Lc I'r.-.1 r ,' r e e \ L F i r e . \ ^ , h I . - L a . e . (
"
naptic terminaLand thus rncreases
the probabilityof exocytosis.
Sl-naplic depression Ls a lr.l sicnt decreasein the elfrL c r ' . ) o L J . . r ' r . 1 . .r-e l . Jr - r r , l .. o r . n , l .c n l ) . J . , o r \
r o n i n I l c , r d - , ' l : t . p o t .n r r r l - r ' e , p n, a . u a f c " . n C J .
frequent nen'e stimuiation. Depressronmay resrLll ftom
tcmporary depleLion ol transmitter l,ladecl vesicles frclm
r L ' . - . d u . L i o r-to I e o u l n availablc
quanta.
Thus. these three temporal
ber of
, h r r o , . r r , ! n , r ) r i . . r , n " t h ' n d c f l i ec ,
fe.. , . nge. Jl JiJlerent-r(f- ^ -)r rlr. r'.. -T -ri' n.
Similaf modulation of slnaptic strength in the CNS pro

' C . - . ' r , . h . ' - r r . f r r , . d o J -or t l n , - . a n d . n hg o , r i r ,

diriclualnene ternrinalsmay learn"(p. 318)

SynapticVesiclesPackage,Store, and Deliver

Neurotransmitters
fne

Dh. . o.",

ol -\ n

r . . r r . r . i . r ' r oonr h . - r r , . r l t h , n c ' r - . b ) . n J o . , n , .

like cellsin animalslrom the most primitive inYerlebraLes
up Lo mammaLs(p. 88). Many o[ Lhc proterns involved in
synaplrc vesrclemo\.en]entand tumo\:er are related to
hr.r .nto-'ed
t c j r t r ' r c e.l r - l n e r . l ' Tt r e - . . r l 1 cr rl o
processesthat take place in alnrost all eukaryoticcells.
This traflicking involves vesicular translocation from the
. r l o I c ( r ^ B c t ' " "r l a n c r - i o r
.rJcl lr.trt. ,r.
1 1r h t h . -- l r - m r n ' ' n b r , i . . ( , r n e l i .. l n l ; - - o r h , 1 , : . .

22O

junction
Transmission
8 / Synaptic
and the Neuromuscular

BIOGENES]S

Vesicleand peptide
neurotransmitterprecursorsand
enzymesarc synlhesizedin ihe
cell and are releasedfrom Colgi.

Endoplasmic
reticulum

Vesiclestravel through the

Nonpeptide neurotransmittersarc
axon on rnicrotubr e tracks
sy1'Ithesized
and transportedinto
via fast axonaltransport.
vcsiclesthe in nerueiern]inal.
Peptidc ncurotransmitters
are alreadyin some vesicles.

N,4yelin
sheath

,/+s.

W)

---.-.

.YY

Nucleus

cts.
trans
Golgi Golgi

Endosomes
(veslcles)

CELLBODY

Nerve
Terminal

EXOCYTOSIS
A nonpeptideneurotransmitteris
svnihesizedin the nerve terminal
and transportedinto a vesicle.

**:;oii
a.)-fl

FIGURE8 14. Slnihesis,rnd ,eclcJmgof sy'naprLr:

resrcLes
andthcif.onLcnL

.., q or. p,rL.\,. L. . iJ. r ['eJ .r o

. f r , . l L ,I ,
!
LhaL are homologous Lo those associatecl \ritlt s).naptic
vesicles ol higl-ter vertebrates.Thus, the processesuncler.lu
.ion
| c t c t 1 L ,.
r Jr ',a
I )nlular exocl,Losisand encLocytosis.
Synaptic r.esrcles are sphelical organelles wtrh a cliame
Ler of '10 to 200 nm. ,\s shou,n rn Flgure 8 14A, syrap,
ric vesicles are procluced in rhe neuronal cel1 boc\, by a
-lhus,
process similar ro Lhc secretory pathway (p. 36).
the membrane proterns ol s)'naptic vesicles arc s1'nthe
. " r'h.r
lr,r i
t
t,
.. ..- L LI
drrecLedto the Golgi netu'ork, lvhere processing,matura
tion. and sorting occur. Nascenl synaptic vesjcLes-\\,hich
r'r, r" ,cl J. r .,1 o ' .'r
.)1 |
. r r . i n . - r ' . -. ,
vesicles once thev reach Lhe ncrve terminal-arc
then
trarsporLed Lo Lhe ncne tenrinal by fast axonal trans-

port (p. 26) mediatedb1.the microtubulesystem,r'hich

alsocarrLes
mitochondriato the ten-ninal.
VesiclesdestinedLo contain pepti,leneurotransmjtters
travcl down the axon with Lhe presynthesizeclpeprLdesor
pcplide precursorsalready l.rside. On arrival at the ner\.c
terninal (Fig. 8-148). the vesicles-now calledsynaprlc
vcsicles Lhat carry pepticle r-reurotransmrtters
become aL
tachedto the acrin basecL
cyLoskeleLaL
net\\'orh.Other ves
. . 1 , . . ' c l . ' l c J , r i h r o n f . . J n ,u o d r - n r , r - c . g . .
ACh) that arc sr,r'rtl.resizecl
locall)' ir the nerve terrrrinal
and liken'ise attach ro rhe actin-baseclc)loskeleral net
lvork. AL this point. the rnatLlrcsymptic vesiclesare functionally' readl' for Ca']* dependent transminer releaseand
becomeclocl<ed
at specilicreleasesitcsin the activezones
of Lhe presynapucmembrane.After exocytoticlusion o[
the slnaptic resicles,endocytosisvia clathrin-coated
vesi

Synaptic
Transmission
andthe Neuromuscular
lunction/ 8
OUTSIDESYNAPTICVESICLE

,-rd

INSIDESYNAPTICVESICLE

Vacuolar
prolonpump

Neuroiransmilter/
transponer

cles (p..12) recolersmembranecompenentslnd recycles

them to an endosornecomparLment
in Lhc terminal.Synap1lcvesiclesmay then be reformedwithin rhe rerminal
for reuse in neurotransmission,or the)' ma)' be transported back ro Lhe ccll bod).for tu].noverand degrada
Lton
, ir. r rL.
b . u r . ,u d . . . L u , n , n l l , " I ' e n o u . - ) s tem zrndtheir-relatively'uniform size and molecularcom
position,synapticvesjclescan be obrainedin largequantrties trom ya ous sourcessuch as the rat brain and the
eleciricorgan ol the loipf,lo ray. The purilicationof syn,
aptrc veslcleshas n.radeit possible to anal)'zeLheir com,
position, r,vhichhas hciLitaLedgene cloning ancl the molecularcharacterizatjon
ol nra[y proteinsthat are intrinsic
to s)'naptic vesicle function. Figure 8 15 sunmarizes
a number ol the n-rajorclassesof s)'napticvesicie proteins.
, n l , l t . J ( t . , l o 1 a ' . r . 1 e - . r , ,u r . .
l .
,Le
phshed b1.the combinaLionol a vacuolartype H' ATPase
't l
t . r ' r r o , I r L t . . n - o t . r l r o . c t n f. p \ a c u o l a r .
type H+ pump is a large, n.rulLjsultur.rir
complex thar
cat;r1,vzes
the inr'varclmovemenl of H- into the vesicle,
coupled Lo the hvdroll,siso[ cyrosolicATP to adenosine
diphosphrte(ADP) and inorgar.ric
plosphaLc(p. 64). The
resulting pH ancl voltage gradients across tlte vesicle
''_

