Neurotransmitter receptors and Second messenger systems
Purves et al., Chapters 6 & 7
Fig. 6.3 A, B, C, D p. 113
Two molecules of acetylcholine bind.
Nicotinic Acetylcholine receptor - so named because of agonist from Nicotinia
found in vertebrate in all (sympathetic and parasympathetic) autonomic ganglia
(the first synapse, not the neuro-effector junction), muscle and other places
Torpedo - electric ray, up to 75 V (not that much) but 20 Amps.
Lots of generator potentials added up (vs. Electrophorus - lots of spikes,
used to isolate the Na+ channel of the action potential).
(There are also fish with electric sense, not just those that stun prey.)
Can be bound by alpha-bungarotoxin - from banded krait Bungarus multicinctus
(snake), 74 amino acids binds receptor irreversibly and thus causes paralysis
by blocking transmission, very useful in studies to label receptor - labeled
by 125I alpha-bungarotoxin.
5 subunits - 2 alpha, beta, gamma and delta
in neurons, 3 alpha, 2 beta (and no alpha bungarotoxin sensitivity)
Here is a transmission electron micrograph
of the neuromuscular junction. Note Schwann cell, nerve terminal and muscle
cell. The subsynaptic muscle cell membrane has invaginations and folds;
the acetylcholine receptor, on the crests, is labeled with alpha bungarotoxin
and horseradish peroxidase.
Fig. 6.7A p. 120
some channel proteins, AMPA receptor shown here, span 3 times
Protein (nicotinic receptor subunit) spans the membrane 4 times.
M2 likely lines the pore.
Nicotine is an agonist; but it seems somewhat like an antagonist because
it blocks transmission at autonomic ganglia by depolarization blockade.
There are pharmacological antagonists (curare, a plant alkaloid from Clondodendron
Important for mechanisms of muscular relaxatants used in surgery (like succinylcholine).
Must relax muscles in surgery but must prove that anesthesia is adequate.
Beverly A Orser, Lifting the fog around anesthesia, Scientific American,
June 2007, 54-61
Obviously, analgesia (absence of pain) is paramount
Interestingly, amnesia (loss of memory)
Dentist William Morton used ether in Boston in 1846
These days, drugs that keep GABA-A channels open (benzodiazepines like Valium
also affect these)
Some additional points about nicotinic receptors:
Developmentally, when nerve-muscle junction is made, diffuse receptors cluster.
Acetylcholinesterase, by contrast, is all over the place.
Receptor molecules are very concentrated at n.m.j. crest, 20,000 - 30,000
per square micron (about as tightly packed as possible in contrast with
punctate voltage gated sodium channels).
Probably water filled pore.
All 4 subunits needed in expression systems to get functioning receptor.
10 to the 6th ACh molecules from one a.p. into n.m.j. cleft.
2.5 x 10 to the 5th channels transiently open.
400 nA n.m.j. end plate current.
1 ms open time.
10,000 Na+'s flow through each channel in this time.
Channel conductances of 25 pS
Box B (Chapter 6) p. 117
Smaller miniature end plate potential.
Great weakness (seen in droopy eyes); here's a picture
I found on the web of the eyelid droop.
Receptors low in mysthenia gravis - autoimmunity to nicotinic receptors.
(Nervous system proteins are generally separated from immune surveillance
by blood brain barrier.)
First noted by Thomas Willis (1685, of circle of Willis fame.)
Treatment by cholinesterase inhibitors - note that action potentials are
increased by neostigmine treatment.
Involvement of thymus.
Fig. 6.3 F p. 113
Glutamate (AMPA, NMDA, Kainate), GABA, glycine, setotonin, purine receptors
can be ion channels.
Keeping in mind that we already discussed ACh (nicotinic) receptors (above),
the notable ligands missing from the channel receptor list are epinephrine
There is a staggering diversity of different types.
Glutamate channel agonists:
NMDA = N-methyl D-aspartate.
blocked by AP5 (2-amino-5-phosphonovalerate)
central excitatory - like inputs to hippocampus
On the basis of the reversal potential, it is inferred that the channel
is nonselective cation channel.
