I.

Receptors
a. Ionotropic Receptors
i. Only a handful of ionotropic receptor types
ii. Nicotinic ACh Receptors (a classic model)
1. Model: electric organ of the Torpedo electric fish
2. This organ has so many AChRs in such a high density on large
electric organs that depolarization leads up to a 100V charge
movement, and this can be used to stun prey
3. High density makes biochemistry and molecular biology easy
4. Structure:
a. 5 subunits 2 alphas, 1 beta, 1 gamma, 1 delta
b. Subunits all have a large amount of conserved amino acids
c. Each subunit has 4 predicted transmembrane regions
(M1-M4) with M2 from each subunit forming the lining of
the pore
d. The alpha subunits contain the binding sites for
neurotransmitters (2 for ACh)
e. The channel will flicker with a brief mean open time when
only one ACh is bound, but open normally when two are
bound
5. There is no regenerative process to activate more channel
opening as there was with voltage-gated Na+ channels. The
probability of opening is increased as the outside concentration of
ACh increases, because this increases the likelihood that ACh will
bind to alpha subunits
6. Binding is blocked by alpha bungarotoxin or curare these
compete at the ACh binding site
7. EqAChR = -10 mV
8. Multi-ion selectivity; nearly equally permeable to Na+ and K+,
but also slightly permeable to Ca++
iii. Glutamate Receptors
1. Unrelated structurally to AChRs
2. Divided into two classes based on sensitivity to chemical analogs
a. NMDA
b. Non-NMDA
i. AMPA
ii. Kainate
3. NMDA (N-Methyl-D-aspartate) Receptors
a. Activated by NMDA
b. High conductance (50 ps)
c. Permeable to Ca++ as well as Na+ and K+
d. Pore is plugged by Mg at resting membrane potential
i. Since plug is removed by depolarization, in
practical terms, this channel is gated by a
combination of glutamate and voltage
e. Relatively slow gating

f. Lots of modulatory sites (glycine, Zn, PCP)

g. Important for LTP and LTD
4. Non-NMDA Receptors
a. Activated by AMPA (Gama-Amino 3-hydroxy-5-methyl-4isooxozole proprionic acid) or Kainate
b. Permeable mostly to only Na+ and K+ (though some Ca++
permeable subtypes)
c. Fast gating
5. These are multiple subunit channels, but the exact mixture and
makeup of them is still not confirmed. These are much larger than
the AChRs and are not structurally related
6. At most synapses that have both NMDA and non-NMDA
receptors, only the non-NMDA channel is available from resting
membrane potential. As a result, the postsynaptic response to a
single stimuli is mediated by non-NMDA channels
7. NMDA channels only contribute when the presynaptic cell fires
repeatedly and the summating non-NMDA potentials depolarize
enough to knock Mg++ out of the NMDA channel through
electrostatic repulsion. At this point, Ca++ can enter through the
NMDA channel and contribute to second messenger activation
(Ca++ dependent enzymes, LTP, etc.) and in extreme cases of
hyperactivity, excitotoxicity, in part mediated by Ca++ dependent
proteases and free radical generation
b. GABA and Glycine Ligand-Gated Ion Channels
i. GABA is most common inhibitory neurotransmitter in the brain; inhibitory
because the receptor channel does not conduct Na+ in and K+ out
(reversal potential ~ 0 mV) but it does conduct Clii. Chloride is distributed such that it is in high concentration outside and low
concentration inside
iii. Equilibrium potential for chloride channels is about the same as for the K+
channel, -70 mV
1. Thus, when these channels open, there is an influx of Cl-, and the
membrane potential moves toward -70 mV (IPSP)
2. If the cell is already at about -70 mV, even then, the opening of this
channel causes an inhibitory action by 2 additional mechanisms:
a. Since the membrane voltage is set by the mixture of
equilibrium potentials that the membrane is permeable to,
even if it is already -70 mV, it will take more open Na+
channels to move it in a depolarized direction significantly.
Blunts function of other channels.
b. The extra increase in membrane conductance (decrease
resistance) will create more pathways for escape of the
charge influx, thus making it harder to depolarize the cell
with these extra channels open creating a current shunt
iv. 5 subunits (GABA)

II.