" ,

. .,.to lr

)ntL erJ inLJ

the \esicle by a unique fami\' ol neurotransmitter transP u r ^ l r . n -* r ' l ' - I n | . c )

p o r t p r o t e l n <l l
\. h" I
tosol for Hf in the r.esicLe.
Thls family' of transporrers
includes members specilic lbr ACh, monoanines (e.g..
serotonin).catccholamines(e.g., norepineplrrine),glr,rta
male, i1nd GABA,/glycine
.
Another clonccl sYnalttic!esicle protein named SV2
(Ior synaptic vesiclc proteln 2) structurallyresemblesa
transpor-tprotein: hou'er.er,a transpor-tsubsLrate
for SV2
l.ursnot been icLentillcclancl iis function is unknor,r'n.
Synaptobrevin is an lg-kDa s).naptjc vesicle prorcin
contalning one transnembrane scgntent. SlmapLobrevin,
which is a v-SNARE(p. 39), ]s essentialfor transmitter
ielease.As discussecl
in the next section,slnapLobrei'in
n n t L . , t i , n r l , r ' ,' - I ' - r ' . . - r, . r ' 1 p | . 1 \ \ ' 1 . \ \ . - s

Synaptobrevin---

(vAruP)
Rab 3 --'---

Synaplotagmin

'prT

on

l.r.nl

SynapsinI

221

Cal\,4kinasetype I

are encLoproteinases
thrt digestsynaptobrevin
and are po,.nt rnhhrn'-nl
- . n ,'' |r r i
..-'. 1, \o
)'o
Rab3 is a mcmber ol a large Ianrily of lou-molecularrveight GTP binding proreinsrhar appearsto be unl\,ersally invoLveclrn cellular membrane trafllcking (p. 39) via
the blnding and hydroLysis
of GTP. SynaptotagminLsrhe
slnaptic \:esicleCaz' recepLor,
a proLein$'ith two external
repetiti\'e domains tl.iaLare homologous lo the C2 clonuin
of protein l<inaseC. The C2 donains appear.to mediarc
bincllng of Caz+. a process Lhat also depends on the
, , - nr

F C U R E B 1 5 . t r l e m b r a n e: l s s o c i e l cpdr o r e i n so l s \ ' l . : r f L i lcc s i c l e s .

,nC ", p- d t\. .c. . ,

r,, . ..I1;.| ,. r1Lr.
f . , c . ' . 1 r , 1 1 t 1 , " 1 , 1 i . 1 , 1, )1: .r '. B , D , l , . n J
{.

, , 1 , , , 1,

n h ^ . ^ h ^ l i '. i

l-

'.

dDu'

m.1

local nse rr.r [Ca2 ], and triggers thc exocytosjsol docked

YesicLes.
Another major constituent,synaptophysin,js an integrai membrane protein $ith [our Lransmembrane
segments that exhibits channel-formin!! acrivity in planar bilayers. It ma,v be irvolved in the formation of a fusion
pore dLLrtnge\ocytosis.The synapsins are a group o1

222

junction
Transmission
andthe Neuromuscular
8 / Synaptic

s).napticYeslcleproteins that are phosphorylatedby both

cyclic adenosine monophosphate (cAMPldependent and
calmoduLin-dependentprotein kinases. Interactions of
sl,napsins wlth cytoskeletalproteins and their inhibition
by phosphorylation have led to the notion thar s)'napsins
normally mediate the attachment o[ synaptic vesicles to
in [( a I ;rrd
l 'tl^ r , rL-edse
l h c a c i n c y t o s l , e l e t oW
subsequent phosphorylation, the synapsin detaches and
p e r m i t s\ e . l c e ' t o r o \ e i o a t . i r e ' i t e s a r , h e ' y . n a o - i ,
membrane.

Neurotransmitter ReleaseOccursby
Exocytosisof SynapticVesicles

A l h o u g . r h e n e c h a - s n b y w n . L s 1 - a p t i cv e s ' .e , f u , e
with the plasma membrane and releasetheir contents is
far from fuLLyunderstood, lve have working models (Fig.
8-16) for the function of various ke)' co-Oon"rlrr und
steps involved in s)'naptic vesicle release.These models
are based on a variety of in vitro experimenls.The use of
speciflc toxins that acr at nerve slnapses and elegant
functional studies of genetic mutanls in Drosphiha, C.
elegans,and gene knockout mice have provided important
information on the roLeo[ various components.
We have already introduced the key proteins located in
the s)'naptic vesicle. Of these, we now focus on the v
S N A R Es y r a p t o b r e r - a n d t h e L a s e n ) o - ) ) m d P l o l a g
min. ln addition, several other protelns-Located in the
rarget area o[ the presynaptic membrane of the nerve
terminal-play an important role in the fusion process.
S).ntaxin is anchored in lhe preslnaptic membrane by a
single membrane-spanningsegment. SNAP-25 (slnaptocome-eqsocir'led
prote 1 -21 kDa) i. tethe-edio tle pre
- ) r - l d p r rm e m h r a n (v i a p c r t o ) , d e c L a i n , B o L h- y r taxin and SNAP-25 are I-SNARES (p. 39). Borulinunr
r o ^ ; r sA a r r d t . r r \ c h a - c e d o p l o L er a ' e ' . ' p e c f i , r 1 l y
cleave SNAP-25, whereas another endoproteinase,botulinum toxin C], specifically cleavessyntaxin. These toxins
b l o c kr h e u s i o no l s y n a p t r 'ce s t c 1 c . .
According to the model shown in Figure 8 16, dock
ing of the vesicle to the pres)-napticmembrane occurs as
n Secl dissoclatesfrom slntaxin. The free ends of s1'naptobrevin, s)'ntaxin, and SNAP-25 begin to coil around
each other. The result is a ternary compLex,an extraordinarily stable rod-shaped structure o[ a helices. As the
energeticaliyfavorablecoiling of the three SNAREScontinues, th vesicle membrane is pulled ever closer to the
pres),'napticmembrane. Car+ enters through voltage-gated
Ca2* channels located in register with the active zone of
. ' o . : l i n .r e r s e i n [ C r ]
L h e p r e . w . r p t i c m e m b r a n eA
triggers the final event, lusion and exocytosis.The synapric vesicle protein synaptotagmin is believed to be the
actual sensor of increased [Ca2+],becauseknockout n-rice
and Drosophilamutants lacking the appropriate isoform of
this prorein have impaired Ca'z-dependent transmitter release.The soluble a-SNAP (solubLeNSF auachment pror e i n ' b r n d : t o h e e " n - t y ( o n ' p l e xr o r r e d b ; t h e i n t " r t $ r n e d 5 \ A R L . a n d p r o m o t e ts\ c S i n d ' n go N : F f a n
ATPase), which uses the energy o[ ATP hydrolysls to
disassemblethe three tightly wouncl SNAREs.The nowfree synaptobrevin presumably undergoes er.rdocytosis,