Fig. (like Fig. 6.6 C p. 119 only older and simpler)
Na+ & Ca2+
Calcium influx - excitotoxicity in injury or stroke.
Voltage, glutamate, calcium cause "vicious cycle of glutamate release."
The general involvement of Ca2+, and its role as a signal transduction "second
messenger" means that a lot of important neural processes, such as
"learning," are attributed to NMDA receptors
AMPA = alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate, also kainate
Kainate (from red alga Digenea simplex) and Quisqualate (from seed of Quisqualis
indica) are excitotoxic amino acids
reversal potential is at 0 mV so it is likely opening for K+ & Na+ channels
Fig. 6.9 A p. 123
GABA-A channel is for Cl-.
Different combinations of 5 different subunits makes for a lot of diversity.
Diazepam (Valium) and chlordiazepoxide (Librium) [tranquilizers] bind to
alpha and delta subunits - enhance GABAergic transmission.
Barbiturates [hypnotics] like phenobarbital bind to gamma subunit.
GABA-A receptors blocked by bicuculline from Dutchman's breeches and picrotoxin
from Anamerta cocculus.
Glycine receptor blocked by strychnine, alkaloid from seeds of Strychnos
nux-vomica - causes seizures.
5HT3 is also an ion channel - maybe the molecule only spans the membrane
Ionotropic vs metabotropic receptors
Earl W. Sutherland, Jr. (US) "mechanisms of actions of hormones,"
father of signal transduction. His major contribution dealt with cAMP as
a second messenger in mediating adrenergic effects on metabolism in the
liver (which mobilizes glucose from glycogen).
When I first took physiology (1969), there was an emphasis on the autonomic
n.s., hence on acetylcholine and norepinephrine.
Acetylcholine nicotinic (ionotropic) at ganglia, muscarinic (now "metabotropic")
at parasympathetic neuro-effector junction (post-ganglionic)
Adrenergic only in sympathetic neuro-effector.
Adrenergic receptors: alpha usually excitatory, e.g. arteriole constriction,
agonist nose decongestant spray like Neosynephrine (phenylephrine).
Beta usually inhibitory but it is excitatory at heart, and beta blocker
propranolol used for hypertension.
About 10 years later, people I knew were involved in showing there were
several alphas & betas.
With hindsight, it is interesting that adrenergic is metabotropic, not ionotropic
(not in Fig. 6.3 F p. 113)
Landmark paper RADixon et al. (RJLefkowitz) Cloning of the gene and
cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin,
Nature 321, 75, 1986, see J NIH Res, 9, 45-52, 1997.
Fig 6.4 AB p 116
7 transmembrane domains called G protein coupled receptors.
By hydrophobicity, they all cross membrane in 7 alpha-helices.
Rhodopsin was the prototype, followed closely by the beta adrenergic receptor.
Then many neurotransmitter and hormone receptors were found.
In the early 1990's, olfactory receptors were found to be G protein coupled
receptors, and there are lots of olfactory receptors; Richard Axel and Linda
B. Buch won the 2004 Nobel
prize for this work.
In summary, there is an enormous diversity! Superfamily (>1000 in mammals).
N terminus outside cell, glycosylation
C-inside (heptahelical) -phosphorylation,
2nd and 3rd loops and C terminus for interaction with a subunit of G protein
Fig 6.4 C p. 116
Here's the example of the huge list for transmitters.
Many types mGluR1-8, NE alpha (6 types), beta 1 , 2, & 3, D1-D5, GABA-B(1&2),
5-HT-12 types, Purines 12 types
And muscarinic (1-5)
Muscarinic receptors (postganglionic parasympathetic) - muscarine - from
poisonous red mushroom (Amanita muscaria) stimulates, atropine (from deadly
nightshade) blocks (belladonna = beautiful lady). SLIDE (Hess, Scientific
American, Nov. 1975, p.111) Women are more beautiful with dilated pupils
muscarinic receptors are at parasympathetic neuro-effector junctions (incl.
Muscarinic "7TD" (G protein coupled receptor, more later).
