3.3 Microphysiology of chemical synaptic transmission

The action of a single tranmitter-gated channel can be studied experimentally

Fig. 10-11
single ACh channels의 patch clamp 실험 (Erwin Neher, Bert Sakmann).
A: patch clamp setup.
B1: in frog muscle fiber, in the presence of 100 nM ACh, at RP -90 mV.
opening of channel---> pulse of inward current (downward deflection).
all-or-none fashion
amplitude fixed,
but duration of the opening varies (mean 1 ms)
single channel open---> 17,000 Na+ ions flow into the cell
somewhat smaller number of K+ ions flow out
B2: amplitudes: average ca. 2.69 pA (elementary current)
C: in the presence of 100 nM ACh, at -130 mV MP.
individual channel currents---> all-or-none increments of -3.9 pA
각개 채널이 동시에 열리면---> amplitude linearly added up (up to 3 channels).

Fig. 10-12
Membrane potential을 voltage clmap로 고정시키면서 2 uM ACh 투여에 의한
single channel의 크기와 direction을 조사.
reversal potential: at 0 mV
negtiave potential에서는 inward current flow
postive potential: outward current flow

current magnitude의 변화: driving force의 변화 때문

Currents for the end-plate potential depend on four factors
1) total number of end-plate channels (보통 200,000 channels)
2) channel open probability (receptor주위의 transmitter 농도)
3) conductance of each open channel
4) driving force

Fig. 10-13
Total end-plate potential: summed average of the currents in thousands of individual ion channels

A: ACh 투여: all channels open rapidly in response to ACh
but closing times are varying
B: Summed average of the currents of individual ion channels

The Nicotinic ACh receptor is an intrinsic membrane protein with five subunits

Directly gated receptor channel has two functions
1) recognize and binds the chemical transmitter
2) opens a channel in the membrane through which ions flow

Questions: where is the binding site located?
where does the channel lie?

ACh receptor:
membrane glycoprotein, MW 275K, five subunits
Fig. 10-15
2 alpha, beta, gamma, delta (four polypeptide chains)
only alpha-subunit binds ACh with high affinity
one ACh bind to each alpha-subunit(extraceullar space에 돌출한
hydrophyilic region의 2 cysteine residues에)--->
conformation change-->open channel.
뱀 독인 alpha-bungarotoxin이 nicotic ACh receptor의
alpha-subunit에 결합한다.
four subunits는 각개의 다른 gene에 의해 encoding된다.
이들 gene들은 하나의 ancestral gene에 의해 유래되었으리라 생각된다.
각 subunit의 amino acids의 sequence를 조사해보면 약 50%가 비슷하다.

Fig. 10-16
ACh receptor-channel 의 각 subunit는 4 개의 membrane-spanning
alpha-helices로 되어있다.
이 membrane spanning (hydrophobic)지역은 약 20개의 amino acids로
구성 되어있다.(M1, M2, M3, M4)
이들 중 M2가 channel lining을 형성한다.

Fig. 10-17 & 10-2
EM 사진---> 3차 구조의 reconstruction by Chikashi Toyoshima
large component (6.0 nm)가 밖으로 돌출,
2.5 nm diameter channel mouth
2.5-3.0 nm narrow region corresponding to the M2 segment

Glutamate Receptors

Fig. 11-4. Four types
Second-messenger linked receptor:
Quisqualate-B: phosphoinositide-linked second messenger system을
activation 시켜서 channel을 Na+ 과 K+에 permeable하게 한다.

Directly gated receptors:
AMPA (alpha-amino-3 hydroxy-5 methyl-4 isoxazole proprionic acid (Na+, K+ ions)
NMDA (N-methyl D-asprtate), (permeable to Na+, K+, Ca2+)
(resting시 Mg2+에 의해 blocked, 막이 20-30 mV depolarize되면 Mg2+가 빠지면서 glutamate가 binding 한다.)
(glycine존재하에서 제대로 기능한다.)

