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BIOL 447 Ion Channels and Disease > Midterm 2 Content - Glutamate Receptors > Flashcards

Midterm 2 Content - Glutamate Receptors Flashcards

1
Q

What is the membrane topology of an ionotropic glutamate receptor like? Name how regions are specific to certain types of glutamate receptors.

A

See notes for drawing; n = 4, tetramer

M2 is reentrant pore loop
M2-M3 is the pore lining domain
S1 and S2 are where ligands bind (can have coagonists)
N terminus is extracellular while C terminus is cytosolic

Q/R (RNA) editing: occurs at M2 pore, only with AMPA and Kainate
Flip/flop: occurs just before M4, only with AMPA

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2
Q

What channel did Armstrong et al (1998) study?

A

A S1S2 flop construct of an AMPA GluR2 subunit

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3
Q

What are key features of glutamate receptor agonists?

A

2 carboxyl groups
1 amine group
Overall negative charge

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4
Q

What is the charge composition of Kainate like?

A

2 negative and 1 positive

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5
Q

What are AMPA channels permerable to? What are agonists and antagonists?

A

Ca2+ impermeable but still permeable to Na+ and K+

Agonists

  • AMPA
  • Glutamate
  • Kainate (partial agonst)
  • Quisqualate

Antagonists:

  • CNQX
  • DNQX
  • NBQX
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6
Q

What are agonists/antagonists of Kainate channels?

A

Agonists:

  • Glutamate
  • Kainate
  • Domoate
  • AMPA (only if GluK2 is present with GluK4/5)

Antagonists:

  • CNQX
  • DNQX
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7
Q

What is NMDA permeable to? What are agonists/antagonists

A

Ca2+, K+ and Na+ permeable

Agonists:

  • Glutamate
  • NMDA
  • L-aspartic acid
  • ESSENTIAL COAGONISTS: glycine or D-serine

Antagonists

  • D-AP5
  • Ketamine
  • MK801
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8
Q

What is an example of a polyamine?

A

Spermine

–> causes channels to be more inwardly-rectifying

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9
Q

What RNA editing is present in AMPA receptors and what 3 key changes does it result in?

A

A posttranslational modification that occurs at the M2 reentrant pore loop of GluA2.

A glutamine (Q; CAG) is mutated to an arginine (R; CIG) at the Q/R site. This leads to…

1) Ca2+ impermability in AMPA receptors with a GluR2
2) Low channel conductance
3) Linear current-voltage relationship: less inward rectification than other GluA channels

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10
Q

What is alternative splicing like in glutamate receptors?

A

Modifying pre-mRNA constructs prior to translation; all glutamate receptors are alternatively spliced at the C terminus.

However, AMPA receptors can be alternatively spliced to either have “flip” or “flop” forms.

  • Flip: desensitizes slower and less profoundly
  • Flop: desensitizes faster and more profoundly
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11
Q

Why are NMDA receptors termed “coincidence receptors”?

A

In order to activate, need…

1) Agonist binding AND coagonist binding.
2) Depolarization to remove Mg2+ block. *Mg2+ hydration energy is high; plugs up pore effectively.

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12
Q

What was the construct made by Armstrong et al (1998)?

A

S1S2 GluR2 flop construct.

Cut GluR2 between the ATD and M2 to get S1, and between M2 and M4 to get S2.
Then attached them using a linker.

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13
Q

What shape does the GluR2 S1S2 and Kainate make? What is the structure like?

A

A clamshell that closes onto the coagonist.

The N-terminus of the GluR2 alpha helices point towards the interdomain crevice to interact with kainate or participate in interdomain interactions.

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14
Q

What are electrostatic (Salt-bridge) interactions?

A

Between side chains of oppositely charged residues

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15
Q

What are hydrogen bonds between?

A

Between the hydrogen attached to an electronegative atom and another electronegative atom.

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16
Q

What are alpha-helix dipole interactions between?

A

Between N and C termini

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17
Q

What are cation pi interactions between?

A

Positive charge and a pi electron cloud (created by aromatic residues like tyrosine, tryptophan, and phenylalanine)

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18
Q

In the GluR2 S1S2 complex with kainate, what stabilizes kainate’s carboxyl groups?

A

Hydrogen bonds –> F helix N terminus

Salt bridges – R485, T655

19
Q

In the GluR2 S1S2 complex with kainate, what stabilizes kainate’s amine group?

A

A Cation-pi interaction with Y450 –> “Lid” on top of Kainate

Also interactions with E705, P478, and T480

20
Q

What are the binding pockets of GluA2 like? Why is this?

A

Binding pockets are negatively charged.

For fast signalling, it is advantageous for the receptor to be quickly released from the ligand for efficient neurotransmission.

21
Q

What was the structure of GluR2 S1S2 and glutamine-binding protein (QBP) like?

A

The clamshell was more closed

–> indicates that partial agonist binding may result in a slightly more open/intermediate degree of closure

22
Q

What is DNQX? What is its position in the binding pocket like?

A

A competitive antagonist for GluR2 receptors.

When bound, the amine groups are to the left of the binding pocket while the nitro groups are to the right.

23
Q

What is Kd?

A

The concentration at which half the receptors are bound.

24
Q

What is IC50?

A

Half maximal inhibitory concentration: the value at which you inhibiting the binding of agonist to 50% of the maximum.

Measures the effectiveness of a compound in inhibiting biological function.

25
Q

What did Armstrong notice when comparing apo and DNQX-bound GluR2 S1S2?

A

Apo = no ligand bound.

DNQX shows more closure than apo.

26
Q

What are major interactions between DNQX-bound GluR2? How does this compare to apo-bound?

