1

Bi 1 Lecture 9

Thursday, April 13, 2006

Synapses: Biophysical Machines

Reminder: Henry Lester’s “office” hoursMon, Thurs 1-1:30 PM, outside the Red Door

2

Video on synaptic transmission

3

Narrated by

Leonard Nemoy

4

Cerebral cortexAxonsDendrites SynapsesSynaptic vesicles (volleyballs), mitochondria (watermelons)

1. Movements do not occur in 1 ms; vesicles are already “docked”.

2. Neurotransmitter diffuses across the synaptic cleft in a few s

3. There is little space between neurons

4. “Slight” errors cause mental illness? Unproven!(But single-codon mutations do cause some neuroscience diseases)

5Nestler Figure 2-2(rotated)

Parts of two neurons

synaptic cleft

direction of information flow

dendrites

Excitatoryterminal

cell body

nucleus

axon

presynaptic terminal postsynaptic

dendrite

Inhibitoryterminal presynaptic

terminal

neuronPresynaptic

neuronPostsynaptic

Greek, “tree”

Greek, “axis”

from Lecture 4

6

presynaptic neuron postsynaptic neuron

The synapse is a point of information processing

An adult human brain contains ~ 1011 neurons,

and each of these might receive 103 synapses apiece,

for a total of 1014 synapses.

Most of these synapses form during the first 2 yr of life.

Thus 1014synapses/108 s = 106 synapses/s form in a fetus and infant!

Nestler Box 2-3 Figure A

Greek, “connection, junction”

from Lecture 4

7

cytosol

receptor

cytosolsynaptic cleft

receptor

receptor

Chemistry is a language of the nervous system, for instance at synapses

presynaptic terminal

postsynaptic dendrite

direction of information flow

from Lecture 4

transmitter moleculesin synaptic vesicles

8

9

Electron microscope image (“micrograph”) of a portion of a synapse

synaptic vesicles

presynaptic terminal

postsynaptic dendrite

It takes 3 different pump / transporter proteins to position the transmitter within the vesicles

10

Na+-coupled cell membrane neurotransmitter transporters:

Antidepressants (“SSRIs” = serotonin-selectivereuptake inhibitors):Prozac, Zoloft, Paxil, Celexa, Luvox

Drugs of abuse: MDMA

Attention-deficit disorder medications:

Ritalin, Dexedrine, Adderall,Strattera (?)

Drugs of abuse: cocaine amphetamine

Na+-coupledcell membrane serotonintransporter

Na+-coupledcell membrane dopamine transporter

NH

HO NH3+

HO

HO

H2C

CH2

NH3+

cytosol

outside

major targets for drugs of therapy and abuse

Presynapticterminals

From Lecture 5

Trademarks:

11

from Lecture 5:

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 m) do indeed store energy as H+ gradients).

12

NeurotransmitterandATP

(1,000 to 10,000 molecules of each)

3. Proton-coupled neurotransmitter transporter

cytosol

2. ATP-driven proton pump

H+

cytosol

cytosol

H +

transmitter

~ isotonic!

How synaptic vesicles fill from the cytosolvesicle interior

vesicle interior

13

Neurotransmitter

and

ATP

kinesin

cell body presynapticterminal

~ 20 distinct proteins

vesicle transport;pumping protons;pumping neurotransmitter; docking;fusion;recycling.

cytosol

50 nm

Lecture 10

Synaptic vesicles have many proteins

14

NeurotransmitterandATP

(1,000 to 10,000 molecules of each)

proton-coupled vesicularneurotransmitter transporter

cytosol

ATP-driven proton pump

pH effects also account for some drug actions on synaptic vesicles

HN

CH3O

H2CO CH3

3,4-methylenedioxymethamphetamine(MDMA, “ecstasy”, XTC) pKa ~ 8.5

15

proton-coupledvesicular serotonin transporter cytosol

ATP-driven proton pump

synaptic cleftNa+-coupledcell membrane

serotonin transporterMDMA

serotonin vesicle

MDMA MDMA-H+

H+

MDMA (“ecstasy”) dissipates the vesicle’s H+ store, preventing the vesicle from pumping serotonin

serotonin

depletedserotonin vesicle

MDMA-H+

Weak acids and weak bases short-circuit many vesicles!

