NMDAs vs AMPARs

Question 1

Glutamate receptors are the primary mediators of excitatory synaptic transmission in the brain (ref?)

Answer 1

Traynelis et al., 2010

Question 2

ionotropic glutamate receptor (iGluR) family members

Answer 2

NMDARs
AMPARs
kainate receptors

Question 3

when and why did resistance to the idea that glutamate could be a neurotransmitter change?

Answer 3

1970s/early 1980s when Neher and Sakmann developed patch-clamp techniques. There is now overwhelming evidence and wide acceptance that glutamate is the major fast excitatory neurotransmitter in the CNS.

Question 4

Most excitatory synapses have a mix of…..

Answer 4

AMPARs and NMDARs, and the excitatory postsynaptic current (EPSC) reflects this heterogeneity.

Question 5

Following a synaptic stimulus, AMPARs mediate the initial…..
(rise time, tau)

Answer 5

fast component, rising rapidly (< 1 ms) and subsiding the fastest (tau = 1 - 3 ms); they determine the onset and maximal amplitude of the EPSC. (Fast kinetics low affinity for glutamate)

Question 6

In contrast, NMDAR currents have a…..

rise time, tau

Answer 6

very slow rise time (~ 10 ms) with and decline much more slowly (τ > 100 ms), making this one of the slowest known ligand-gated ion channels. (Slow kinetics high affinity for glutamate)

Question 7

AMPARs activate with little depolarisation acting as a gate for…..

Answer 7

the NMDAR by providing further depolarisation, facilitating the voltage-dependent expulsion the Mg2+ ion blocking the channel pore. (Mayer et al., 1984)

Question 8

Glutamate concentration in vesicle?

Answer 8

100mM

Question 9

Glutamate concentration in synaptic cleft during a) synaptic transmission b) at rest

Answer 9

a) mM range

b) 1uM

Question 10

Structural components of NMDARs and AMPARs, what do they both contain?

Answer 10

Both have a large ectodomain composed of N-terminal (NTD) and ligand-binding (LBD) domains, a short transmembrane domain (TMD) and cytoplasmic C-terminal domain (CTD), the latter being structurally unresolved.

Question 11

AMPAR subunits?

Answer 11

GluA1 (GRIA1), GluA2 (GRIA2), GluA3 (GRIA3), and GluA4 (GRIA4), which combine to form tetramers. All subunits bind glutamate. Each subunit differs in its contribution to channel kinetics, ion selectivity, and receptor trafficking properties, heteromerization also adds considerable functional diversity.

Question 12

All AMPAR subunit proteins have…..

Answer 12

an extracellular NTD (containing the S1/S2 domains which from a clam shell around the agonist (LBD)), an intracellular CTD, and three TMDs (M1, M3 and M4) and an intracellular re-entrant loop (M2) that forms the pore lining domain.

Question 13

AMPARs tetramers are formed in the…..(and what interactions hold the tetramer together)

Answer 13

endoplasmic reticulum (ER) as dimers of dimers. Dimerization of two subunits that is dependent on the interactions in the NTD. Second dimerization step mediated by associations at the LBDs and TMDs, and the formation and stabilization of the tetramer is further promoted by NTD interactions.

Question 14

AMPAR subunit splice variant…..

Answer 14

alternative splicing of 38 amino acids in the extracellular loop between M3 and M4 of AMPARs generates two variants, flip and flop, conferring distinct biophysical properties.

Question 15

Which splice variant predominates in developing tissues?

Answer 15

Flip isoform (slower to desensitise)

Question 16

Which splice variant predominates in mature tissues?

Answer 16

Flop isoform (faster to desensitise)

Question 17

AMPARs are widely expressed throughout the central nervous system an are found in…..

Answer 17

neurons and in glia.

Question 18

Majority of CNS AMPARs exist as…

Answer 18

heteromers containing GluA2, but can also function as homomeric assemblies of subunits.

Question 19

In forebrain (hippocampus and neocortex) the predominantly expressed AMPAR subunits are…..

Answer 19

are GluA1 and GluA2, with low levels of GluA3 and GluA4 (Craig et al., 1993)

Question 20

Major neuronal population express AMPARs comprised of?

Answer 20

heterotetramers of GluA1 and GluA2 (Sans et al., 2003)

Question 21

Which GluA subunit makes the receptor impermiable to calcium?

Answer 21

GluA2

Question 22

Why are GluA2 impermeable to calcium?