Because Acetylcholine from Vagus (X cranial nerve) slows heart, poisoning
with organophosphate (acetylcholinesterase inhibitor such as insecticide
malation or nerve gas) would stop heart; atropine, by blocking receptor,
would save your life.
Fig. 7.4 ABCD p. 144
Channel, enzyme (many for development), G protein coupled receptor, intracellular
(like for steroid).
Receptor like the beta adrenergic receptor binds G protein (alpha, beta
and gamma subunits).
Signal transduction cascades
Fig. 7.5A p. 145
G protein so named because it (alpha subunit) binds GTP
AGGilman and M Rodbell, Nobel
heterotrimer alpha, beta and gamma
alpha and beta are about the same size, gamma is smaller
alpha and gamma linked to membrane by fatty acid
alpha subunit affects effector and is GTPase
Fig. 7.6 p. 146
cascades with different second messenger (signalling) systems (effectors)
For beta adrenergic receptor, Gs (stimulatory) activates adenylyl cyclase,
to make cAMP, which, in turn, activates protein kinase (PKA) for phosphorylation
For glutamate, Gq (q is designation here) activates Phospholipase C (PLC)
Recent paper SKEKoekkoek et al., Cerebellar LTD and learning-dependent
timing of conditioned eyelid responses, Science 301, 1736-1739, 2003, see
also D. J. Linden, From molecules to memory in the cerebellum (Perspectives),
Science 301, 1682-1685, 2003.
LTD=Long term depression, a model for learning.
Conditioned eyelid response is a paradigm for learning.
Transgenic with a Purkinje-specific promotor was used to express a molecule
to inhibit PKC.
These mice lack LTD for this learning.
For dopamine, a Gi (inhibitory) inhibits the cAMP cascade
The phosphoinositide cascade
Fig. 7.7D p. 148
My real name is phosphatidylinositol-4,5-bisphosphate but my friends call
me PIP2 (apologies to Charles Dickens) (special membrane lipid) is cleaved
to DAG (diacyl glycerol) and IP3 (inositol trisphosphate.
IP3 causes release of Ca2+ from nonmitochondrial stores, IP3 receptor.
Ca2+ is a real wide ranging intracellular messenger.
Calcium binds to calcium binding proteins like calmodulin
DAG activates PKC (protein kinase C) [kinase is an enzyme that phosphorylates].
In 1970, I took "membrane biochemistry" - lipids seemed boring,
By early to mid 1980's, lipids shown to turn over and to be signaling precursors.
NorpA (no receptor potential) Drosophila have rhodopsin but lack phospholipase
I did not isolate the mutant or make this discovery, but I did work on norpA.
Eventually, I did some research
on lipids and fatty acids in Drosophila with reference to phototransduction.
Fig. 7.8 p. 151
How phosphorylation (by kinase) could affect protein - (activate it by binding
phosphate onto it).
Need a phosphatase to take phosphate off.
Fig. 7.7C p. 148
ATP -> adenylyl cyclase -> cAMP -> phosphodiesterase -> 5'AMP.
Caffeine and theophylline inhibit PDE (phosphodiesterase for cAMP), thus
potentiating the "upper" action of norepinephrine by making its
second messenger longer lasting.
Fig. 7.9 A p. 152
A-Kinase (PKA) - catalytic (C) and regulatory (R) (inhibitory) subunits.
2 cAMP's each bind 2R's, pull them off of 2C's
Fig. 7.11 p. 156
CREB = cAMP response element binding protein affects gene transcription
Chapter 7 (Molecular signaling within neurons) is difficult because, following
Chapter 6 (Neurotransmitter receptors and their receptors), one would hope
it was restricted to metabotropic transmitter mechanisms, but it expands
to signal transduction in general, a broad topic indeed, and the subject
of an entire course
I have taught).