16

These proteins have evolved in a natural—perhaps necessary--way to provide that

• The resting potential arises via selective permeability to K+

This selective permeability also leads to the Nernst potential. Transient breakdowns in membrane potential are used as nerve signals.

• Neuronal and non-neuronal cells also signal via transient influxes of Na+ and Ca2+.

3 classes of proteins that transport ions across membranes:

Little Alberts 12-4© Garland

Ion channels that flux many ions per event

Ion-coupled transporters

“Active” pumps that split ATP

from Lecture 5

17

How does the electric field across a biological membrane compare with other electric fields in the modern world?

1. A “high-voltage” transmission line1 megavolt = 106 V.The ceramic insulators have a length of ~ 1 m.The field is ~ 106 V/m.

2. A biological membraneThe “resting potential” ~ the Nernst potential for K+, 60 mV.The membrane thickness is ~ 3 nm = 30 A.The field is (6 x 10-2 V) / (3 x 10-9 m) = 2 x 107 V/m!

Dielectric breakdown voltages (V/m)

Ceramic 8 x 107

Silicone Rubber 3 x 107

Polyvinyl chloride 7 x 106

from Lecture 6: Introduction to Voltage-Gated Channels

18

K+ ions lose their waters of hydration and

are co-ordinated by backbone carbonyl groups

when they travel through a channel.

H2O K+ ion

carbonyl

From Lecture 5

19

H2O K+ ion

carbonyl

From Lecture 5

As of 13 April 2006, there are no crystal structures of voltage-gated Na+ and Ca2+ channels.

But the similarities in sequence allow us to assume that the secondary and tertiary structures resemble those of K+ channels.

A voltage-gated Na+ channel can be changed to a voltage-gated Ca2+ channel by mutating . . .just 2 out of 1800 amino acids

20

docked vesicle

nerve impulse

voltage-gated Ca2+ channel

neurotransmitter

Electricity, then chemistry triggers synaptic vesicle fusion

21

voltage-gated Ca2+ channel

Electricity, then chemistry triggers synaptic vesicle fusion

Ca2+

docked vesicle

neurotransmitternerve impulse

22

fused vesicle

Ca2+

neurotransmitter

Electricity, then chemistry triggers synaptic vesicle fusion

23

A conotoxin:25 amino acidsheld together by disulfide bondsindividual conotoxins specifically block individual ion channels

This conotoxin blocks Ca2+ channels.Slightly modified, it is now the drug, ziconotide.It suppresses transmission at pain synapses in the spinal cord.

(Swiss-prot viewer must be installed on your computer)

http://www.its.caltech.edu/~lester/Bi-1/conotoxin-annotated.pdb

from Lecture 8:

24

V

measured postsynaptic response

1 ms 5

mV

-60

+60

large“synaptic potential” leads to postsynaptic action potential

subthreshold synaptic events(revealed in low Ca2+)

stimulus to presynaptic neuron, producing action potential

Electrophysiological analysis of

quantal synaptic transmission(slide 1)

Nestler Box 2-3 Figure A

25

V

we stimulate the presynaptic neuron, producing an action potential

we measure thepostsynaptic responses,“postsynaptic potentials”

repeated identical stimuli to the presynaptic neuron . . .

. . . yield variable postsynaptic responses!

5 mV

5 ms

Electrophysiological analysis of

quantal synaptic transmission(slide 2)

26

no stimulus; spontaneous “miniature” postsynaptic potentials

repeated stimuli to presynaptic neuron

5 mV

50 - 1000 channels (differs among types of synapse).

This is the contents of a single vesicle.