Fig. 11-5. 쥐의 hippocampus의 cell culture에서
left: bathing soution에 Mg2+가 없을 때 채널의 열고 닫힘이voltage변화와 무관하다.
right: Mg2+이 정상 농도 (1.2 mM) 있을 때:
at RP(-60 mV): closed
30 mV depolarization 시키면 channel opening

Fig. 11-6. NMDA와 non-NMDA receptors를 갖는 cell에서 RP 때 NMDA-activated
channel은 Mg2+에 의해 block되어 있다.
Cell이 non-NMDA receptor의 activation에 의해 더 depolarize되면서 NMDA-activated channel이 열린다.(late current).
NMDA receptor blocker: APV(2-amino-5-phophonovalerate), phencyclidine (PCP)

따라서 NMDA-activated receptor-channel은 chemically gated될뿐만 아니라 gated by voltage.
Ca2+ influx에 따른 Ca2+-dependent second messenger cascade를 acivates---> long lasting synaptic modification에 중요

Glutamate toxicity: excessive amounts of glutamate
---> acts on NMDA-activated receptor에 작용해서
---> excessive inflow of Ca2+
---> activating Ca2+-dependent protease
---> production of free radicals toxic to the cell
(간질병, cell death의 원인일 가능성)

GABA and Glycine

Fig. 11-8
Channel opening by GABA (10 uM) and Glycine (10 uM)
---> produce similar elementary pulses of outward currents
(transmitter-gated Cl- inhbitory channels in a mouse spinal neuron)

Fig. 11-9, rat hippocampal neuron에서
Glutamate single channel current VS. GABA single channel current
: different reversal potentials
A: Glutamate current
MP---> depolarizing direction--->
Glutamate (inward) current smaller
at 0 mV MP: current pulses are nullified
(averaged Ek + ENa at 0 mV MP)
at 30 mV MP: reversed
B: GABA current
at -60 mV MP: reversal potential for IPSP
(near the ECl-)
at more depolarizing levels: current pulses are outward

Transmitter Release
Transmitter release에 관여하는 channel은?

Fig. 13-1. Tetrodotoxin (TTX): Na+ channel blocker
No effects on
K+ channel,
postsynaptic receptor
channel controled by postsynaptic receptor
따라서, at cholinergic synapse:
TTX blocks the presynaptic Na+ spike
그러나 postsynaptic receptor에 직접 투여한 ACh은 EPSP 생성
(Na+ channel activated by AP is different from
the channel permeable to the both Na+ and K+)

A: recording electrodes in both pre- and post-synaptic membrane of the
squid giant axon

B: Presynaptic cell: AP 약 110 mV---> transmitter release---> large EPSP
if TTX 투여---> presynaptic AP 이 점점 작아짐---> EPSP 감소
presynaptic AP이 40 mV 정도 되면--->EPSP 소멸
C: TTX로 presynaptic Na+ channel을 block했을 때 transmitter release의
input-output curve를 볼 수 있다.
presynaptic potential이 40 mV의 threshold level 이하에서는 EPSP불능
40 mV를 조금 넘어서면 steep increase in EPSP

Fig. 3.17 (TEXT)
Steep increase in postsynaptic current as the [glutamate]
(정상적인 대사과정에서도 agonist와 transmitter가 있음으로 이들이
receptor에 작용해서 noise를 나타내는 것을 막는다)

Fig. 13-2. Transmitter release는 K+과 Na+에 의존하지 않는다.

B: TTX로 presynaptic Na+ channel을 완전히 block하고 current pulse
injection을 증가 시키면 presynaptic terminal이 점점 크게
depolarize되면서 postsynaptic potential이 점진적으로 증가한다.
(Na+ influx가 transmitter release와는 무관하다)

C: TTX로 Na+ channel을 block한후 다시 TEA로 voltage-dependnet
K+ channel을 block.
presynaptic cell에 current pulse injection--->
presynaptic depolarization--->
postsynaptic potential이 current pulse injection에 비례적으로 증가
(neither Na+ nor K+ is required for effective transmitter release)
D: input-output curve

Calcium influx is essential

[Ca2+]o increase---> enhances transmitter release
lowering---> reduces the transmitter release
Mg2+ : blocker of Ca2+ channels

Fig. 13-3
squid giant axon, Na+ and K+ channels blocked by TTX and TEA
depolarize presynaptic cell---> increase in Ca2+ current--->
increase in postsynaptic potential
Ca2+ chan