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Bi / CNS 150 Lecture 12
Friday, October 25, 2013
The G Protein Pathway in Neuroscience:
A Whirlwind Journey From Neurotransmitter to Gene Activation
Henry Lester
Chapter 11 (Alberts Chapter 15)
kinase
phosphorylatedprotein
cAMPCa2+
intracellularmessenger
receptor
tsqiG protein
enzymechannel effector
2
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 smoked drum
The diffusible substance:
acetylcholine acting on
muscarinic ACh receptors
From previous lectures
3
cytosol
vesicles containingserotonin
vesicles containing dopamine
NH
HO NH3+
HO
HO
H2C
CH2
NH3+
synapticcleft
G protein-coupled(muscarinic)
acetylcholine receptor
G protein-coupleddopamine receptor
cytosol
vesicles containing acetylcholine
N+(CH3)3O
O
G protein-coupledserotonin receptor
Some postsynaptic membranes contain G protein-coupled receptors
(“metabotropic” receptors) rather than ligand-gated channels
4
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
5
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.
Plasma Membrane Components of the G Protein Pathway
GTP GDP + Pi
Effector: enzyme or channel
outside
Neurotransmitter or hormonebinds to receptor
activatesG protein
inside
Rasmussen et al., Nature 2011PDB file 3SN6
How fast?100 ms to 10 s
How far?Probably less 1 m
7
1. All have 7 -helices
2. There are about 1000 G protein-coupled receptors in the genome.
(Most are still “orphans”; their ligands are unknown)
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)
G protein-coupled receptors receptor
tsqiG protein
cAMPCa2+
intracellularmessenger
enzymechannel effector
Note the “propeller” in the subunit which caps the subunit, preventing either
subunit from interacting with the effector (There is no effector in this structure):
receptor
tsqiG protein
effector
intracellularmessenger
Structure of a heterotrimeric G protein:a molecular switch
PDF file: 1GOT
GDP
α subunit
β subunit
γ subunit
9
Note the “propeller” in the subunit
which caps the subunit, preventing
either subunit from interacting with the
effector (There is no effector in this
structure):
Viewer required
receptor
tsqiG protein
effector
intracellularmessenger
http://www.its.caltech.edu/~lester/Bi-150/G protein-alpha-beta-gamma.pdb
Structure of a heterotrimeric G protein:a molecular switch
http://www.its.caltech.edu/~lester/Bi-150/G protein-beta-only.pdb
10
acetylcholine in the pipette opens channels in the pipette
2. Chemically tight
The seal compartmentalizes molecules.
Molecules outside the pipette do not mix with molecules inside the pipette
acetylcholine outside the pipette opens channels outside the pipette
How ”tight” is the gigaohm seal? From previous lectures
11
Gi protein effectors include some K+ channels
3b. Mechanically tightUse weak suction.Excised “inside-out” patch allows access to the inside surface of the membrane
no transmitter
no additionsn= 0 (closed)
no channel openings
receptor
tsqiG protein
cAMPCa2+
intracellularmessenger
enzymechannel effector
+GNormally:
released from Gi;Here: added byexperimenter
+G
n = 0 (closed)
n = 1 (open)
1 ms 5
-60
+60
mV
G protein-gated K channels inhibit neuronal (& cardiac) firing
Capacitance
outside
cytosol = inside
EK
-90 mV
GK
Resting
EEPSP
~ -5 mV
GEPSP GCl
ECl
-80 mV
Ligand-gated
ENa
+50 mV
GNa
EK
-90 mV
GK
Voltage-gated
EK
-90 mV
GK
G protein-gated
additional K+ channels keep the membrane potential away from threshold, and therefore
decrease firing rates
VE G E G
G GK K EPSP EPSP
K EPSP
E GCl Cl
GCl
E GNa Na
GNa
receptor
tsqiG protein
cAMPCa2+
intracellularmessenger
enzymechannel effector
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.
13
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
14
Enzyme
Ca2+
in cytosol
Gq, Gs, and Gt protein effectors include some enzymes
Ca2+ in endoplasmic
reticulum
Gq
receptor
tsqiG protein
cAMPCa2+
intracellularmessenger
enzymechannel effector
phosphatidyl inositol4,5 bisphosphate = PI(4,5)P2
Alberts et al., Molecular Biology of the Cell, © Garland Science
Our first example of intracellular ligand-gated channels
16
Figure on p.242
17
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.
KCNQ channels
Figure 11-11
18
Gs
Gq, Gs, and Gt protein effectors include some enzymes:
Gs-coupled receptors often stimulate neurons & other cells
cyclic AMP (cAMP)ATP
N
NN
N
NH2
O
OHO
HH
O
P-O
O
cyclic AMP (cAMP)
N
NN
N
NH2
O
OHOH
HHCH2
H
OP
O-
O
OP
O-
O
-O OP
O-
OMg2+
ATP
cyclase
Mg2+
receptor
tsqiG protein
cAMPCa2+
intracellularmessenger
enzymechannel effector
See Figure 11-3
19
cyclase
N
NN
N
NH2
O
OHO
HH
O
P-O
O
cyclic AMP (cAMP)
phosphodiesterase
AMP
N
NN
N
NH2
O
OHOH
HHCH2
H
OP
O-
O
OP
O-
O
-O OP
O-
OMg2+
ATP
caffeine prolongs the intracellular messenger cAMP
Inhibited by caffeine
receptor
tsqiG protein
cAMPCa2+
intracellularmessenger
enzymechannel effector
20
cAMP ATP
cyclase
phosphodiesterase
AMP
Inhibited by caffeine
intracellularmessenger
Ca2+ cAMP
cGMP GTP
cyclase
phosphodiesterase
GMP
cGMP
Phosphodiesterase inhibitors prolong the life of intracellular messengers
Inhibited by . . .