1. Alpha and beta; beta is the binding site for GABA; benzodiazepine
binds at the alpha site
Metabotropic Receptors
a. Overview
i. Some hormones and transmitters act slowly, result in enzyme activation
ii. 1950s Sutherland et al., studied the effect of epinephrine (now known
as a NT in many neurons; 1st known as a hormone released from the
adrenal cortex in an endocrine fashion)
iii. Epinephrine increased activity of a cytoplasmic enzyme, liver
phosphorylase (used in glycogen metabolism)
iv. How was this happening? A chemical in the blood is binding to a
membrane receptor to cause an intracellular change in metabolism
b. Experiment with Liver Phosphorylase
i. Homogenize liver Add Epi Activate enzyme (in cytoplasm)
ii. Centrifuge membranes from cytoplasm in test tube add Epi to
cytoplasm no effect (i.e. Epi has no direct effects on enzyme)
iii. Two test tubes: 1 liver phosphorylase in cytoplasm; 2 homogenate that
contains both membrane and cytoplasm. Add Epi to tube 2 centrifuge
membranes down add treated cytoplasm to tube 1 activate enzyme
iv. Thus, there must be a soluble messenger that transduces the signal
between the membrane and the cytoplasmic enzyme
v. Epi 1st messenger receptor activates membrane associated with
adenylyl cyclase
vi. cAMP soluble 2nd messenger activates liver enzyme
vii. Generalized to neurons (i.e. its a conserved pathway)
1. If you look a an NMJ, you find that NE can increase EPP and
mEPP size
a. e.g. AChRs become more sensitive to ACh when
phosphorylated
c. Alteration of Synaptic Transmission
i. Using a second messenger, how could this transmitter alter synaptic
transmission? There are 4 ways:
1. Alter Ca++ channel function (e.g. phosphorylation can change
NT release)
2. Alter K+ channel function (block channel slower
depolarization of pre/change in K+ resting membrane potential
alters Vm and resistance in post)
3. Alter presynaptic active zone protein function (CORE and
modulatory proteins)
4. Alter postsynaptic sensitivity to transmitter (change sensitivity
of nicotinic AChRs)
ii. Metabotropic receptors are probably present at every synapse and have
critical roles to play
iii. Postsynaptic effects paracrine actions
iv. Presynaptic effects autocrine actions
d. Examples:

i. Sympathetic Ganglia
1. Transfer of electrical signal from one neuron to the next (like
iono), but usually still only modulatory for EPSP mediated by
ionotropic receptors
2. Two nerve inputs (from sympathetic chain connecting nerves and
from spinal nerve roots) onto large B cells and small C cells in
the ganglion
3. Slow synaptic potentials present under some stimulus conditions
a. When you stimulate the two inputs to the ganglion
record intracellularly from B cells in the ganglion 3
different postsynaptic potentials fast, slow, late slow
4. The slow and late slow are only seen with trains of APs
a. Single shock to sympathetic chain fast EPSP
i. Nicotinic AChR response; block these, AP wont
occur
b. Single shock to spinal nerve root no effect in B cell
c. Train to the sympathetic chain slow EPSP
i. Not seen if muscarinic AChRs are blocked
d. Train to the spinal nerve root late slow EPSP
i. Still seen if muscarinic AChRs are blocked, but not
if LHRH receptors (peptide receptor) are blocked
5. Slower EPSPs are NOT sufficient to bring the cell to threshold, but
they do make it easier for the next synaptic activity to bring it to
threshold
6. Late slow response can be mimicked by LHRH (luteinizing
hormone releasing hormone), a hypothalamic hormone
a. Analogs of this peptide could have very potent effects, but
these analogs were not in the nerve terminals directly
onto the cells under study. Rather, they were released from
neighboring synapses, acting in a paracrine fashion.
b. LHRH-like peptide blocks a K+ channel that is voltage
activated near resting membrane potential (i.e. M type
channels are open at rest)
c. When blocked, results in depolarization (resting Na+ influx
is not balanced by K+ efflux) Vm moves towards EqNa
to create a subthreshold depolarization
7. Both muscarinic receptors and the LHRH-like peptide block these
K+ channels
a. Muscarinic effect has a different time course due to source
of ACh (local) and the metabolism of ACh as compared
with LHRH-like peptide (diffuses from neighbor and not
metabolized as quickly)
8. When these slow indirect ionic effects are in place, another
synaptic AP will produce a fast EPSP that will be
a. Larger (due to increased membrane resistance, which will
increase the depolarization resulting from injected current)

