Transcript No Slide Title

receptor
G protein
i q s t
effector
channel enzyme
intracellular
messenger
Ca2+ cAMP
kinase
Bi / CNS 150 Lecture 12
Friday, October 27, 2014
phosphorylated
protein
The G Protein Pathway in Neuroscience
Bruce Cohen
Kandel Chapter 11 (Alberts Chapter 15)
1
From a previous lecture
Early evidence for 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
Several neurotransmitters are agonists for both ligand-gated
channels & GPCRs in 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
3
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.
(Most are still “orphans”; their ligands are unknown)
intracellular
messenger
Ca2+ cAMP
3. Individual receptors respond to:
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)
5
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):
Three types of G proteins are involved in
neurotransmitter signal transduction
• Gi proteins are typically involved in neuronal
inhibition (hence the “i” subscript)
• Gq proteins bind to enzymes that ultimately induce
Ca2+ release into the cytoplasm
• Gs proteins bind to enzymes that catalyze the
synthesis of intracellular messengers such as cAMP
and cGMP
7
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
8
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
V=
GK + G EPSP + G Cl + G Na
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
10
phosphatidyl inositol
4,5 bisphosphate = PI(4,5)P2
An example of an intracellular, ligand-gated channel
Alberts et al., Molecular Biology of the Cell, © Garland Science
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.
Figure 11-11
12
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+
Mg
Mg2+
NH2
N
N
O
O
O
-O P O P O P O CH2 O
H
H
OOOH
OH OH
ATP
cyclic AMP (cAMP)
Gs
NH2
N
N
N
N
N
cyclase
O
O
O
P
-O
O
N
H
H
OH
cyclic AMP (cAMP)
See Figure 11-3
13
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
14
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
intracellular
messenger
Ca2+ cAMP
kinase
phosphorylated
protein
kinase
phosphorylated
protein
Nucleus
16
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
18
End of Lecture 12
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