Answer 22

Most mature GluA2 protein contains an arginine residue (R) within the re-entrant M2 membrane loop region at position 586 in place of the genomically encoded glutamine (Q) (Verdoorn et al., 1991). The positivity charge on R residue repels divalent cations.

Question 23

In GluA2 how is Q changed to R?

Answer 23

The change is the result of hydrolytic editing of a single adenosine base in the pre-mRNA to an inosine by the adenosine deaminase enzyme ADAR2 (Higuchi et al., 1993)

Question 24

The additional positive charge introduced into GluA2 subunits in the pore by the presence of R586 prevents what 3 things?

Answer 24

The passage of divalent cations (including Ca2+) and block by endogenous, positively charged, intracellular polyamines, and reduces single-channel conductance.

Question 25

Channels containing edited GluA2 subunits have.....

Answer 25

a linear current-voltage relationship, are impermeable to Ca2+, and exhibit a relatively low single-channel conductance GluA2 containing

Question 26

Channels lacking edited GluA2 subunits are.....

Answer 26

Ca2+ permeable, of higher conductance, and are inwardly rectifying due to a voltage-dependent block by endogenous intracellular polyamines (Bowie and Mayer, 1995). However, although GluA2-lacking AMPARs exhibit significant Ca2+ permeability, they are less permeable to Ca2+ than NMDARs.

Question 27

Polyamine block: what is spermine at physiological conditions?

Answer 27

Spermine is positively charged at physiological pH due to 4 nitrogens becoming protonated.

Question 28

Polyamine block: what kind of AMPARs are blocked by spermine?

Answer 28

GluA2 lacking receptors

Question 29

Polyamine block: why does spermine block GluA2 lacking receptors?

Answer 29

They are attracted by the negatively charged ring of carbonyl-oxygen groups provided by the glutamine (Q) residue (present in GluA1, GluA3 and GluA4 subunits) but are repelled by the positively charged arginine residue in the GluA2 subunit.

Question 30

Polyamine block: when is block highest?

Answer 30

At positive membrane potentials

Question 31

Polyamine block: At what positive membrane potential does current pass again and why?

Answer 31

Approximately +55mV current flows again due to the fact that polyamines pass right through the channel from the inside to the outside when the driving force is large enough (outside very negative)

Question 32

Polyamine block: what happens at low membrane potentials?

Answer 32

when the inside is more negative than outside, the block is decreased as the positive polyamines are more attracted to the larger negative charge intracellularly than the negative charge on the glutamine (Q). As the membrane hyperpolarises less polyamine block

Question 33

Polyamine block: block occurs between?

Answer 33

-20mV to +50 mV

Question 34

NMDAR subunits?

Answer 34

NMDA receptors function exclusively as heterotetramers of seven genetically encoded, differentially expressed subunits: GluN1, which is processed into eight molecularly distinct splice variants, four GluN2 (A-D), and two GluN3 (A-B).

Question 35

What does GluN1 bind?

Answer 35

Glycine and d-serine (endogenous) | sarcosine (exogenous)

Question 36

What does GluN2 bind?

Answer 36

Glutamate

Question 37

What does GluN3 bind?

Answer 37

Glycine (makes receptor partially permeable to Ca2+ and insensitive to Mg2+ block

Question 38

favoured NMDAR subunit dimers?

Answer 38

combination being two GluN1/GluN2 dimers, diametrically apposed. tetramers cannot be homomeric.

Question 39

What subunit is required in an NMDAR

Answer 39

Functional glutamatergic NMDARs contain at least one GluN1 subunit, providing a functionally-required glycine-binding site.

Question 40

where is the proton binding site? and what does it do?

Answer 40

The proton-binding site on the GluN1 subunit operates under normal physiological conditions to block approximately 50% of NMDARs (Traynelis and Cull-Candy, 1990)

Question 41

GluA subunits D1/D2 rotation ligand binding

Answer 41

DNQX (antagonist 0 degrees) Kainate (partial agonist) 12 degrees) glutamate (full agonist) 20 degrees (Armstrong et al., 1998) no rotation observed in NMDAR subunits

Question 42

what did Jin et al., show?

Answer 42

Jin et al., (2003) have shown that a series of AMPAR partial agonists, varying by a single atom, induce a differential degree of domain closure that determines the coupling efficiency of the receptor subunits, which contributes to the incremental increase in single channel conductance.

Question 43

how does AMPAR conductance relate to subunit ligand binding?

Answer 43

a non-concerted mechanism by which individual AMPA receptor subunits control channel conductance in a manner related to the nature of the induced fit (i.e. degree of domain closure) of each ligand with its binding site and the number of binding sites that are open.