w h e r e a sr h e , l , n r a ^ i na n d S N A P2 5 o n l h e p r e s ) T a p l i c
membrane are availabLefor the next round of vesicle
fusion.
The modeLjust presentedleavesunanswered some important queslions. For example, whal is the struclure of
the fusion pore detected by electrophysiologicalmeasuremenLsas a primary event in membrane fusion? Also, the
model does not fuily explain the basis for the rapid catalysis of lusion by Ca'z*.Neuroscientistsare very interested
in the deLailso[ synaptic vesicle lusion becausethis exocytotic process might be a target lbr controlling synapllc
srrengrh and may thus play a role in the synaptic plasricity that is responsiblelor changesin animal behavior.

Re-Uptakeor Cleavageof the

NeurotransmitterTerminates
Its Action
Eflective transmission across chemical syeapsesrequires
not only releaseof the neurotransmitter and aciivaiion of
the receptor on the postsynapticmembranebut aLsorapid
a n d e l h . i e n tm e c h d n i , mfso r r e m o v i n gL h et r a n . m . L t eAr t
synapseswhere ACh is released,thls removal is accomplished by enzymatic destruction of the neurotransmitter.
However, the more general mechanism in lhe nervous
- r . r e r r r o l r e s - e - r . t r L e n f t h e n e J r o L - d n strL' te rn e d . ated by specific, high-affinity transporl systemslocated in
the presynaptic plasma membrane and surrounding glial
cel .. fl-ese secordary aclive r-d->po-Lsys.emsu5e lhe
normal ionic gradients of Na+, K*, H*, or Cl to achieve
concentraliveuptake of transmilter. Vertebrateshave two
p r o L en >
o t s i n c t [ a r ' r e s o f n e L - o L _san t r e r I r d n 5 p o r L
The first family is characterizedby a common motif of 12
membrane-spanningsegments and incLudes transporte$
w i l n > p e cF c i t l f o c r r e c l - o l a n i n e<se. r o r o n , nG. A B A .g 1 c i n e . a n d , h o l i n e . r n e r g y c o u p l i n go I l r a n s p o l i n l h i c
classo[ 'v,rems is generallybasedon tor"ansporto[ the
substratewith Na* and Cl . The second farnily is represented by transportersfor the excitatory amino acids glu
l a m a L ed n d a s p a r L a Llen. i l - e ' e l a t t e ' . 1. t e ' r ' . s u b ' t - a t e
t r r n s p o r tB e n e r r l l .)o r p l " , t o c o l r a r < p or o f \ a a n d H ' .
and io exchangeof K*.
At the neuromuscular junction and other cholinergic
syrapses, immediate termination of the action of ACh is
accomplishedenzlrnatically by the action of acetylcholinesterase (AChE). AlLhough AChE is primarily lound at
r h e n e J r o r - ' r L > c Ljdurn c t , o r . A L h F a c l \ i l ) c a n b e d e tected throughout the nervous system.The enz).meoccurs
in a variety of physical forms. The globular or G forms
exist as monomers, din-rers,or tetramers of a common,
approximately 72 kDa glycoprotein catalytic subunit.
These molecules can be lound either in soLublelorm or
bound to cer m.nb-a-e.via a CPI Lnlage(p. t5) in
which a post translationalmodification attachesthe C ter
minus of the protein !o a glycolipid moiety. The asymmetdc or A forms consist of one to three tetramers of
the globuLarenzyme coupled through disulftde bond link
a g et o : c o l l a g e n - l r l.et r u c t u r apl r o t e i n .l h e l a r g e ' ta . ; . m metric form, which l.ras 12 cataiytLcsubunits attached to
the collager.r-liketail, is the major specieslocated at the
neuromuscular junction. The triple-helicaL,collagenJike

and the Neuromuscular

SynapticTransmlssion
lunction / 8
E

INITIALSTATE

The three SNARE5

synaptobrevin,

syntaxinand SNAP-25-continue
to form a tight bundle of o helices,
draliring the vesicleand presynaptic
membranesinto closeapposition.
TIGHTENING
OF TERNARY
SNARECOI\,4PLEX

FORI\,1ATION
OF TEBNARY
COIVIPLEX
OF SNARES

Synaplotagmin

Synaptic
vesrcle_
-

It

Zt

n sec 1 dissociatesfrom syntaxin,allowing

the syntaxinard SNAP-25to Iorm a
complex.The distal end of synaptobrevin
beginsto l\rind around the syntaxin/SNAP25 complex,fonning a temary conplex.

Vesicleswith svnaptotagrrinand
synaptobrevin(a V-SNARE)move
to the nerve terminal membrane,
h.hich containssyntaxinand
SNAP-25(both t SNAREs).

I
|

,,,,l(
. ,,,.,,,,
.-t

.,F

''

a"/

''

"

) .:: :....

. . .-t:. -. - \ l
.,...:::::.: - '

;hravin

n-Sec-1

l*

n'Sec 1

Syntaxin

memorane
OFSNARES
RECYCLING

o-SNAP
DISASSEI\,lBLY
OF TERNARY
SNARECOMPLEX

FUSION
ANDEXOCYTOSIS

D
&
NSF

.)
0-SNAP\

fr

,-il

]!

With the endocytosisof the

vesicle,ihe synaptobrevin
is effectivel)'recycled.The
syntaxinand SNAP-25are
now ftee for an additional
cycleof vesiclefusion.

q-SNAP and the ATPaseNSI bind to

the ternary SNAREcomplexand use
the energyof ATP hydrolysis to
disassemblethe SNARES.

