Transcript G - Caltech

receptor
G protein
i q s t
effector
channel enzyme
intracellular
messenger
Ca2+ cAMP
kinase
Bi / CNS 150 Lecture 12
Friday, October 26, 2015
phosphorylated
protein
The G Protein Pathway in Neuroscience:
A Whirlwind Journey From Neurotransmitter to Gene Activation
Henry Lester
Chapter 11 (Alberts Chapter 15)
1
From previous lectures
Proof of chemical synaptic transmission, 1921.
Many details of the G protein pathway were first worked out for
neuronal control of the heart
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
Some postsynaptic membranes contain G protein-coupled receptors
(“metabotropic” receptors) rather than ligand-gated channels
cytosol
vesicles containing
serotonin
NH 3+
HO
N
H
G protein-coupled
serotonin receptor
vesicles containing
acetylcholine
O
O
N+(CH 3)3
G protein-coupled
(muscarinic)
acetylcholine receptor
vesicles containing
dopamine
HO
HO
H2
C
C
H2
NH 3+
synaptic
cleft
G protein-coupled
dopamine receptor
cytosol
3
Several small-molecule transmitters
serve as agonists for both ligand-gated channels & GPCRs
(among vertebrates)
Transmitter
Ligand-gated channel
GPCR
ACh
nicotinic AChR
muscarinic AChR
GABA
GABAA
GABAB
glutamate
iGluR
mGluR
serotonin
5-HT3
5-HTn, n = 1,2, 4-7
histamine
(invertebrates only)
Hn
dopamine
(invertebrates only)
Dn
4
On a time scale of seconds (perhaps minutes),
the language of the nervous system is still electricity;
and we are still describing a set of mechanisms that manipulate impulse
frequencies in individual neurons.
5
Plasma Membrane Components of the G Protein Pathway
How fast?
100 ms to 10 s
How far?
Probably less 1 mm
Neurotransmitter or hormone
binds to receptor
activates
G protein
Effector:
enzyme or channel
outside
a
Rasmussen et al.,
Nature 2011
PDB file 3SN6
GTP
b g
a
GDP + Pi
b g
inside
G protein-coupled receptors
receptor
G protein
i q s t
1. All have 7 a-helices
effector
channel enzyme
2. There are about 1000 G protein-coupled receptors in the genome. intracellular
messenger
(Most are still “orphans”; their ligands are unknown)
Ca2+ cAMP
3. Individual receptors respond to:
(a) a low-molecular weight neurotransmitter
such as serotonin, dopamine, or acetylcholine
(b) a short protein (8-40 amino acids, a “peptide”) such as an endorphin
(c) a relatively insoluble lipid such as anandamide, the endocannabinoid
(d) an olfactory stimulus;
or
(e) light, in the eye (receptor = rhodopsin)
7
Structure of a heterotrimeric G protein:
a molecular switch
receptor
β subunit
G protein
i q s t
effector
α subunit
intracellular
messenger
GDP
γ subunit
PDF file: 1GOT
Note the “propeller” in the b subunit which caps the a subunit, preventing either
subunit from interacting with the effector (There is no effector in this structure):
receptor
Structure of a heterotrimeric G protein:
a molecular switch
G protein
i q s t
http://www.its.caltech.edu/~lester/Bi-150/G protein-alpha-beta-gamma.pdb
Viewer required
effector
intracellular
messenger
Note the “propeller” in the b subunit
which caps the a subunit, preventing
either subunit from interacting with the
effector (There is no effector in this
structure):
http://www.its.caltech.edu/~lester/Bi150/G protein-beta-only.pdb
9
From previous lectures
How ”tight” is the gigaohm seal?
acetylcholine in the pipette
opens channels in the pipette
2. Chemically tight
acetylcholine
outside the
pipette opens
channels
outside the
pipette
The seal compartmentalizes
molecules.
Molecules outside the pipette
do not mix with molecules
inside the pipette
10
receptor
Gi protein effectors include
some K+ channels
G protein
i q s t
effector
channel enzyme
intracellular
messenger
Ca2+ cAMP
no transmitter
+Gbg
Normally:
released from Gi;
Here: added by
experimenter
no additions
n= 0 (closed)
b g
b g
b g
3b. Mechanically tight
Use weak suction.