Exam questions form 2005 - 2011 relating to this outline
A poisonous snake, the banded Krait (Bungarus) bites its prey. Answer
either (1) What effect does the venom have on the prey? (2) It has this
effect by binding what specific neurotransmitter receptor? Or (3) Where
are these affected receptors located?
paralyse, nicotinic, neuromuscular junction
A extensive table with all the possible components for ionotropic receptors
for lots of neurotransmitters was shown to you. Why were dopamine and norepinephrine
not on this table?
there are no channel receptors for dopamine or norepinephrine
The NMDA receptor (answer either) (1) is for what transmitter? Or (2) passes
sodium, potassium and (what other important ion?).
Answer either: The second and third cytoplasmic loops (and what terminal?)
of the beta adrenergic receptor interact with (What is the next downstream
molecule in this cascade?).
C terminal, G protein
The activated alpha subunit activates adenylyl cyclase. Answer either (1)
Why would you expect it to remain attached to the inside of the membrane?
Or (2) What has to happen before the activated alpha subunit reassociates
with the beta-gamma subunits?
it is bound to the membrane, it turns ATP into ADP
"In this situation, atropine would save your life." What situation?
And why would it save your life? (Answer both.)
poisoning with malathion, block acetylcholine receptors on tyhe heart, prevent
it from stopping
"IP3 is a ligand for a channel." Answer either (1) For what ion?
Or (2) On what membrane?
calcium, smooth endoplasmic reticulum
Whereas cAMP is known for actions such as gating a channel, it can also
regulate the transcription of DNA into mRNA. Name one of the proteins intermediate
between cAMP and RNA polymerase.
Caffeine inhibits what enzyme?
What would 125I - labeled a-bungarotoxin label?
the nicotinic receptor on muscle
What is the next in line downstream of the metabotropic glutamate receptor?
Describe, in terms of chemical structure, how cAMP interacts to activate
PKA (protein kinase A).
4 cAMPs bind 2 inhibitory subunits releasing catalytic subunits
Linda B. Buch and Richard Axel won the 2004 Nobel Prize for discovering
the nature of the olfactory receptor. What type of molecule is it?
G rotein coupled receptor
In what way is the nicotinic receptor different for muscle vs. neurons?
different subunits for pentamer
The Nobel laureate Earl Sutherland is considered the founder of signal transduction
and is associtated with identifying what "second messenger" for
What is the transmitter for the NMDA (N-methyl D-aspartate) receptor?
Why is atropine from deadly nightshade called "belladonna alkaloid."
women used pupil dilation to look beautiful
What would you give a patient with myasthenia gravis to relieve the symptoms?
Give the name (that relates to the pharmacological agonist) for the ionotropic
(channel) receptor for acetylcholine.
Inositol trisphosphate is a ligand for what kind of channel located where?
calcium on reticulum
"Atropine could save your life if your heart were stopping from Malathion
poisoning." This relates to what type specific receptor on the heart
(name should include pharmacological agonist plus neurotransmitter)?
"Transgenic mice in which a Purkinje cell-specific promotor drove expression
of a molecule that inhibits PKC lack LTD for the conditioned eye blink."
Explain the significance, including what is PKC and LTD.
This was a way to show that protein kinase C mediated long term depression,
a model of synaptic learning is a specific cell type, the Purkinje cell
A vesicle contains a pretty standard amount of acetylcholine. Then why is
the mepp (miniature end plate potential) smaller in myasthenia gravis?
there are fewer nicotinic receptors
How do you make an NMDA receptor from subunits NMDA-R1 and NMDA-R2A through
Lots of possibilitits since receptor is a multimer (4 or 5) mixed from that
The lecture and outline indicated that "the second and third loops
and C terminus are for interaction with the alpha subunit of heterotrimeric
G protein." Loops and C terminus of what type of protein, and where,
in the cell's topology, are the loops and C terminus?
G protein coupled receptor is on membrane and those places are, obviously,
on the cytoplasmic side
In the G protein signal transduction cascade, which specific molecule or
subunit has GTPase activity?
alpha subunit of heterotrimeric G protein
Where does CREB (cAMP response element binding protein) bind, and what does
it do by binding there?
promoter, affect transcription
How does caffeine have its stimulatory effects?
inhibits cAMP phosphodiesterase
In the good old days (1980's), gene cloners started with a tissue that expressed
the gene product abundantly. What was this tissue for the nicotinic receptor?
electric organ of Torpedo
Why would you use alpha-bungarotoxin labeled with radioactive iodine in
label nicotinic receptor
Curare is most famous for blocking (what specific kind of receptor) in (what
nicotinic motor end plate
For ionotropic receptors, e.g. NMDA receptors, there are "a large number
of receptor isoforms." Why?
many possible subunits in many different conformations
Librium, Valium and barbiturates bind to subumits of (what type of receptor)
for (what transmitter)?
