1 Bi / CNS 150 Lecture 12 Friday, October 25, 2013 The G Protein Pathway in Neuroscience: A Whirlwind Journey From Neurotransmitter to Gene Activation

1

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+



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


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


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

http://www.its.caltech.edu/~lester/Bi-150/G%20protein-alpha-beta-gamma.pdb

http://www.its.caltech.edu/~lester/Bi-1/G%20protein-beta-only.pdb

http://www.its.caltech.edu/~lester/Bi-1/G%20protein-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+



+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+



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+



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+



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+



20

cAMP ATP

cyclase

phosphodiesterase

AMP



Ca2+ cAMP

cGMP GTP

cyclase

phosphodiesterase

GMP

cGMP

Phosphodiesterase inhibitors prolong the life of intracellular messengers

Inhibited by . . .

receptor

tsqiG protein


21

Intracellular messengers bind to proteins

kinases


A few ion channels(olfactory system, retina)

N

NN

N

NH2

O

OHO

HH

O

P-O

O

cyclic AMP (cAMP)

Ca2+ and

cAMPCa2+


22

kinase


cAMPCa2+


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


cAMPCa2+


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


cAMPCa2+


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+



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+



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


kinase


cAMPCa2+


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


cAMPCa2+


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


cAMPCa2+


receptor

tsqiG protein


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

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cAMP ATP

cyclase

phosphodiesterase

AMP



Ca2+ cAMP

cGMP GTP

cyclase

phosphodiesterase

GMP

cGMP

Phosphodiesterase inhibitors prolong the life of intracellular messengers

Inhibited by Viagra, Cialis, Levitra

receptor

tsqiG protein


Documents

1 Bi / CNS 150 Lecture 12 Friday, October 25, 2013 The G Protein Pathway in Neuroscience: A Whirlwind Journey From Neurotransmitter to Gene Activation