Henry Lester’s “office” hours Mon, 1:15-2 PM, Fri 1:15-2 outside the Red Door
Bi/CNS 150 Lecture 5
Wednesday, October 9, 2013
Revised after lecture 10/9/13
Presynaptic transmitter release
Chapters 9, 12 (co-written by T. Sudhof, one of this week’s Nobel Prize awardees)
1
Proof of chemical synaptic transmission, 1921
Vagus nerve
runs from the head to the heart
Spontaneous
heartbeats in both
hearts are
stopped by stimuli
to the “upstream”
vagus
The diffusible
substance:
acetylcholine
acting on
muscarinic
ACh receptors
smoked drum
2
electrical
transmission in
axons:
electric field
open
closed
Past lectures:
V-gated Na+ channels
V-gated K+ channels
Today: V-gated Ca2+ channels
chemical
transmission at
synapses:
[neurotransmitter]
closed
open
Friday: ACh-gated excitatory cation (Na+ / K+ / Ca2+) channels
& GABA- and glycine-gated inhibitory anion (Cl- channels
Next week: Glutamate-gated excitatory (Na+ / K+ / Ca2+) channels
3
Many basic principles of
chemical transmission
and
developmental neuroscience
were discovered at the
neuromuscular junction
(nerve-muscle synapse);
acetylcholine is the
transmitter.
Figure 9-1
4
Fine structure of the NMJ
0.3 µm
Incl. acetylcholinesterase
ACh receptors
Figure 9-1
5
Life cycle of a synaptic vesicle
Figure 12-10
6
Caught by
flash-freezing,
invented at Caltech
~ 50 yr ago
A. Van Harreveld
Presynaptic terminal
postsynaptic cell
Like Figure 12-7
7
Proteins associated with synaptic vesicles, slide 1
Vesicles can be isolated from brain
tissue by cell biological methods
A. Homogenize brain in isotonic sucrose.
B. Isolate synaptosomes
(cut-off nerve terminals)
by differential and sucrose gradient
centrifugation
C. Lyse synaptosomes in hypotonic solution
to release vesicles.
D.
Isolate vesicles
by glass bead column chromatography.
8
Proteins associated with synaptic vesicles, slide 2
Synaptophysin
Synaptotagmin (the Ca2+ sensor)
Snares (residents of either the vesicle [v-snare]
or the target membrane [t-snare])
VAMP (also called synaptobrevin), a v-snare
Syntaxin, a t-snare that also associates with Ca2+ channels
SNAP-25, a t-snare (~25 kD)
ATP-driven proton pump creates concentration gradient that drives
neurotransmitter uptake against concentration gradient
(one of three transporters that function in transmitter release)
Lecture 1 asked,
“Could cells utilize plasma membrane H+ fluxes?”
“Probably not.
There are not enough protons to make a bulk flow, required for robustly
maintaining the ion concentration gradients.
(but some very small organelles (~ 0.1 mm) and bacteria do indeed store
energy as H+ gradients).”
Mary
Kennedy’s
work
9
vesicle interior
How synaptic vesicles fill from the cytosol
H+
Transporter #1: ATP-driven proton pump
cytosol
Transporter #2: Proton-coupled
neurotransmitter transporter
Neurotransmitter
and
ATP
cytosol
(3,000 to 10,000 molecules
of each)
vesicle interior
~ isotonic!
See Figure 13-1A
10
From Lecture #1
Transporter # 3. Na+-coupled cell membrane neurotransmitter transporters:
Antidepressants
(“SSRIs” =
serotonin-selective
reuptake inhibitors):
Prozac, Zoloft, Paxil,
Celexa, Luvox
Drugs of abuse:
MDMA
Na+-coupled
cell membrane
serotonin
transporter
Attention-deficit
disorder medications:
Ritalin, Dexedrine,
Adderall
Trademarks:
Drugs of abuse:
cocaine
amphetamine
Presynaptic
terminals
Na+-coupled
cell membrane
dopamine
transporter
cytosol
NH3+
HO
outside
HO
N
H
See Figure 13-1B, C
HO
H2
C
C
H2
NH3+
11
From a previous recent lecture
Atomic-scale structure of (bacterial) Na+ channels (2011, 2012)
As of fall 2013, there are no
crystal structures of voltagegated Ca2+ channels.
From the similarities in
sequence, we expect the
secondary and tertiary
structures to resemble those of
K+ and Na+ channels.
A voltage-gated Na+ channel
can be changed to a voltagegated Ca2+ channel by mutating
...
just 2 out of 1800 amino acids
See Table 12-1
12
Electricity, then chemistry triggers synaptic vesicle fusion
docked vesicle
nerve impulse
Na+ and K+ channels
neurotransmitter
voltage-gated
Ca2+ channel
We’ll show a more complete
animation in a few minutes
See 1st part of Chapter 12
13
Electricity, then chemistry triggers synaptic vesicle fusion
docked vesicle
nerve impulse
Na+ and K+ channels
Ca2+
neurotransmitter
voltage-gated
Ca2+ channel
We’ll show a more complete
animation in a few minutes
See 1st part of Chapter 12
14
Electricity, then chemistry triggers synaptic vesicle fusion
1. The Na+ channels have produced
the voltage change (depolarization);
the K+ channels
have rendered it brief (~ 1 ms)
fused vesicle
Ca2+
2. The Ca2+ channels
produce some depolarization,
but their main function:
to introduce the
intracellular messenger Ca2+
See 1st part of Chapter 12
neurotransmitter
We’ll show a more complete
animation in a few minutes
15
Synaptotagmin is the calcium sensor
Synaptotagmin has as many as 40
Ca2+-binding sites. Perhaps
binding of more Ca2+ increases the
rate of fusion and/or pushes the
vesicle toward the “slow track” and
full fusion.