The entry of Ca2*and its binding

to synaptoiagminiriggers fusion.

F|cURE8-16'Nlode]o|s]na]rric'!esic]elusionanc|eroc\)Sis,{DP.
scnsiri|r lacLori-sN.{l'-25.slnaptosone-associaredprorein 2i l{Da: d S\AP. solublc NSF rt(dchlnenr pforern: SN-\RF. SNAP r.LtPio

tail attachesthe as).mmetricAC}rE compiex to cxtracellular- nratrix components of thc synaptlc basal lamir-ra.The
various phtslcai forms ol AChE are a result o[ the alterna
rh lr n.tfl. 'n ^ r 'rr,Al
* t i . e ' o l i , i n g n . . lo . r ' - , n
I@ r\ChE genc
l\c er-) np A.Ll -;'id'r l-rlro u,. \, I t ' .holrre
ancl acetale1n a t\\rc step process:

Equation 8-5

/\
ACLE+ ACh I'

acetyL-AChE -J acelate+ AChE

choline
cho
ln the first step ol the reacLion,the enz,vnecLeaves
line from ACh, rvhich results in Lhe lbtmaLion ol an

8 / SynapticTransmission
and the Neuromuscular
lunction

DISEASES
OFTHEHUMANACETYLCHOLINE
RECEPTOR:
MYASTHENIA
GRAVISAND A CONGENITAL
MYASTHENIC
SYNDROME
The term "myasthenia"meansmuscleweakness
(from
the Creek mys and asthenia)and is used clinicallyto
usuallymeanweakness
in the absenceof primarymuscle
disease,
neuropaLhy,
or centralnervoussyitem d'isorder.
Myasthenia gravls, one specifictype of myasthenia
and the most common adult form, afflicts25 to 125 of
every1 millionpeople.lt can occur at any age but hasa
bimodaldistribution,with peakincidences
occurring
among peoplein their 20s and 60s.Thosealflictedat an
early age tend to be women with hyperplasiaof the
thymus, whereasthose who are older are more likelyto
be men wilh coexistingcancerot the thymusgland:The
cellsof the thymus possessnicotinic acetylcholinereceptors (AChRt, and the diseasearisesas a resultof antibodiesdirectedagainstthesereceptors.
The antibodiesthen
leadto skeletalmuscleweakness
causedin Dartbv competitiveanLagonism
of AChRs.Symptomsinclude'fatigue
and weaknessof skeletalmuscle.Two maior forms of the
diseaseare recognized:
one that involvesweakness
of
only the extraocularmusclesand anotherthat resultsin
generalized
weakness
of all skeletalmuscles.In either
(ase,myasthenia
gravisis typiliedby flucLuating
sympgrealeslLowardthe end ot the day
toms,with weakness
or after exertion. In severecases,paralysisof the respiratory musclescan lead to death. Treatment directed at
enhancingcholinergictransmission,
aloneor combined
with thymectomyor immunosuppression,
is highlyeffective in most patients.
Progress
toward achievingan understanding
of the
causeof myastheniagraviswas first made when electrophysiologicanalysisof involved muscle revealedthat the
amplitudeof the miniatureend-platepotentialwas decreased,although the frequencyof quantal eventswas
normal.Thisfinding suggestedeithera defectin the
postsynapticresponseto ACh or a reducedconcentration
of ACh in the synapticvesicles.
A majorbreakthrough
occurredin 1973,when Patrickand Lindstromfound that
symptomssimilarto thoseof humanswith myasthenia
developedin rabbitsimmunizedwith AChRpiotein purilied trom the eleclriceel.This tindinqwas shortlvlollowed by the demonstration
of anti-AchRantibodiesin
human patientswith myasthenia
gravisand a severereductionin the surfacedensityof AChRin the iunctional
folds.Theseanti-AChRantibodiesare directedagainst
one or more subunitsof the receptor,wherethey bind
and activatecomplementand accelerate
desLruction
of
the receptors,The most common target of these antibodies is a regionof the AChRd subunitcalledMIR (main
immunogenicregion).
gravisis now recognizedto be an acquired
Myasthenia
autoimmunedisorderin which the spontaneous
DroducLionol anti-AchRantibodiesresulGin proqressive
lossof
muscleAChRsand degeneration
of poatju;ctionalfolds.
Treatmentis aimedat eitherreducingthe potencyof the
immunologicattackor enhancingcholinergicactivity
within the synapse.
The former is achievedby the useof
(mostcommonlycorticosteroids)
immunosuppressants
or
plasmapheresis
(removalof antibodiesfrom the

patient'sserum).Somepatientswith myasthenia
gravis
havea thymoma (a tumor of the thymusgland)that is
often readilyseenon routinechestradiographs.
ln these
patients,removalof the thymoma leadsto clinicalimprovement in nearly 75o/oof the cases.Enhancementof
cholinergicactivityis achievedthrough the useof AChE
inhibitors,with pyridostigmine
being the most widely
usedagent.The dosageof thesedrugsmust be carefully
monitored to prevent oyerexposureof the remaining
AChRsto ACh. Overexposurecan lead to overstimuiation
of the postsynapticreceptors,prolonged depolarizationof
the postsynaptic
membrane,inactivationof neighboring
Nat channels,and thus synapticblockade.
Another condition characterizedby progressivemuscle
weaknessand fatigue is the Lambert-Eaton syndrome
(seebox on p. 193). Lambert-Eaton
syndromeis caused
by antibodiesthat attackthe presynaptic
Ca2"channel
and can be distinguished
from myasthenia
gravisin severalways.First,it primarilyattacksthe limb muscles,not
the ocularand bulbarmuscles.Second,repetitivestimula,
tion of a particularmuscleleadsto enhaniedamplitude
of the postsynapticaction potential,whereasin patients
with myasthenia,repetitivestimulation leadsto progressive lesseningof the action potential.Thus, repeated
musclestimulationleadsto increasinq
contractilestrenqth
in patientswith Lambert-Eaton
syndr-ome
and to decreising strength in patientswith myasthenia.
The term congenital myasthenic syndromes (CMS)
refersto a variety of inherited disorders,presentat birth,
lhal aflectneuromuscular
transmission
in a varietvof
ways.Because
specificcasescan involveacetylcholinesterasedeficienc, abnormal presynapticreleaseof ACh, or
defectiveAChRfunction (without the presenceof antireceptorantibodies),
the signsand symptomscan alsovary
widely.In 1995, an unusualexampleof a CMS d;sorder
was tracedto a mutationin the o subunitof the human
AChR.Single-channel
recordings
from biopsiedmusclefibers of a young myasthenicpatient revealeda profound
alterationin AChRkinetics.The burstdurationof AChR
openingswas greatlyprolongedin comparisonwith that
of normalhumanAChRchannels.The moleculardefectis
a point mutationof Thr to Proat position264 in the
adult subunitof the AChR.This bmino acid residue
corresponds
to an evolutionarily
conservedpositionin the
M2 membrane-spanning
segment,which is involvedin
Jormationof the channelpore.Thus,a human mutation
in the pore regionof the AChRproteinresultsin failureof
the channelto closenormall, therebycausjngexcessive
depolarization
and pathologicconsequences
at the muscle end plate.
The aforementioned
mutationis only one of at least53
mutationsin 55 differentkinshipsthat have been identified in the AChR.Someof the other mutationsresultin
electrophysiologic
changessimilarto thosedescribedearlier.Thus,failureof neuromuscular
transmission
may be
inducedby multiplemechanisms,
and eventhose felaLed
to the AChRcan havemany causes.