What drug slows the termination of cAMP activity and how?
caffeine blocks cAMP phosphodiesterase
"The beta blocker Propranolol is used to treat hypertension."
What is the neurotransmitter whose receptor is blocked and where?
norepinephrine, SA node
Why would you think, even before it had been shown, that the N-terminal
part of the G protein coupled receptor was not the part that binds the G
it must be on the outside of the membrane
Atropine causes the pupil to dilate. With that hint, tell me what part of
the nervous system, using what transmitter, causes the pupil to constrict.
The alpha subunit of the heterotrimeric G protein has GTPase activity. Why
is that important?
to terminate its action
IP3 causes an increase in cytosolic Ca2+. How?
bind IP3 receptor (Ca2+ channel) in smooth ER
It takes 4 cAMP molecules to activate PKA. How (biochemically) do they do
2 each bind and remove 2 inhibitory subunits from 2 catalytic subunits
In the cAMP pathway, protein kinase A (PKA) can phosphorylate CREB (cAMP
response element binding protein). What type of molecule does CREB bind
How would alpha bungarotoxin help the snake get its prey?
What is missing and where in myasthenia gravis?
nicotinic receptors at end plate
Some anesthetics, Valium, and barbiturates affect what kind of receptor
for what neurotransmitter?
channel for GABA (GABA-A receptor)
With dopamine as the ligand and the inhibitory G protein, what happens to
the level of what famous "second messenger?"
cAMP goes down
The blind Drosophila mutant norp A has no photoreceptor potential and lacks
phospholipase. Name either one of the two products of this enzyme or the
precursor. Abbreviations will suffice
PIP2 -> IP3 + DAG
Torpedo was useful Answer one (1) To do what? (2) Why was it particularly
Clone the acetylcholine receptor, which is plentiful in T's electric organ
What would you use radioactively labeled (125I) alpha-bungarotoxin to do?
To isolate the acetylcholine receptor
Anesthetics, benzodiazepines and barbiturates affect the GABA-A receptor.
Describe or draw what this receptor looks like.
Describe or draw what a dopaminergic receptor looks like.
G protein coupled receptor crosses membrane 7 times
Related to adrenergic receptors, answer either (1) Why would an alpha agonist
unstuff a stuffed nose? Or (2) Why would a beta antagonist help alleviate
Stimulate arteriole (precapillary sphincter) smooth muscle, decrease rate
and contractility of heart
Why would atropine save your life if you were dying of acetylcholinesterase
Block muscarinic receptors that are making the heart stop when there is
too much acetylcholine
Tell me one of the things the alpha subunit of the G protein does when it
is activated by binding GTP.
Dissociate from beta-gamma, activate the next molecule in the cascade, break
When a G protein signals to PLC (phospholipase C), answer either: (1) What
is the next effect for one product of that enzyme (DAG=diacyl glycerol)?
Or (2) What is the next effect after the production of the other (IP3 =
DAG activates protein kinase C, IP3 is the ligand for a calcium channel
on an intracellular cistern (smooth ER) of calcium
For cAMP answer either: (1) How, molecularly, were 4 cAMP molecules used
to achieve their effect? Or (2) What drug would keep cAMP present for a
longer amount of time?
Two each bind to and remove 2 inhibitory subunits from two catalytic subunits,
caffeine or theophyline
How does a famous "second messenger" activate protein kinase A
4 cAMPs bind 2 inhibitory subunits and pull them off of 2 catalytic subunits
With the assistance of CREB (cAMP response element binding protein), Answer
either (1) what enzyme acts on (2) what macromolecule to medeiate transcription.
RNA polymerase acts on DNA
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