Like Figure 12-13
Animation of “full collapse fusion”:
http://stke.sciencemag.org/content/vol2004/issue264/images/data/re19/DC2/slowtrack2.swf
16
Other proteins that act on synaptic vesicles
II.
Peripheral membrane proteins
A.
Synapsins anchor vesicles to cytoskeleton.
B.
Rab 3A is a GTPase perhaps involved in vesicle trafficking
III.
Soluble proteins that participate in vesicle fusion and release
A.
SM proteins

Munc-18-1 binds to the N-terminus of syntaxin and participates
in vesicle docking and priming.

Munc-13 - essential for all forms of synaptic vesicle fusion,
participates in vesicle priming.
B.
Complexins interact with SNARE complex and stabilize SNARE
complex.
C.
NSF and its associated proteins are needed for SNARE recovery.
17
An alternative form of Ca2+-dependent vesicle fusion,
termed fast tracking, or “kiss and run”
predominates at low frequency stimulation
Animation:
http://stke.sciencemag.org/content/vol2004/issue264/images/data/re19/DC2/newFasttrack2.swf
18
Transmitter release depends strongly on extracellular Ca concentration
Experiments at the squid giant synapse, which excites the giant axon (See Figs. 12-1, 12-2, 12-3)
Cooperative processes cause
nonlinear relation between
[Ca2+] and transmitter release
HAL’s first paper, Nature 1970
19
Timing of synaptic events
“Synaptic delay”, between the
peak of the action potential and
the start of transmitter release, is ~
0.5 ms.
Delay between the peak of the Ca2+
current and the beginning of the
EPSP is ~ 0.2 ms (more at lower
temperature).
Most of the “synaptic delay” is
caused in opening of Ca2+
channels during the action
potential.
mV
The size and timing of the
EPSP’s can be modulated by
prolonging the action potential.
Figure 12-1
20
stimulus to
presynaptic motor
axon, producing
action potential
measured postsynaptic response
V
large
“synaptic potential”
leads to muscle
action potential
+60
mV
-60
1
Electrophysiological analysis
of
quantal synaptic transmission
(slide 1)
ms
5
subthreshold synaptic events
(revealed in low Ca2+)
(Figure 12-6, Box 12-1)
21
stimulus to
presynaptic motor
axon, producing
action potential
measured postsynaptic response
V
repeated identical
stimuli to the
presynaptic
neuron . . .
5 mV
Electrophysiological analysis
of
quantal synaptic transmission
(slide 2)
(Figure 12-6, Box 12-1)
5 ms
. . . yield variable postsynaptic responses!
22
Electrophysiological analysis of
quantal synaptic transmission
(slide 3)
repeated
stimuli to
presynaptic
neuron
5 mV
Fraction of
Observations
Analysis of Quantal Synaptic Transmission
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Stimulated
Spontaneous
01
12
23
34
45
56
Amplitude of Postsynaptic Response (mV)
no stimulus;
spontaneous
“miniature”
postsynaptic
potentials
50 - 1000 channels
(differs among types of synapse).
This is induced by the transmitter in a single vesicle.
(Figure 12-6, Box 12-1)
23
Electrophysiological analysis of quantal synaptic transmission
(slide 4):
Binomial statistics of vesicle release
N vesicles per terminal (3 in this example)
p probability of release per vesicle
what is the probability P of releasing n vesicles?
(n = 2 for this action potential)
N n
P(n)    p 1  p N  n
n
As N   and p  0,
binomial distribution becomes Poisson distribution
(Figure 12-6, Box 12-1)
N and p sometimes change during
memory, learning, and drug addiction
24
Electrophysiological analysis of quantal synaptic transmission (slide 5):
Summary of the classical evidence:
1. Stimulated postsynaptic potentials (psp’s) have variable amplitudes
2. Spontaneous “miniature” postsynaptic potentials occur with only
modest amplitude variability.
3. The amplitudes of the stimulated psp’s are integral multiples of the
spontaneous “miniature” psp’s
(Figure 12-6, Box 12-1)
25
A more direct electrical measurement of quantal release:
Measuring the presynaptic capacitance increase due to vesicle fusion
fused vesicle adds capacitance
inside
outside
Na+
inside
K+
Cl-
G
DC
C
E
outside
See Figure 12-8
26
Measuring the presynaptic capacitance increase due to vesicle fusion
DC ~ 1 femtofarad
Na+
= 1 fF = 10-15 F
K+
Cl-
G
DC
C
E
To measure the conductances, we set IC = CdV/dt = 0, but DG/dt  0.
To measure capacitance, we set IC = CdV/dt  0, but DG/dt = 0.
Phys1 reminders, as usual
See Figure 12-8
27
On a time scale of seconds,
Signaling at synapses occurs via 2 classes of mechanisms
Discussed today
1. Chemical signaling is the dominant form in mammalian nervous systems.
A. A chemical transmitter is secreted by the presynaptic terminal and
diffuses within the gap or “cleft”, binding with specialized receptors in the
membrane of the postsynaptic cell.
B. The bound transmitter receptor can electrically excite or inhibit the
postsynaptic cell. It sometimes also “modulates” the action of other
transmitters.
Not discussed today:
2. Electrical signaling results when current generated in one cell spreads to
an adjacent cell through low resistance channels called “gap junctions”
(see pages 178 – 185)
28
Reminder: Henry Lester’s “office” hours
Mon, 1:15-2 PM,
Fri, 1:15-2 PM
outside the Red Door
End of Lecture 5
29