and the Neuromuscular

SynapticTransmission
Jonction/ 8

FICURE B 17. Pharma.oLogr of

rhe rcrlebrate neuronuscular jun.
lion N1:uy of thc pnneins rhal are
jnvohed in slraplic lransmissron
at rhe mammalian neuromuscLrlar
lLrncriona.c thf 1.rr8ersol narLrnll)
occurnng or slnLLleLi. drugs The
'
anugonEts rfe sbown :rs
signs
highlithtd jn fcd Thc agonisLsare
shown as +' signs hiilhlighted in
gr..n

Acetylcholine
Nicotine
E d-Tubocurarine
E.r-Bungarotoxn

.ern\d,rc
.lrl

,o

'

I
"h,.

d\e.aLe
l f o u p r . o . - r e n L) . . r .-

- rine urnrrn nn

,hn

nn;.,n

fhF

-F ^.d

',

is the hydrolysLs and release ol this acetate. as \\,eLl

--

rhe

tcp

pn/!.n.

"h-.fF.

rr

-.o n,,i

uplake syslem, the nerve lerminal recoversthe chollne

formed in Lhrsreactionand uses it lor the svnthesisof
ACh.

TOXINSAND DRUGSAFFECTING
SYNAPTICTRANSMISSION

entire process.As cliscussecl

in Chapter7, Lhe depoLarlzing phaseol LheacLionpotentialis mecliatedby voltage
dependentNa' channelsthat are specificallybJockedb,v
nanomolarconcentratiolrs
ol the small guanidiniun neu
rotorins tetrodotoxin (TTX) and saxitoxin (STX) (see
F i g .7 5 C ) .
The mamba snaLe to)iin dendrotoxin (p. 19.1) has an
effccLLhaLis preciselyopposiLeLhatol TTX: it facilitates
tl'le releaseof ACh that is evoked by nerve stimulation.
1 p r ' .r ^ r ^ 1 \ / '
/ '|
, ) o \ r n r t e \ , r . l. e r . t L r "f
proteinsuJiih three disulfidebonds that block certainiso
f.m.,f

.h'nnl-

h.

nrn

-g.e8.7
h, ) p-r
lle-r l f r. i"n;l di -' oro
illustrates the reLativesyl.Iapliclocation and corresponding
pharmacology of AChE, as well as several ion channeis
irnd pfoteins involved 1n exocytosis.

L6 .l[ , r r - . . ] I ' r l i , rr . . n . h P r . . , J . , t r n
"
t
\
e
c
l
.
' "h... nr -.- In
r
. , 1r . . . l ' a . e ,
in terminatingthe processo[ transmltterrelease.Blockadc
ol preslnapticK clrannelsbl dendrotoxinlnhibiLsrepo
l a r z , to n o l i \ , 1 - , - 1 , f l . r r ' n ' h , r c L; 1 r " l " r r g
Ingl.J
r ' i o n o , c . , t o p t ( r . , r d f u . r t - t . gL l r c
releasc o[ transmittel in responsc to the entry of extra
Caz- inLothe nen'e termLnaL.

GuanidiniumNeurotoxinsSuchas
Tetrodotoxin Prevent Deoolarizationof the
NerveTerminal,Whereai Dendrotoxins
lnhibit Repolarization

By InhibitingCa2*Channelsin the Active

Zone of the NerveTerminal,Molluscan
Toxins Such as o-Conotoxin Preventthe
Triggeringof Exocytosis
by IntracellularCa2*

..h^r
^^r--r.l
,. tL"
f i.' .
. ,l e p I n ' r d ' : m . 5 l u n : ,
neNe action DolentiaL alri\,ins at the telminal initiates the

.h r^o.'., i. f'-.,.
n u, ) tr.
...-po"l
Lioned at presynapticactive zones and the subsequent

Much ol our knou'ledge of the s)'naptic physiology of the

neuromuscularjunction and the identltresof its various
molecular components have been derived from experi
, r lf.

Tl-,-

.F,

m,

nlnor

. o p n ,- , . , 1

, \ n-

L-i

226

junction
8 / SynapticTransmissionand the NeuromLrsculaf

TABLE A_2

"

TARGET

Tetanus

Synaptobrevin

BotulinumB, D, F, C

Synaptobrevin

BotulinumA/E

5NAP.25

BotulinumC1

Syntaxin

protein 25 kDa.
synaptosome-associated
SNAP-25,

hrn,

inlp, inn

Lr

hece loq1q 1, cdn ledd to

death becausethe toxins that they slnthesize are potent

inhibitors of neurotransmitter release.This inhibition occum becauseboth tetanus and botulinum toxin proteins
h a r e z ' - t - d e p e d e n Le n d o p r o r e i n aa. e- r i \ i l y f l a b l e 8 2 )
These toxins enter nene terminals and specificallycleave
three different proteins required for slnaptic vesicle exocytosis. Tetanus toxin and botulinum toxils B, D, F,
and G cleave s1-naptobrer.in,an inlegral membrane protein of the s)'naptic vesicle membrane. Botulinum toxins
(1
a r r . " . - . . , , , . " t . . t . , , . c y r t d y r na n J S \ A l 2 5 .
"-A
two proteins associatedwith the preslrraptic membrane.
These neurotoxins can have uselul medical applications.
For example, botuLinum toxin is used Lo treat cenain
disorders characterizedby muscle spasms.Injection of a
smal1amount of botulinum toxin into the eye muscles of
a patient with strabismus(a condition in which both eyes
cannot focus on the same object because of abnormal
hyperactivity of particular eye muscLes)is able to suppress
aberrant muscLespasmsand restorenormal vision-