III.

b. Longer (slower repolarization because it is sped up by M

current. This longer fast EPSP can lead to multiple
postsynaptic APs riding on top of each EPSP because they
can stay above threshold longer)
c. Closer to threshold (since riding on top of a slow EPSP)
9. Slow EPSPs function in modifying transmission
ii. Modulation of Presynaptic Function
1. Modulation of presynaptic function alters transmitter release
2. Many metabotropic receptors alter the probability that Ca++
channels will open in the nerve terminal
3. If you decrease the probability that Ca++ channels will open, you
have strong affects on NT release
4. This is often in the form of autocrine inhibition of the nerve
terminal that released the NT
MECHANISMS OF METABOTROPIC SIGNALING
a. Overview
i. Majority of modulatory functions are metabotropic
ii. There are 1000s of different receptors with different binding to modulators
(peptides and transmitters)
iii. If we count odorant receptors, there are specific receptors in the brain for
thousands of different chemical modulators or transmitters that act through
indirect mechanisms
iv. Different subset probably present in every cell and affects every synapse
v. Cells do NOT have 1000 different types of ion channels and kinases; in
fact, many of these receptors target a small subset of cellular proteins that
usually include only a few protein kinases and ion channels (called
effectors)
vi. So do we have 1000 different second messenger systems? No, waste of
energy.
vii. One conserved aspect is that these receptors are all 7 TMRs (7transmembrane proteins). They have a characteristic 7 transmembrane
segments
viii. ALL of the 1000s of modulators bind to 7TMRs; considering the diversity
of functions, that is an incredible conservation of form
ix. They ALL tap into a common cytoplasmic second messenger mechanism:
G-proteins
x. The common receptor structure is essential for association with this
second messenger
b. G-Proteins
i. GTP biding proteins
ii. Several families of GTP-binding proteins
1. e.g. small GTP-binding proteins, Rab3, dynamin
iii. The class that regulates indirect second messenger modulation are
heterotrimeric GTP-binding proteins\
iv. Essentially molecular switches with an on/off state governed by a GTPase
cycle

v. In the resting state, all 3 subunits associate with receptor and one another,
and alpha binds GDP (high affinity for this less abundant form)
vi. Modulators binding to their receptor alter receptor conformation, causing
GDP to be released from alpha (decreased affinity). Since GTP is much
more abundant, it takes the place of GDP and the whole complex comes
apart
vii. The alpha GTP and beta-gamma are now considered active. They are
actually functional dimers since the beta-gamma are not thought to
dissociate. Active state as long as GTP is bound to alpha
viii. The alpha subunit turns itself off because it is a slow GTPase. There is
some evidence that binding of alpha-GTP to an effector increases GTPase
activity.
ix. Once GTP is cleaved to GDP, the alpha subunit switches conformation
the alpha, beta, and gamma complex is re-associated and returns to the
inactive state
x. Heterotrimer conformation required for association with receptors
c. Experimental Evidence that GTP is Used
i. These patch clamp experiments will prevent GPCRs from activation:
1. Remove GTP from the patch pipette (washes out the endogenous
GTP from the cell)
2. Replace GTP with GTP-gamma-s (non-hydrolysable form)
persistent activation
a. Many GTP binding proteins are active when bound to GTP
and deactivated by hydrolysis of GTP
3. Replace GTP with GDP-beta-s (high affinity binding to the alpha
subunit of the G-protein) block of signaling
ii. How can this conserved pathway lead to both diversity of signaling (tap
into all the possible 2nd messengers) and specificity of action (avoid cross
talk between two different receptors in the same cell)
d. Diversity and Specificity of Actions
i. Determined by:
1. Receptor signaling compartmentalization
2. Recruitment of other diverse 2nd messenger cascades
ii. Alpha, beta and gamma are families of proteins (different GPCRs use
different family members)
iii. Compartmentalization (each receptor may have a different environment)
iv. Alpha or Beta-Gamma can mediate different actions
e. Specific GPCR Interactions
i. Some of the specificity of action can be derived from differences in
specific G-proteins which are assembled for interactions with specific
receptors
ii. There are at least 20 alpha, 5 beta, and 7 gamma subunits. Theoretically,
this allows for an amazing number of combinations
f. Antisense Oligonucleotides
i. Used to knock out specific G protein subunits

