Glutamate Anaesthetics

Question 1

How can the properties of glutamate receptors be recorded?

Answer 1

Transfect epithelial cells with the cDNA encoding for the ionotropic glutamate receptor subunits and for GFP

1-2 days later, record from the GFP cells using whole cell patch clamp technique

Agonist-induced inward current

Question 2

What are the 3 types of glutamate receptors?

Answer 2

AMPA
Kainate
NMDA

Question 3

What is the main flow of current at -60mV in terms of a neurone and glutamergic receptors

Answer 3

Na+
Ca2+

Under these conditions, the Mg+ enters and blocks the NMDA open ion channel

Question 4

Describe the NMDA receptor

Answer 4

Conducts Na+, Ca2+ and K+ BUT is blocked by Mg 2+

The block by Mg2+ is both voltage and agonist dependent

For the NMDAR to open it needs to be activated by glutamate (or NMDA) and by the co-agonist glycine

Phencyclidine (PCP) and ketamine are blockers of the NMDAR ion channel

Question 5

Describe the main flow of current at +20 mV in terms of a neuron and glutamatergic receptors

Answer 5

Outward mainly carried by K+ leaving the cell

Under these conditions, Mg2+ does not enter or block the channel

Question 6

Does NMDA require just glutamate to activate?

Answer 6

No; requires glutamate and the co-agonist glycine (D-serine)

Question 7

What are the binding sites for glutamate and glycine on NMDA?

Answer 7

Glutamate at GluN2

Glycine at GluN1

Question 8

Describe the impact of Mg+, APV (NMDA antagonist) and CNQX (AMPAR antagonist) on EPSCSs

Answer 8

Standard solution (containing Mg2+) there is a fast ESPC 
This is not influenced by APV but is eliminated by CNQX

In a Mg2+ free solution, the EPSCs exhibit a slow component. This slow component is inhibited by APV, however CNQX, the fast component is inhibited but there is no effect on the slow component of EPSC

Question 9

Describe the synergistic interplay of synaptic AMPARs and NMDARs at an excitatory synapse

Answer 9

Neurally released glutamate activates synaptic AMPAR, but although glutamate binds to NMDARs, the assoc ion channel does not conduct due to blockage via Mg2+

The Na+ influx results in a depolarisation of the neuronal spine. If the presynaptic glutamergic nerve fires at high frequencies, then the depolarisation may be sufficient to unblock the Mg2+ NMDAR; allowing for a slow prolonged synaptic depolarisation

Question 10

Describe the conductance of NMDARs

Answer 10

Inward Na+/ Ca2+
Outward K+
At neg membrane potential, blocked by Mg2+

Question 11

What is EPSP integration?

Answer 11

Neurons require many excitatory postsynaptic potentials to summate to produce an EPSP sufficient to fire an action potential

Question 12

Describe the difference between temporal and spatial summation

Answer 12

Spatial; 2 or more presynaptic inputs are simultaneously active causing their individual EPSPs to summate

Temporal; the same presynaptic fibre fires action potentials in quick succession, causing the individual EPSPs to summate

Question 13

What do NMDA, AMPAR and KA receptors have in common?

Answer 13

Composed of 4 transmembrane subunits
Large N terminal domain, crosses membrane at TM1, re-entrant loop, Q/R site, TM2, extracellular loop, TM3, intracellular carboxyl tail

NMDA; GluN1, GluN2 (A,B,C or D), GluN (3A or B)

AMPA: GluA (1,2,3,4)

KA: GluK (1,2,3,4,5)

Question 14

Describe the ability of AMPAR to be calcium permeable or impermeable

Answer 14

Majority of AMPARs are heteromers containing GluA2 subunit

AMPARs containing GluA1, 3 or 4 are calcium permeable

The GluA2 subunit is critical in determining receptor function. Incorporation of GluA2 impairs calcium permeability

The permeability to calcium is dictated by a single amino acid located on the ion conducting pore (glutamine Q (permeable), or arginine R (impermeable)

The GluA2 RNA may be subject to RNA editing, which will determine the GluA2 will contain a Q (calcium permeable) or an R (impermeable) at this key ion channel location

Question 15

Describe the role of RNA editing and ion permeability of the AMPA receptor

Answer 15

GluA2 undergoes Q to R editing (adenosine deamination)

CAG = Q 
CGG = R 

In adult; 95% of GluA2 is edited i.e. GluAR

GluA2Q = Ca2+ permeable 
GluA2R = Ca2+ impermeable 

Note, GluA1 (Q), GluA3 (Q), GluA4 (Q) and unedited GluA2 (Q) are ALL Ca2+ permeable

Question 16

Where is the glutamate binding site on AMPAR glutamate receptors?

Answer 16

S1 and S2

Evidence:

1) S1 and S2 show sequence homology to bacterial amino acid binding proteins
2) bacterial proteins bind amino acids between 2 lobes of a clam shell that are in a dynamic equilibrium between open and closed states; ligand binding stabilises the closed state
3) swapping S1 and S2 domains between GluA3 (AMPAR) and GluK2 (KAR) causes appropriate changes to agonist pharmacology

Question 17

Describe the structure of NMDA receptors

Answer 17

Mostly composed of GluN1 and GluN2

The NMDAR subunits have a similar topology to AMPA and KA subunits

GluN1 subunit binds Glycine/ D-serine
GluN2 binds glutamate

At the equivalent AMPAR AMPAR Q/R site, NMDARs have an asparagine residue (N) - site of Mg2+ block

Question 18

Describe the effects of ketamine upon IV injection

Answer 18

Dissociative anaesthetic state; marked sensory loss, amnesia and analgesia without complete loss of consciousness

Structurally related to PCP

Little to no effect on GABAARs

Acts as a “use-dependent” voltage-dependent blocker of NMDAR assoc cation channel

Question 19

Describe the blockade of NMDA via ketamine

Answer 19

Blocks the channel in the open state (2ARD*)

Voltage dependent; the more negative the membrane, the more likely the blockade

Agonist dependent; channel has to be in open state for ketamine to access binding site

Question 20

Describe the uses for nitrous oxide

Answer 20

Occasional inhalational anaesthetic
Analgesic in short, painful procedures; labour, dentistry

Evidence emerging for ability to demonstrate rapid antidepressant properties

Rapid onset and offset

Question 21

What is the basic mode of action of NO?

Answer 21

Non-competitive NMDAR antagonist

Question 22

Describe xenon as a GA

Answer 22

Rapid induction, short duration
Lack of CV effects
Powerful analgesic effects
Exhibits neuroprotective effects

Unfortunately, very expensive therefore limiting use

Question 23

What is the mode of action of xenon?

Answer 23

NMDA receptor antagonist by inhibiting the glycine binding site on the NR1 subunit