Excised “inside-out” patch
allows access to the inside
surface of the membrane
no channel openings
n = 1 (open)
+Gbg
n = 0 (closed)
11
receptor
G protein-gated K channels inhibit
neuronal (& cardiac) firing
Resting
GK
EK
-90 mV
effector
channel enzyme
Voltage-gated
Ligand-gated
G protein-gated
outside
GEPSP
EEPSP
~ -5 mV
G protein
i q s t
GCl
ECl
-80 mV
GNa
GK
GK
ENa
+50 mV
EK
-90 mV
EK
-90 mV
intracellular
messenger
Ca2+ cAMP
Capacitance
cytosol = inside
+60
additional K+ channels keep
the membrane potential away
from threshold, and therefore
decrease firing rates
mV
-60
1
ms
5
G protein gated K+ channels
(GIRKs) are inward rectifiers.
When activated, they “latch” the cell
quiet until excitatory stimuli finally
succeed in depolarizing to threshold.
E K GK + E EPSP GEPSP + E Cl GCl + E NaGNa
DV =
GK + G EPSP + G Cl + G Na
Gi-coupled receptors usually inhibit neurons
Gi directly activates some K channels
Gi directly inhibits some voltage-gated Ca channels
Gi directly inhibits adenylyl cyclase
All these actions slow neuronal firing and decrease transmitter release
13
receptor
Gq, Gs, and Gt protein effectors
include some enzymes
Gq
G protein
i q s t
effector
channel enzyme
Enzyme
Ca2+ in
endoplasmic
reticulum
Ca2+
in cytosol
intracellular
messenger
Ca2+ cAMP
14
phosphatidyl inositol
4,5 bisphosphate = PI(4,5)P2
Our first example of intracellular ligand-gated channels
Alberts et al., Molecular Biology of the Cell, © Garland Science
Figure on p.242
16
KCNQ channels
PIP2 is necessary for keeping some K channels open.
Gq activation leads to less PIP2
Result: some K channels close.
These are called “M” channels, and are now termed the KCNQ family.
because they were first discovered downstream from muscarinic receptors . . .
A different muscarinic receptor subtype from the one that opens K channels in
heart.
Part of Figure 11-11
17
receptor
G protein
i q s t
Gq, Gs, and Gt protein effectors
include some enzymes:
effector
channel enzyme
Gs-coupled receptors often stimulate
neurons & other cells
intracellular
messenger
Ca2+ cAMP
ATP
2+
Mg2+
Mg
NH 2
N
N
O
O
O
-O P O P O P O CH 2 O
H
H
OOOH
OH OH
ATP
cyclic AMP (cAMP)
Gs
NH 2
N
N
N
N
N
cyclase
O
O
O
P
-O
O
N
H
H
OH
cyclic AMP (cAMP)
See Figure 11-3
18
receptor
G protein
i q s t
caffeine prolongs the intracellular messenger cAMP
effector
channel enzyme
intracellular
messenger
Ca2+ cAMP
ATP
cyclic AMP (cAMP)
NH 2
N
N
Mg2+
N
O
O
O
-O P O P O P O CH 2 O
H
H
OOOH
OH OH
NH 2
N
N
N
N
cyclase
O
O
O
P
-O
O
N
H
H
OH
Inhibited
by caffeine
phosphodiesterase
AMP
19
receptor
G protein
i q s t
cyclase
cAMP
ATP
effector
channel enzyme
Inhibited
by caffeine
phosphodiesterase
AMP
intracellular
messenger
cAMP
Ca2+ cGMP
Phosphodiesterase inhibitors prolong the life of intracellular messengers
cyclase
cGMP
GTP
Inhibited by . . .
phosphodiesterase
GMP
20
intracellular
messenger
Ca2+ cAMP
Intracellular messengers bind to proteins
kinases
A few ion channels
(olfactory system, retina)
phosphorylated
protein
NH 2
N
N
Ca2+
and
O
O
O
P
-O
O
N
N
H
H
OH
cyclic AMP (cAMP)
21
Ca2+ or cAMP binds to
kinase;
this activates the kinase.
intracellular
messenger
Ca2+ cAMP
kinase
phosphorylated
protein
Alberts 11-31
© Garland
serine
O
N CHC O
H
CH2
OH
Residue in
target protein
kinase
phosphatase
O
N CHC O
H
CH2
O
-O P O
O
22
Example of ion channel phosphorylation:
β-adrenergic receptors regulate
accommodation in hippocampal neurons
intracellular
messenger
Ca2+ cAMP
kinase
Apply norepinephrine
phosphorylated
protein
Norepinephrine inhibits the SK (smallconductance, Ca2+ -activated K+) channel.
Apply 8-bromo-cAMP
Therefore the after-hyperpolarization (AHP)
is smaller and spike trains are longer.
Apply forskolin (then apply glutamate in the presence of TTX)
epsp
The norepinephrine effect is also
mimicked by agents that mimic
or increase cAMP.