IV.

ii. General protocol: inject into cells, sequence of about 20 nucleotides that
are complementary to (antisense) and will selectively bind a particular
mRNA that codes for the G protein subunit of interest. When bound, these
mRNAs are no longer single stranded in this region, and will not be
translated into protein. After several hours to days, that particular protein
is depleted.
iii. EX: Effects of two different modulators on the same effector.
1. Both somatostatin and ACh (acting through muscarinic
receptors) decrease the size of the Ca++ current that flows
through the channel when the rat pituitary cell membrane is
depolarized
a. After KO of each type of subunit expressed in these cells
and assaying the effects of neuromodulators on calcium
current:
i. SST uses alpha 01/ACh uses alpha O2
ii. SST uses B3/ACh uses B1
iii. SST uses gamma3/ACh uses gamma4
b. Although each results in at least one identical effect
(reduced Ca++ current), they use different G-proteins.
c. THIS MAY REDUCE CROSS TALK
g. Specificity of Action
i. Another mechanism to avoid cross talk and preserve specificity: receptor
signaling compartmentalization
ii. The receptor, G protein, and interacting proteins (effectors, ion channels)
are all within a macromolecular complex in a restricted region of the cell
h. Another experiment proving transmitter action is through GPCRs
i. Block with pertussis toxin inactivates a subset of G alphas (0 and 1)
1. Toxin covalently modifies G proteins ADP-ribosylates a
carboxyl terminal cysteine on the alpha subunit of only G0 or G1,
and uncouples the G protein from the receptor
SIGNALING PATHWAYS
a. 4 Major Signaling Pathways that Couple Receptors to Cell Changes
i. 1st messenger receptor transducer primary effector 2nd
messenger secondary effector
ii. PRIMARY EFFECTORS DETERMINE THE PATHWAYS NOT THE
TRANSMITTER OR RECEPTOR
iii. G proteins can have these indirect functions of cells either by coupling
directly to ion channels or acting through an enzyme system in the cell
b. Direct Action of G-Proteins on Ion Channels
i. One or both of the activated G proteins can have physiological effects
ii. Initially, the alpha GTP was thought to be the only active subunit, with
beta-gamma serving to inactivate the alpha by rebinding following
GTPase activity; it is now clear that both can regulate effector function in
flexible and perhaps complex ways
c. Direct Action of Beta-Gamma Subunits in the Activation of K+ Channels