A

E705: in apo, forms a salt bridge with another residue; in DNQX-bound, flips 135 degrees and points to aromatic rings of DNQX

Y450: forms pi-stacking interactions with DNQX.

Incorporated sulfate interacts with F helix and 2 water molecules.

27
Q

What did Armstrong et al notice when comparing GluRS1S2 glutamate-bound and kainate-bound forms?

A

Structures were similar, but L450 moves substantially further into the cleft in the glutamate-bound state.

Therefore, the clamshell is more closed when bound to the full agonist glutamate in comparison to the partial agonsit kainate.

28
Q

What did Armstrong et al notice when comparing GluRS1S2 glutamate-bound and AMPA-bound forms?

A

They are both full agonists, but occupy different site of the agonist-binding site (AABCDEFG)

AMPA: aabcEFG; more to right

Glutamate/kainate: aabcDE; more to left

29
Q

What did Armstrong et al notice when comparing GluRS1S2 kainate-bound and AMPA-bound forms with an apo form?

A

Kainate shows 12 degrees more closure than apo, while AMPA shows 20 degrees more closure than apo.

30
Q

What can be concluded by Armstrong et al’s comparison of different ligands bound to the AMPA GluR2 S1S2 complex?

A

Full agonists have the greatest degree of closure (20 degrees)
Partial agonists follow (kainate; 12 degrees)
Antagonists are next (4 degrees)
Apo form is last (2 degrees)

31
Q

What is the mechanism of glutamate binding to AMPA GluR2? Name specific interactions and residue movements, and compare with the apo form.

A

In apo form:

  • ligand binding cleft is open
  • Domains 1 and 2 of gluR2 are separate
  • E705 interacts with the domain 2 F helix and makes a salt bridge with K730

When glutamate binds:

  • Glutamate binds to domain 1 residues (R485, T480, Y450, E750)
  • -> E750 breaks its salt bridge with K730 and flips 135 degrees.
  • Domain 1 and 2 close in together (clamshell closes)
  • Glutamate is further stabilized by domain 2 (F helix N terminus)
32
Q

What channel did Sobolevsky study?

A

GluR2

33
Q

What are the 3 main regions of the GluR2 receptor? What shape does it make?

A

“Capital Y”

Amino terminal domain: splays outwards to form V of Y
Ligand-binding domain: binds ligand; sandwiched in between.
Transmembrane domain: membrane-spanning regions; trunk of Y

34
Q

What is the symmetry of the GluR2 receptor like?

A

Overall, has 2 fold symmetry.

ATD and LBD have 2 fold symmetry
TMD has 4 fold symmetry

35
Q

What is the pre-M1 cuff? What residue is responsible and what is its function?

A

In the GluR2 M1-M2 linker region, there is a sharply bent kink in the helix produced by a P520 “elbow”.

This proline makes close association with M3 and M4 to lock M3 into a closed conformation and must be removed in order for M3 to splay open.

36
Q

What domains are the equivalent conformation pairs in the GluR2 domain?

A

A/C and B/D

A/C have the same conformation, and B/D have the same conformation, but the A/C conformation is different than the B/D conformation.

37
Q

Explain domain swapping in a GluR2 receptor, naming which subunits are dimers and where they are situated in each domain.

A

ATD: A/C are distal, while B/D are proximal. A/B and C/D are dimers.

LBD: B/D are distal, and A/C are proximal. A/D and B/C are dimers.

TM: there is four fold symmetry.

38
Q

Explain domain swapping in a NMDA receptor, naming which subunits are dimers and where they are situated in each domain.

A

NMDA receptors contain 2 GluN1 and 2 GluN2A. GluN2A binds main agonist while GluN1 binds coagonist.

ATD: GluN1 subunits are distal, and GluN2A subunits are proximal.
LBD: GluN2A subunits are distal, and GluN1 subunits are proximal.

39
Q

What shape does the AMPA receptor GluR2 change to when domain swapping? How did Sobolevsky et al. observe this? What linking peptides are involved?

A

Transition from parallelogram, two-fold symmetry to square, four-fold symmetry.

Switch from two fold to four fold symmetry can be seen by observing seleium (2-fold) and mercury (4-fold) site on a labelled receptor and measuring root mean square difference. Lower values mean closer to 4-fold symmetry, while high values are closer to 2 fold symmetry.

Three linking peptides make the transition:

  • M1-S1 linker
  • M3-S2 linker
  • S2-M4 linker
40
Q

What is the structure of (specifically) the ION CHANNEL of GluA2 like? What is the greatest constraint of the ion channel?

A

Has 4-fold symmetry (TMD) and is shaped like a Mayan temple/upside down KcsA channel.

Largest constraint is the bottlneck at the top of the channel, caused by M629 and T625.

41
Q

What could be the mechanism for the gating hinge for the GluR2 channel?

A

The gating hinge is located near the extracellular side of the membrane at the crossing/bundling of the M3 helices.

There are 3 glycines within M3 that can bend; this may be the mechanism behind the gating hinge.

42
Q

What are 3 similarities between GluR2 and KcsA channels? What is a difference?

A
  • iGluR channels resemble upside-down KcsA channels
  • GluA2 has a large central cavity like KcsA
  • M1, M2, and M3 segments of GluA2 overlap well with equivalent portions of KcsA (pore-lining regions and pore helices are somewhat aligned).

Difference: selectivity region is different (KcsA is more selective)

43
Q

What is the mechanism Sobolevsky proposed for GluR2 gating?

A

Domain 2 of the clam shell closes 25 degrees and moves 20 Angstroms up and outwards.

This pulls up on M3 at the bundle crossing, splaying the channel and opening the pore.

BIOL 447 Ion Channels and Disease (4 decks)

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