receptor
tsqiG protein
enzymechannel effector
21
Intracellular messengers bind to proteins
kinases
phosphorylatedprotein
A few ion channels(olfactory system, retina)
N
NN
N
NH2
O
OHO
HH
O
P-O
O
cyclic AMP (cAMP)
Ca2+ and
cAMPCa2+
intracellularmessenger
22
kinase
phosphorylatedprotein
cAMPCa2+
intracellularmessenger
Alberts 11-31© Garland
Ca2+ or cAMP binds to kinase;
this activates the kinase.
serine
kinase
phosphataseResidue in target protein
NH
CHC
CH2
O
O
-O OP
O
O
NH
CHC
CH2
O
O
OH
Example of ion channel phosphorylation:β-adrenergic receptors regulate
accommodation in hippocampal neurons
Apply forskolin (then apply glutamate in the presence of TTX)
kinase
phosphorylatedprotein
cAMPCa2+
intracellularmessenger
Norepinephrine inhibits the SK (small-conductance, Ca2+ -activated K+) channel.
Therefore the after-hyperpolarization (AHP) is smaller and spike trains are longer.
epspThe norepinephrine effect is also mimicked by agents that mimic or increase cAMP.1. phosphodiesterase.does not hydrolyze 8-bromo-cAMP2. Forskolin activates cyclase
Apply norepinephrine
Apply 8-bromo-cAMP
Example of ion channel phosphorylation:β-adrenergic receptors regulate
accommodation in hippocampal neurons
kinase
phosphorylatedprotein
cAMPCa2+
intracellularmessenger
Apply norepinephrine
Apply forskolin (then apply glutamate in the presence of TTX)
epsp
Apply 8-bromo-cAMP
The norepinephrine effect is also mimicked by agents that mimic or increase cAMP.1. phosphodiesterase.does not hydrolyze 8-bromo-cAMP2. Forskolin activates cyclase
Norepinephrine inhibits the SK (small-conductance, Ca2+ -activated K+) channel.
Therefore the after-hyperpolarization (AHP) is smaller and spike trains are longer.
25
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)
All this amplification and indirect coupling requires energy!Limitations of the pathway:(a)Speed(b)co-operativity
Further advantages / limitations? Suggestions in class:
Discussion
receptor
tsqiG protein
cAMPCa2+
intracellularmessenger
enzymechannel effector
26
Genomic diversity of the G protein pathway
G proteins all have 3 subunits There are ~ 18 subunit genes
in 4 major classes i, q, s, t ~ 5 subunits ~ 3 subunits
There are 2 major types of effectorsChannels affected by G proteins:
~5 known K channel genes~4 Ca2+ channels
Enzymes3 major classes, each with 2 to 10 members
and many“accessory proteins”.
Now we discuss
one
~ 1000 G protein-coupled receptorsAll have 7 helices
receptor
tsqiG protein
cAMPCa2+
intracellularmessenger
enzymechannel effector
27
RGS4
Gαi
GTP
Regulators of G protein Signaling tune the kinetics of effector (GIRK channel) activation/deactivation
Expressed: muscarinic ACh Receptor + GIRK . . .
. . .+ RGS
CHO CHO
25GTP GDP + Pi
RGS
28
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
fluorescent lamp
“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
Seymour Benzer’s early Drosophila learning
mutants
30
cAMP ATP
cyclase
phosphodiesterase
AMP
rutabaga
dunce
Two of Seymour Benzer’s early Drosophila learning mutants involve the cAMP system
31
Nucleus
kinase
phosphorylatedprotein
kinase
phosphorylatedprotein
cAMPCa2+
intracellularmessenger
Many genes have a DNA sequence called
“cAMP-Ca2+ responsive element” (CRE)
CRE
The transcription factor that binds to this CRE:
“cAMP-Ca2+ responsive element binder” (CREB).
pCREB
-O OP
O
O
kinase
phosphorylatedprotein
cAMPCa2+
intracellularmessenger
Target or reporter gene
Alberts et al., Molecular Biology of the Cell, © Garland Science
33
cytosol
The pathway from GPCR to gene activation
nucleus
How fast?10 s to days
How far?Up to 1 m
kinase
phosphorylatedprotein
cAMPCa2+
intracellularmessenger
receptor
tsqiG protein
enzymechannel effector
membrane
from Lecture 12outside
inside
outside
inside
34
A typical schematic drawing
See also Figure 11-15
35
End of Lecture 12
Henry Lester’s “office” hours continue all term Monday & Friday 1:15-2 PM
Outside the Red Door
36
cAMP ATP
cyclase
phosphodiesterase
AMP
Inhibited by caffeine
intracellularmessenger
Ca2+ cAMP
cGMP GTP
cyclase
phosphodiesterase
GMP
cGMP
Phosphodiesterase inhibitors prolong the life of intracellular messengers
Inhibited by Viagra, Cialis, Levitra
receptor
tsqiG protein
enzymechannel effector