1. phosphodiesterase.does not
hydrolyze 8-bromo-cAMP
2. Forskolin activates cyclase
Example of ion channel phosphorylation:
β-adrenergic receptors regulate
accommodation in hippocampal neurons
intracellular
messenger
Ca2+ cAMP
kinase
Apply norepinephrine
phosphorylated
protein
Norepinephrine inhibits the SK (smallconductance, Ca2+ -activated K+) channel.
Apply 8-bromo-cAMP
Apply forskolin (then apply glutamate in the presence of TTX)
epsp
Therefore the after-hyperpolarization (AHP)
is smaller and spike trains are longer.
The norepinephrine effect is also
mimicked by agents that mimic
or increase cAMP.
1. phosphodiesterase.does not
hydrolyze 8-bromo-cAMP
2. Forskolin activates cyclase
receptor
Discussion
G protein
i q s t
Selective advantage of such a complex pathway?
The neurotransmitter or hormone does not
directly influence the response--from the viewpoint of
(a) Chemistry
(b) Speed
(c) Localization (to some extent)
effector
channel enzyme
intracellular
messenger
Ca2+ cAMP
All this amplification and indirect coupling requires energy!
Limitations of the pathway:
(a) Speed
(b) co-operativity
Further advantages / limitations? Suggestions in class:
25
receptor
Genomic diversity of the G protein pathway
~ 1000 G protein-coupled receptors
All have 7 helices
G proteins all have 3 subunits
There are ~ 18 a subunit genes
in 4 major classes i, q, s, t
~ 5 b subunits
~ 3 g subunits
G protein
i q s t
effector
channel enzyme
intracellular
messenger
Ca2+ cAMP
and many
“accessory
proteins”.
Now
we discuss
one
There are 2 major types of effectors
Channels affected by G proteins:
~5 known K channel genes
~4 Ca2+ channels
Enzymes
3 major classes, each with 2 to 10 members
26
Regulators of G protein Signaling
tune the kinetics of effector (GIRK channel) activation/deactivation
CHO
CHO
Expressed: muscarinic ACh
Receptor +
GIRK . . .
. . .+ RGS
RGS4
GTP
b g
a
a
b g
RGS
GTP
Gαi
GDP + Pi
25
27
On a time scale of seconds (perhaps minutes),
the language of the nervous system is still electricity;
and we are still describing a set of mechanisms that manipulate impulse
frequencies in individual neurons.
Now we proceed to effects on a longer time scale
(hours to days).
Classical “Outside-in” Mechanisms
for
Long-term Actions on G Protein Pathways
28
Seymour Benzer’s early
Drosophila learning
mutants
“Normal Drosophila learn to
avoid an odorant associated
with electric shock. A . . .
mutant, dunce, has been
isolated that fails to display
this learning in spite of being
able to sense the odorant and
electric shock and showing
essentially normal behavior in
other respects.”
Quinn, et al., PNAS 1974;
Dudai et al., PNAS 1976
fluorescent lamp
Two of Seymour Benzer’s early Drosophila learning mutants
involve the cAMP system
cyclase
rutabaga
cAMP
ATP
phosphodiesterase
dunce
AMP
30
intracellular
messenger
Ca2+ cAMP
kinase
phosphorylated
protein
kinase
phosphorylated
protein
Nucleus
31
Many genes have a DNA sequence called
“cAMP-Ca2+ responsive element” (CRE)
intracellular
messenger
Ca2+ cAMP
kinase
Target or reporter gene
CRE
pCREB
phosphorylated
protein
The transcription factor that binds to this CRE:
“cAMP-Ca2+ responsive element binder” (CREB).
O
-O P O
O
Alberts et al.,
Molecular Biology of the Cell,
© Garland Science
from Lecture 12
outside
receptor
membrane
b g
G protein
i q s t
a
b g
a
inside
effector
channel enzyme
The pathway from GPCR
to gene activation
intracellular
messenger
Ca2+ cAMP
cytosol
kinase
phosphorylated
protein
nucleus
How fast?
10 s to days
How far?
Up to 1 m
33
A typical schematic
drawing
See also Figure 11-15
34
Henry Lester’s “office” hours continue all term
Monday & Friday 1:15-2 PM
Outside the Red Door
End of Lecture 12
35
receptor
G protein
i q s t
cyclase
effector
channel enzyme
cAMP
ATP
Inhibited
by caffeine
phosphodiesterase
AMP
intracellular
messenger
cAMP
Ca2+ cGMP
Phosphodiesterase inhibitors prolong the life of intracellular messengers
cyclase
cGMP
GTP
Inhibited
by Viagra, Cialis, Levitra
phosphodiesterase
GMP
36