i. In cardiac cells, activation of muscarinic receptors (ACh released from

vagus nerve) opens K+ channels, causing hyperpolarization and decreased
heart rate
ii. The ACh effect requires 100 msec not direct ligand-gated ion channel;
too slow; but pretty fast for a metabotropic response
iii. To prove the role of beta-gamma in a direct effect, people have used a
relatively clean reconstitution system to demonstrate direct effect:
1. Xenopus oocytes injected with RNA from muscarinic-activated
atrial K+ channel (GIRKI) and G-protein subunits
2. Observed co-expression of K+ channel with G-protein subunits
assay basal K+ channel current increases only with beta-gamma
iv. Manipulations of GIRK Channel Function
1. Beta-Gamma + GIRK channel activated
a. Could still be endogenous alpha that mediates the effect
i. But exclusion of GTP or inclusion of GDP-beta-s
(high affinity for alpha subunit) do NOT block betagamma effect
2. Alpha-beta-gamma + GTP-gamma-s + GIRK channel
activated
a. No activity with just alpha-beta-gamma; need GTPgamma-s to dissociate the Heterotrimer
b. Further, inside-out with K+ channel and add alpha-betagamma + GTP-gamma-s to cytoplasmic face
i. Induced K+ channel activity, but this was blocked
when GTP-gamma-s was removed
ii. Increased activity again when recombinant betagamma was added
iii. NO activity when G-alpha-GTP-gamma-s was
added
iv. Active beta-gamma K+ channel could be silenced
by adding G-alpha-GDP (high affinity for beta
gamma)
3. Beta-gamma has 2 binding sites on the K+ channel hypothesized
to exist due to in vitro binding and displacement with peptides
4. Activated alpha-GTP can bind to the K+ channel, but this binding
just increases the rate of GTP hydrolysis by speeding up the
termination of the response
5. The resting muscarinic receptor G-protein heterotrimer may also
bind to the k+ channel. Subcellular localization of the receptor-Gprotein-effector
d. Indirect Pathways for Metabotropic Signaling
i. Primary effector usually defines the signaling pathway
ii. Experimentally, either block the primary or secondary effector to
prevent the action of a transmitter that works through the path, or
stimulate or mimic the second messenger to mimic the action of the
transmitter and occlude further transmitter modulation

iii. PI Pathway: Model for presynaptic metabotropic receptors modifying

synaptic activity by altering the magnitude of NT release (NE effects on
NMJ)
1. DRG neurons modulation of Ca++ current by NE and GABA
2. Direct action of G-protein subunits on specific subtypes of Ca++
channels
a. In sympathetic neurons, N type channels are the
predominant channel type and regulate transmitter release
in this system
b. Example here shows a direct effect of G proteins on Ca++
channel by one transmitter and an indirect effect through
the phosphoinositol system by the other transmitter
c. In chick DRG sensory neurons, NE and GABA both inhibit
Ca++ current
i. A selective PKC activator, oleooylaacetylglycerol
(OAG), was shown to inhibit N type Ca++ current
1. OAG inhibits Ca++ current
a. + NE no further change
b. + GABA further inhibition of
current
ii. Pseudosubstrate inhibitor PKC 19-31 peptide
which binds to the kinase specifically and blocks
function
1. Blocked function of NE (and OAG) but had
no effect on GABA mediated inhibition
2. This NE effect appears to act through the PI
system:
a. G protein phospholipase C
cleavage of PIP2 to IP3 and DAG
IP3 induced Ca++ release DAG +
Ca++ activate PKC
phosphorylation of channel, and
decreased function
d. Remember pathway is only determined from primary
effector on down
e. Just because this pathway for NE involves a kinase, it
doesnt mean that there is a soluble cytoplasmic
messenger that travels a long distance
i. Whole cell dialysis does not effect inhibition
ii. Cell attached recordings dont show inhibition when
transmitter is added to the outside
f. But this does NOT mean that this uses the direct pathway.
Often kinases and phosphatases that regulate proteins are
co-localized by tethers or anchors often no long range
actions are needed DAG only needed to locally
activated the PKC that is anchored to the Ca++ channel

g. Evidence to date indicates that this GABA-initiated Gprotein mediated inhibition appears to work in the absence
of any other known second messenger intermediary (i.e.
GABA works through direct pathway)
i. Addition of modulators for any of the known
second messenger systems has no effect
ii. Proof of this would require reconstitution of the
known elements into a clean system (bilayer) and
demonstration the effect is preserved