. ee , , - co ' A C h - e c u i e h p e l n e L a - i n t o L h e n e r v e
terminaL. Ca2* enters the presynaptic terminal through
voltage-gatedCa'?- channels rhat are activatedby the depolarization of an incoming action potential. One type of
voltage-gatedCa'znchannel, the Ntype isoform, has been
localrzedro Lhe reg.o ol Lhe ac ive zone ol t\e frog
neuromuscularjunctlon. Vohage-clampexperimentsdemonstrate that a class of molluscan pepiide toxins called
u r c o n o t o \ i n s( o I q l ) b l o L k N r y p e t a - c l r f l p n r cI n d
Both Agonists and Antagonistsof the
practically irreversible fashion. Exposure of a frog nerve
Nicotinic AcetylcholineReceptorCan Prevent
muscle preparation to (d conotoxin lhus inhibits the reSynapticTransmission
lease of neurotransmitter. This effect is manifested as an
The ionotropic (nicotinic) AChR channeLlocated in the
a o o lL . o r o l r r u > t l e F P P w h e n r h e p r e p a r a r i o inc s l i m L posts)'napticmuscle membrane (seeFig. 8- 17) aLsohas a
lated via the nerve. The (d-conotoxinsare 24 to 29 restrich and diverse pharmacology thar can be expLoitedfor
dues long and contain three disulfide bonds. Imaging
clinical applications,as weLl as for elucidating many funcwith confocal laser scanning microscopy l-rasshown that
t o - a l a . p e c r .o f t h e n e u r o . r u s , u l a1r . - r nt .,o . . F i g u r eB higl.rest
binds
at
density
to
voltage-dependent
@-conotoxin
lB shows the chemical structures of two classesof agents
Ca2* channels in rhe preslnaptic nerve terminal, directly
act on the nicotinic AChR. Theseagentsare cLassifred
that
acrossthe s)naptic clelt liom AChR channels.This obseras agonistsor antagonistsaccording to whether they actiyation implies that Ca2* channels are located precisely at
l u c i o n l h i . a r r a n g e - \ a l e o p e n i n go 1 t h e . h a n n e l o r p r e v e n ti r c a c l i v a l i o n .
I e a . r i v e - o n e <o r s \ . n a oct v e s r c t e
Many agonists have a structure similar to that of the
ment provides for focal entry and short-rangedilfusion of
' , l u r J l n e u r o L r d n ) r r . t LAeerh . I n g e n e r a ls. u c h a g o n i s t '
p
r
r
p
a'2
ino he nerrc le ninal to the e\.t.t ,ite, in
activate the opening of AChR channels with the same
volved in promoting Caz*-dependenttransmitter release.
d 1 A ( h . b u r r ^t h
u r i t a r y, o n d u , t a n cae. t h o . ea . n v a t e b
k el'c, ol channe ope- rg a-d co, ng rhe
d.fferenL
syntherlc drugs carbamylcholine (or carbachol) and sucBy Cleaving Proteins lnvolved in Exocytosis,
cinylcholine contain the choline moiety of ACh that is
BacterialToxins Such as Tetanus and
required for receptor actiyation. Carbamylcholineis a car
Botulinum Toxins Prevent Fusionof the
bamyl ester of choline, whereassuccinylcholine (or succi
SynapticVesicles
nyldicholine) is dimer of ACh linked together r.ia the
Anotller class of neurotoxins that specificallyinhibits neuacetyl methyl group. Both of these agents are resistanrto
h'/ir^lv-,(
h\
m i<.lp Afh]hrrr c,r
invlehnl,np
i- <rr<
rotransmitter releaseincludes the tetanus and botulinum
(
.
,
5
0
l
a
r
g
e
p
r
o
L
e
i
n
e
s
p
e
c
roxins
k D a )a r e
r o x i n , .I h e s e
ceprible to hydroLysisby plasma and liver esterases.Thls
property allows for prolonged activation of AChRs.
dveLy produced by lhe bacteria Clostidium tetqni and C.
c , , i n v l c l n p . , r p d r o n r n d rC e : . s t a r n e dn u , c l e
baLulinum(see the box titLed Clostridial Catastrophes).C.
relaxation or "flaccid paralysis,"which is useful in certain
ieldni is the causatrveagent of lelanus ("lockjaw"), which
is characterizedby a general increase in muscle tension
types of surgery ln which rt is rmportant to prevent exciand muscle rigidlty, begrnning most often with the mustation and contraction of skeletal muscLes.Thrs paralytlc
cles o[ mastication. The reason fbr this paradoxical enaction occurs becausesuccinylcholine prolongs the openhancement of muscle aclion is that the toxins have their
ing of AChR channels and thereby depolarizesthe muscle
greatest eflect on inhibitioil o[ synaptic transmission by
membrane in the vicinity o[ the end plate. Such depolari
inhibitory neurons in the spinal cord, neurons that rvould
zation results in iniiial repetitiye muscle excitation and
normally inhibt muscle contraction. C. botulinum causes rremors, folLowedby relaxation secondaryto inactivation
botulism, which is characterizedby weaknessand paralyof Na- channels in the vicinity of the end plate. This
sis of skeletal muscle, as well as a variety of sl.mptoms
latter effect prevents the spread of muscle action potenthat are related to inhibition ol choLinergicnerve endings
tials beyond the end-plate region. On a longer time scale,
rn the aulonomLcner,/oussystem.
such agents also lead to desensitization of the AChR to

Synaptic
Transmission
andthe Neuromuscular
/ 8
Junction

CLOSTRIDIALCATASTROPHES
Botullsm, althoughhardlyone of the most common
causesof food poisoningtoday, is stillthe illnessthat
many people think of when food-borne disordersare
discussed.The neurotoxin of Aostridiumbotulinumis
very potent,and only a smallamountof contamination can leadto death.The most common sourceof
botulism is homemadefoods. The sporesof this organismcan surviveboilingtemperatures
for a number
of hours, and if the cooked food is allowed to stand at
room temperaturefor more than 16 hours,the clostridialsporescan germinateand producetoxin. Symptoms of the illnessmay appearfrom severalhours to
more than a week after jngestion,although most cases
occurwithin 18 to 36 hours.Patientsbegin to complainof symptomsattributableto inhibitionof synaptic vesiclereleasein the autonomic nervoussystem
(seeChapter15), suchas dry mouth, doublevision,
and difficultyswallowingand speaking,and laterbegastrointestinal
gin to experience
complications,
includingvomiting,pain, and diarrhea.Symptomsattributable to inhibition of synapticvesiclereleaseat
junctionsuchas weakness
and pathe neuromuscular
ralysisof the limbs may soon follow; ultimatel, paralysis of the respiratorymuscles(seeChapter 26) can be
fatal. Prompt interventionwith mechanicalventilation
hasreducedthe mortalityfrom botulismdramatically,
and the figuretoday standsat about 20yo.Almostall
deathsoccuramong the firstvictimsof a contaminated ingestion becausethe diseaseis not quickly recognized; those who fall victim later, when the diagnosisis much easier,do much better.
Vaccinationhasreducedthe numberof casesof
tetanus reported in the United Statesto only about
100 eachyear,almostall occurringin inadequately
The diseaseis causedby a neuvaccinatedindividuals.
rotoxin (tetanospasmin)produced by Aos dium tefori The organism gains entry to its host through a
cut or puncturewound. The toxin then travelsalong
the peripheralnervesto the spinalcord, the major site
of its attack. There,the toxin inhibits synaptic-vesicle
that normallyinhibitfiring of
releaseby interneurons
the motor neuronsthat, in turn, activateskeletalmusinhibitionof
cle.Thus,becausethe toxin suppresses
the normal reflex arc, muscularcontraction leadsto
of the jaw
profound spasms,most characteristically
musclesbut potentiallyaffectingany musclein the
body. Symptomscan commenceon the day of the
injuryor as long as 2 months later.Complications
arrest,aspirationpneumonia,rib
includerespiratory
fracturescausedby the severespasms,and a host of
other pulmonaryand cardiacmanifestations.

