Signalling

Neurotransmitter receptors and Second messenger systems
Purves et al., Chapters 6 & 7

Channels

Nicotinic receptors

Fig. 6.3 A, B, C, D
Two molecules of acetylcholine bind.
Nicotinic Acetylcholine receptor - so named because of agonist from Nicotinia tabacum nicotine
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.

Protein subunit structure likely 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 tomentosum).
Important for mechanisms of muscular relaxatants used in surgery (like succinylcholine).
Must relax muscles in surgery but must prove that anesthesia is adequate.

Recent paper
Beverly A Orser, Lifting the fog around anesthesia, Scientific American, June 2007, 54-61
Obviously, analgesia (absence of pain) is paramount
Unconsciousness (hypnosis)
Immobility
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

Fig Box B (Chapter 6)
Myasthenia gravis
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.

Other channels

Fig. 6.4 A B C
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 and dopamine.
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. 6.7 A
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 B
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 3 times.

Ionotropic vs metabotropic receptors

1971 Nobel 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).

personal reflection:
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. 6.4 C)

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 6.5 A
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 6.5 B
Here's the example of the huge list for transmitters.
Many types mGluR1-8, NE alpha 1 & 2, beta 1 , 2, & 3, D1(AB)-D4, GABA-B(1&2), 5-HT-1- 7, Purines 1, 2 (a&b), 3, P 2(x,y,z,t,u)
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. smooth muscle)
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.

Metabotropic receptors

Fig. 7.4 ABCD
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
G protein so named because it (alpha subunit) binds GTP
AGGilman and M Rodbell, Nobel 1994
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
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 (more below)

For glutamate, Gq (q is designation here) activates Phospholipase C (PLC) or PLA2.
(more below)

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
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].

personal reflection:
In 1970, I took "membrane biochemistry" - lipids seemed boring, hold proteins.
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 C.
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
How phosphorylation (by kinase) could affect protein - (activate it by binding phosphate onto it).
Need a phosphatase to take phosphate off.

cAMP

Fig. 7.7C
cAMP mechanism
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
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
CREB = cAMP response element binding protein affects gene transcription

Summary

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 recently taught).

Exam questions form 2005 - 2007 relating to this outline

What would 125I - labeled a-bungarotoxin label?

the nicotinic receptor on muscle

What is the next in line downstream of the metabotropic glutamate receptor?

G protein

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 adrenergic transmission?

cAMP

What is the transmitter for the NMDA (N-methyl D-aspartate) receptor?

glutamate

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?

neostigmine

Give the name (that relates to the pharmacological agonist) for the ionotropic (channel) receptor for acetylcholine.

nicotinic

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)?

muscarinic acetylcholine

"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 NMDA-R2D?

Lots of possibilitits since receptor is a multimer (4 or 5) mixed from that list

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 research?

label nicotinic receptor

Curare is most famous for blocking (what specific kind of receptor) in (what specific location)?

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)?

GABA

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 protein?

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.

parasympathetic, acetylcholine

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 that?

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 to?

DNA


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