Question 44

how does NMDAR conductance relate to subunit ligand binding?

Answer 44

NMDA receptors show a concerted pore opening only after all subunits are bound by agonist and all pregating steps have been accomplished.

Question 45

what does conserted opening of NMDARs show?

Answer 45

These findings are consistent with the much slower rise and longer bi-exponential deactivation of NMDAR-gated currents (as opposed to AMPAR-gated currents), which dominate and control the slow decay phase of the EPSC.

Question 46

do NMDAR LBD (D1/D2) rotate?

Answer 46

the LBD of the GluN1 subunit shows no relationship between the degree of agonist-induced domain closure and agonist efficacy

Question 47

What have recent crystallography analysis identified in NMDARS?

Answer 47

Recent crystallographic analyses have identified a tyrosine residue in the heterodimer interface that appears to be critical in regulating the rate of deactivation and prolongs the time-course of the synaptic signal (Furukawa, 2005).

Question 48

What blocks the channel pore of NMDAR?

Answer 48

Mg2+ binding in the channel pore directly blocks NMDAR-gated currents in a voltage-dependent manner.

Question 49

Currents recorded from Xenopus oocytes expressing wild-type NMDARs in the presence of differing concentrations of extracellular Mg2+ and at different voltages showed different subunits were more sussceptable to Mg2+ block which were blocked more?

Answer 49

Channels containing GluN2A and GluN2B subunits were blocked more strongly by Mg2+ than those with GluN2C or GluN2D subunits (Kuner and Schoepfer, 1996) Less extracellular Mg2+ required to block current through GluN2A/GluN2B containing receptors (IC50=2.4 and 2.1μM, respectively), and does so at lower voltages, compared to those containing GluN2C/GluN2D (IC50=14.2 and 10.2μM, respectively)

Question 50

what make NMDARs have an outwardly rectifying current?

Answer 50

rapid depolarisation mediated by AMPARs repels divalent cations (Mg2+) from the pore, which is blocked at hyperpolarized membrane potentials

Question 51

NMDARs can act as a coincidence detector what does this allow to happen?

Answer 51

Following the presynaptic release of glutamate AMPARs depolarise the membrane sufficiently Mg2+ block is removed from the NMDAR pore allowing the influx of Ca2+ (Mayer et al., 1984).

Question 52

What is the calcium hypothesis?

Answer 52

According to the calcium hypothesis (Artola and Singer, 1993), depending on the stimulation frequency, two distinct types of Ca2+ signals can arise, that lead to the activation of separate downstream pathways.

Question 53

Calcium hypothesis: High frequency stimulation (HFS)

Answer 53

The rapid, large intracellular rise in Ca2+ after HFS promotes the activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and is followed by the induction of LTP

Question 54

Calcium hypothesis: Low frequency stimulation (LFS)

Answer 54

LFS results in a more gradual, modest rise in Ca2+ that leads to the recruitment of phosphatases, such as protein phosphatase 1 (PP1), and calcineurin, which are necessary for LTD (Yang et al., 1999)

Question 55

Calcium hypothesis: High frequency stimulation (HFS) mechanism

Answer 55

A proposed mechanism for the former involves the phosphorylation of the GluA1 (Ser831) subunit of AMPARs by CaMKII which results in an enhancement of their transmission and an associated strengthening of the activated synapse (Malinow and Malenka, 2002).

Question 56

Calcium hypothesis: what other factor has been reported to be involved in synaptic modification?

Answer 56

Another factor that has been reported to be involved in synaptic modification is the presence of tropomyosin-related kinase B (TrkB) receptors in the postsynaptic terminal, which can increase the NMDAR conductance to upregulate the influx of Ca2+ (Wörgötter et al., 2018)

Question 57

what downstream signalling pathways are also activated by Ca2+ influx?

Answer 57

downstream signalling pathways such as the positive feedback loop following mTOR-dependent translation of the neurotrophin BDNF which stimulates presynaptic neurotransmitter release, while postsynaptically promoting the phosphorylation of NMDARs. Effectively, this leads to an increase in the open probability of the channels while facilitating synaptic clustering and AMPA receptor upregulation (Caldeira et al., 2007).

Question 58

the combined voltage-sensitivity of the NMDAR and requirement of postsynaptic depolarisation, which occurs only during tetanus, for the induction of LTP suggests that?

Answer 58

the Hebbian mechanism underlying LTP resides in the NMDAR itself, laying a mechanistic foundation for LTP as a compelling, logical link to associative learning.