agonist, which further inhibits neuromuscular transmLssionAnother lmporlant agent acting on AChRs is nicotine,
a n a l u r a cl o " t ) t i L U eonIt L o b a . c o1 6 2 1. ' r s . p 6 n .b l e f o t t r e
stimulant action and at least some of the addictive elfects
of smoking. The selective ability of nicotine to activate
AChR channeLsis the basis of the ciassifi.cationschemeof
"nicotr.nrc"AChRs versus "muscarinic" AChRs (seeFig. I
3). Nicotine is not an agonist ol the muscarinic or G

227

protein-llnked receptors, which instead are activated by

the mushroom alkaloid muscarine. Although nicotine is
able to activate the AChR at the neuromuscuLarjunction,
the physiological effects of smoking are primarily manifested in the CNS and autonomic gangha, where other
neuronaLisoforms of nicotinic AChRs are located.
A cLassicexampLeof a nicotinic AChR antagonist is dtubocurarine (see Fig. I 1B), the active ingredient of
curare. a poison extracted from plants of the genus
Srrychnos.The indigenous tribes of the Amazon region
used curare to poison arrows for hunting. d-Tubocurarine is a competitive inhibitor of ACh binding to two
acrivation sites on the a subunits of the AChR. This
action leads to flaccid paralysis o[ skeLetalmuscle from
inhibition of the nicotinic AChR. However, curare does
not cause depolarizarion.A hallmark of the action oI dtubocuradne is that it can be reversedby an increasein
concentrationof the natural agonistACh by binding competition. A Largeincreasein locaLACh concentmlion can
be produced indirectLy by an inhibitor of AChE such as
neostigmine(seeLater).
Figure 8 18 also shows the structure of pancuronium,
which is a tnthetic bis-quatemary ammonium steroid
d e r r v a l r v1, .n . s d r l g 1 5a l s ou s e [ u [ o r t L e p r o d u . t i o no f
neuromuscular blockade in surgery, and it is actually a
more potent, selectivecompetitive antagonistof the mus
cle nicotinic AChR than D-tubocuranne$.
Another class of nicotinic AChR inhibitors is a lamily
of approximately 8 kDa proteins present in the venom o[
Elapidae snakes (e.g., cobras). These toxins include cr
bungarotoxin (cv-Bgt)and homologous c toxins, whrch
b r n d ' e r y s t r o n g l yL o n i c o t . -L r e L e p l o r r 1 [ p . p e . , f ,
binding of a-Bgt to the nicotinic AChR of skeletalmuscle
is virtually irreversible.When a-Bgt binds to the nicotinic
AChR, it obstructs the agonist-brndrngsite and prevents
, " r' . i n n r r ' h p - p , . n . r t ' q C h . I h e r a d r o r o d r n a r e d
derivative Dillabeled a-Bgt has been widely used as a
ligand for punfying the nicotinic AChR from various tis
sues. FluorescenLderivativesof a-Bgt can also be used as
- recF, lr.pl- ar la"a z no Aa\Rs at the mus(le end
plate. The same snake venom (Bungair.ismulticinctus)that
contains a-Bgt also contains a homologous protein toxin
called r bungarotoxin (r-Bgt). This toxin has little elfect
on nicotimc AChR channeis at the neuromuscularjuncrro- \rr' r d^e- l-ihr Aa.hR, hannelsrn neuroral tis
sue. The differential elfecLof a-Bgt and rc-Bgton muscle
and neuronal curents activated by both ACh and nico
tine led to the recognition that different classesof nicorinic receptors exist in the CNS versus skeletal muscle.
The basis for these isoforms is the differential expression
of multipLe genes for homologous nicotinic AChR subunils.

Prolong
Inhibitors of Acetylcholinesterase
and Magnify the End-PlatePotential
A variety of specific inhibitors of anticholinesterasehave
h c e r h e l n l ' r l - d e l n r n o - h e c o n t r i b . r u o no [ A u L I t o
- e c n ^ r - e - r ' r h e n u . , p e n d r ' l a i e .I n n , b i L i o no f q r h L
generally increasesthe amplitude and prolongs the duration of the postqmaptic response to ACh; thus, the en

8 / SynapticTransmission
and the Neufomuscular
Junction
AGONISTS
Acetylcholine

H3c-

Carbamylcholine(carbachol)

!"'
ttl

N+- cH2cH2-

o -a

H3C-

cH3

CH:

i"'
N*

cH2cH2-

o -a

NHz

CHs
Acetyl

Choline

Carbamyl

Succinylcholine

:"' CH2CH2- ?
?
C -CH2CH2 -C -O

H3C-N+-

Nicotine

CH2CH2

:"' at
T*
CH:

CH:
Choline

,A\

\./'1lr/-N
,z*\
CHg

Succinyl

ANTAGONISTS
Pancuronium

o-Q-.",
H3CO
H'/"
CHs
FIGURE8-18.