Electrophysiological analysis of quantal synaptic transmission

(slide 3)

Analysis of Quantal Synaptic Transmission

00.10.20.30.40.50.60.70.80.9

1

1 2 3 4 5 6Amplitude of Postsynaptic Response (mV)

Fra

ctio

n o

f O

bse

rvat

ion

s

Stimulated

Spontaneous

0 1 2 3 4 5

27

nNpnpn

NnP

1)(

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 and p sometimes change during memory, learning, and drug addiction

Electrophysiological analysis of quantal synaptic transmission(slide 4):

Binomial statistics of vesicle release

28

1. Stimulated postsynaptic potentials have variable amplitudes

2. Spontaneous “miniature” postsynaptic potentials occur

• The amplitudes of the stimulated psp’s are integral multiples

of the spontaneous “miniature” psp’s

Electrophysiological analysis of quantal synaptic transmission (slide 5):

Summary of the classical evidence:

29

fused vesicle adds capacitance

C

E

G

Na+ K+ Cl-inside

outside

C

inside

outside

A more direct electrical measurement of quantal release:Measuring the presynaptic capacitance increase due to vesicle fusion

30

To measure the conductances, we set IC = CdV/dt = 0, but G/dt 0.

To measure capacitance, we set IC = CdV/dt 0, but G/dt = 0.

C

E

G

Na+ K+ Cl-

C

Measuring the presynaptic capacitance increase due to vesicle fusion

C ~ 1 femtofarad

= 1 fF = 10-15 F

like Lecture 6

http://www.srl.caltech.edu/sal/ph1bc/phys1c.html

http://www.theory.caltech.edu/people/politzer/web2.html

Phys1c reminders. Anal:

Practical:

31

The light chain of botulinum toxin is an enzyme that cleaves synaptic vesicle fusion proteins

32

Popular press:http://search.news.yahoo.com/search/news/?p=botox

What’s the latest new use for Botox ?

Scientific journals:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=Details&DB=PubMed

((((("botulinum toxins"[MeSH Terms] OR botulinum toxin[Text Word]) AND hasabstract[text]) AND Clinical Trial[ptyp]) AND English[Lang]) AND (("human"[MeSH Terms] OR "hominidae"[MeSH Terms]) OR "Human"[MeSH Terms]))

33

Membrane trafficking, fusion and sorting: crucial to many aspects of cell function.

© Garland publishing

34

Membrane trafficking, fusion and sorting: crucial to many aspects of cell function.

© Garland publishing

35

Date: Thursday, 13 April 2006 11:38:53 -700To: Henry Lester lester@caltech.eduFrom “Mary B. Kennedy” kennedym@its.caltech.edu

Subject: I wish to interrupt Bi 1

Hi Henry,

I wish to tell the Bi 1 students about the biochemistry of postsynaptic signalling and regulation.

Mary

Mary B. Kennedy, 4/14/05, I wish to interrupt Bi 1

36

Email from Professor Mary Kennedy

http://www.search.caltech.edu/CIT_People/action.lasso?-database=CIT_People&-response=Detail_Person.html&-layout=all_fields&person_id=11610&-search

37

How do synapses Learn?

The answer is mostly Biochemistry

38

The NMDA receptor conducts only when 1. The membrane potential is more positive than -30 mV2. Glutamate is present

outside

inside

Action potential plus glutamate functioning channel

Na+, Ca2+

A molecular coincidence detector leading to Na+ and Ca2+ influx,with many intracellular effects (lectures 12, 14)

-30 mV

(intracellular concentrations of glutamate and Mg2+ are nearly irrelevant)

39

Calcium ion is a biochemical trigger

(Often called a “Second Messenger”)

40

41

42

43

Ca2+-dependent protein kinase (12 subunits)

44

Protein kinases are enzymes that catalyze transfer of phosphate to hydroxyl groups on proteins.

The phosphate moiety is transferred from ATP to serine, threonine, or tyrosine side chains

45Phosphorylation of synaptic proteins can change the behavior of synapses

46

End of Lecture 9

Reminder: Henry Lester’s “office” hoursMon, Thurs 1-1:30 PM, outside the Red Door

47

Electrical studies on synapses

Nestler Box 2-3 Figure A

presynaptic neuron postynaptic neuron

48

no stimulus; spontaneous events

repeated stimuli to presynaptic neuron

5 mV

50 - 1000 channels (differs among synapses).This responds to the contents of a single vesicle.

Analysis of Quantal Synaptic Transmission

0

0.1

0.2

0.3

0.4

1 2 3 4 5 6

Amplitude of Postsynaptic Response (mV)

Fra

ctio

n o

f O

bse

rvat

ion

s

Stimulated

Spontaneous

1.0

0 1 2 3 4 5

Electrophysiological analysis of

synaptic transmission(slide 3)