Agonists and anlagonislsof rhe nicorinic acerylcholinerecepror (AChR).

zyme plays an important role in limiting rhe excilatory

action of ACh under normal physiological condltions. ln
the absenceof ACh breakdown by AChE, the prolonged
decay of the EPP reflects the underl1.'rngkinetics of activated receptors and slow depletion of the agonist in the
vicinity of the junctional foLdsby diffusion of ACh.
The pLantalkaloid physostigmine (also knowr as eserine) is the protot),?ic anticholinestemse(Fig. B- 19). Neostigmine (also called prostigmine), a s)'nrhetic anti-AChE
drug that is partially analogousto physostigmine,is used
to treat myastheniagravis. As discussedin rhe box on p.
1 r 4 t h r q d q e a - ei c c a . r < e dh v r - p a u t o r m n u n ed e , t r u c
tion and loss of nicotinic AChRs at the muscle end plate.
BecauseACh is an acetyl ester, the catalytic cycle of
AChE results in the formation o[ an intermediate ln
r r h i c h a . e r i n e g r o u o o f r h e A C \ f i . a c e r l l a r e dP. r ; ' o
stigmine and neostigmine, on the other hand, have a
carbamyl ester linkage that produces a carbamoylatedin
termediate lorm of AChE. However, slow hydrolysis ol
the carbamoylatedenzyme results in an approximately 4,
h o u r d u r a t i o no f e ' t e r a s ien h i bt r o r
Another impo ant class of syrthetic AChE inhibitors
consistsof organophosphorus compounds, which are irreversibieinhibitors. This group is typified by diisopropyl-

fluorophosphate (DFP) (see Fig. B-19). These compounds react with the aforementioned serine residue of
AChE and form an essentiallyirreversiblecovalentmodifi,
calion of the enz)'me.Such agenr rank highly among the
most potent and lethal of toxic chemicals.Their devastat
ing effect is causedby excessiveenhancementof choliner
gic neurotransmission,mediated by both muscarinic and
nicotinic receptor pathways throughout the body. For example.exposurero tor.c organophosphorus
agerl> res..r
ts
in the facc'd parallsisof re>pirdronnuscles becauseol *
i n i t a m - > c l e ) r r n u l a r i o n b l l o u e d b 1 o e p o l a r i z a r i oW
r
blockade. The devastatingaction of these compounds dramaticaLlyunderlines the essentialrole of AChE in termi
n a t i n gc h o J i n e r g ince u r o r r a n iin> s o - A t r a g r .a p p lc a L i o n
o [ v a r i o u ' v o - a t i ] e[ o r m . o f t h e . ec o m p o u n d si s t h e . r : - e
ar chemrcal r,rar'ire agenls /i e . -nene ga5 sLcl^ as
sarin). Related compounds, such as malathion (see Fig.
g - 1 9 ' r , . h i r ha - e ' e l e c r r v e itl o \ t . l o i n ) e . L >a. r e w i d e l y
used as agricuLturalinsecticides.
A n a t u r a ol r g a n o p h o ' o h o r ur e. u r o r o r . n s p r o d u r e ob y
Anaebenaf.os-aquae,a toxic cyanobacterlum (blue-green
K n o w n a s a n a r o x i n - a ( s )t.h i s r o r i n . s r p o t e n L
"iga).
i n l - i b i t o ro A L h E a n d . s r e . p o n . r b l eo r i L e p o i s o n . n go
dogs and farm animals that drink from contaminated

SynapticTransmissionand the Neuromuscular)lndion / 8

REVERSIBLE
Physostigmine(eserine)
CHe

'-]L_r^r-o-c-N

(A,JV
NN

,/H

l!

tatu

Neostigmine(prostigmine)

IRREVERSIBLE
Diisopropylf luorophosphale (DFP)

F-

cr.

9| . /

P-O-CH

t\

cHs

CH
HSC

CHg

O,O-dimethyl
S-(1,2-dicarbethoxyethyl)
phosphorodilhioate
(Malathion)
I

ill

COOC,H.

cH3o- P-s -cH

tl
ocH3 cH2cooc2Hs
(AChE)inhibitors.
FICURE8-19. Strucuresof acetylcholinesrerase

ponds. Alother interesting class of natural inhibitors includes the fasciculins, a family of small protein toxins
present in mamba snake venom that inhibir AChE with
very high affinity and specificity.

229

By InhibitingMuscleNa+Channels,
Certain
NeurotoxinsBlockSpreadof the Postsynaptic
Action Potentialand MuscleContraction
An action potential is not only the first but also the last
step in transmission at the neuromuscular junction: the
production of an action potential in rhe muscle fiber
membrane signals the successfulcompletion of qmaptic
tmnsmission. As discussed earlier, action potentials, including those in muscle, can be blocked by TTX and
STX. Selectiveblockade of the muscle action potential can
be achieved with a unique toxin called p-conotoxin (p.
187), which is obtained from a marine snall (Conusgeographu't. p-Conotovinis a 22 residue.ba,ir pepride;lh
a discoidal, starlike three-dimensional structure that is
stabilized by three disulfide bonds. lt is an especially
potent blocker of the particular isoform of the voltagedependent Na* channel that is present in adult mammalian skeletal muscle, but it has little effect on the Na+
channel isoforms of nerve or heart. lf an intact nervemuscle preparation is exposed to ;r,-conotoxin, stimulation of the newe still evokes the releaseof ACh, but the
muscle action potential is complerelyeliminated.
@
REFERENCES
Books and Reviews
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zini-Armstrong C (eds): Myology, Basic and Clinical,2nd ed. New
\ork. M.6.aq Hil..l9q4 pp 76q-17o7
Ha]l Z!V, SanesJR: Slraptic struclure and development: The newomuscular junction. CeI l0 (Suppl):gg-r21, 1993.
Jahn R, Hanson Pl: SNARIS line up in new environmenr. Narure 393:
t4-15, 1998.
KaE B: Newe, Muscle, and S).napse.New York, Mccraw Hill, 1966.
Nicholls JG, Maftin AR. Waliace BG: From Neuron to Brain, 3rd ed.
5 r n d e - l a n d .M A . . r n a u e rf u s o d a t e s , o q 2
Sridhof T: The s)'naptic vesicle cycie: A cascade of protein-prorein interactions. Nature 375:645 653, 1995.
Van der Kloot W, Molg6 J: Quantal acetylcholine release at the vertebrate neuromuscuiarjunction. Physiol Rev 7,1:899 989,1994.

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Del Castillo J, Kaiz B: lnteraction at end plate receplors between different choline derivadves. Proc R soc Lond B Biol Sci 1461369-381,
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Fatt P, Katz B: Spontaneoussubthreshold acriviry at moror nerve end,
i n g s .J P h i s i o l 1 1 7 : 1 0 9 - I 2 8 , 1 9 5 2 .
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crayfish.J Physiol 145:289-325, 1959.
Magleby KL, Stevens CF: A quantilative description of end-plate currents.J Physiol 233:173-197, 1972.
Noda M, Takahashi H, Tanabe T, et al: Srructural homologl of Tal-pedo
.alfomicc acetylcholinereceptor subunits. Nalwe 302:528, 1983.
Ohno K, Hutchinson DO, Milone M, et al: Congenital myasthenic slndrome caused by prolonged acerylcholine receptor channel openings
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Acad Sci U S A 92:758 762, 1995.