Glutamate Receptors: • An unusual Glutamate-activated Cl channel

(parasitic helminths): Cys-loop receptor

Glutamate-activated cation channels:

• Glutamate as a neurotransmitter

• Glutamate receptor structure: different from Cys-loop receptors

• 3 Classes of glutamate receptors: pharmacology & physiology

Glutamate Receptors

Glutamate-activated Cl channels:a target of anti-helminthic drugs

• Roundworms (nematodes) and flatworms are responsible for parasitic infections, that usually involve complex parasite lifecycle with multiple hosts

• Onchocerciasis (causing river blindness), lymphatic filariasis(causing elephantitis), and canine heartworm are caused by filarial nematode infections transmitted by black flies or mosquitoes

• World Health Organization Initiatives have targeted these conditions, resulting in improvement in worldwide health

• Antihelminthic drugs act by preventing muscle contraction in the parasite larvae or altering metabolism (e.g. interfering with microtubule function)

• Another approach is antibiotics to kill symbiotic Wolbachiabacteria, rendering the female worms sterile

Approaches to inhibit worm larvae:• Unlike vertebrates that have only

excitatory synapses at the NMJ, roundworms have both excitatory and inhibitory (GABA-activated Cl channels) synapses onto skeletal muscle

• Also, Glutamate-activated-Cl channels inhibit muscle in pharnyx directly, and inhibit skeletal muscle via inhibitory interneuron

• Glutamate-activated Cl channels are Cys-loop receptors, like nAChR, GABAAR & Glycine R

• Ivermectin is a Glutamate-Cl receptor allosteric agonist used to kill microfilariae that cause river blindness, and to lesser extent to treat elephantitis

• Piperazine, a GABA agonist at invertebrate NMJ, also is effective

• Other therapeutics include inhibitors of microtubule function or arachidonic acid metabolism (see126C next quarter)

Worm Glutamate-activated Cl channel

Hibbs & Gouaux 2011

Glutamate• The major excitatory neurotransmitter in mammalian brain• A family of 3 types of ligand-gated cation channels;

permeable to Na+, K+. NMDA subtypes also permeable to Ca2+

What does glutamate do?• Involved in learning & memory, and long-term potentiation, a

cellular correlate of learning• But…too much glutamate causes neural toxicity.What happens if inhibit glutamate action?• Depress excitation in brain, anesthesia, but has psychotropic

actionsCan glutamate pathways be targeted to enhance learning/memory, control Alzheimer’s disease, or inhibit effects of stroke/brain trauma? How is glutamate action terminated?

Does this require energy? EAAT (Excitatory Amino Acid Transporter co-transports Na+ and glutamate

EAAT

2 Families of Structurally Different Ionotropic Receptors :

Nicotinic ACh R5-HT3 RGABAA RGlycine R(& Glu-Cl R: worms)

Glutamate R(mammalian)

5 subunits: N-ACh R, 5-HT3 R, GABA R, Glycine R, (&invertebrate Glutamate R)

4 subunits: Glutamate R (mammalian)

Glutamate Receptor Structure (mammalian):

• Ligand binding site: N terminus & M3-M4 linker

• Pore region:Re-entrant loop M2& M3

• Channel is a tetramer of homologous subunits

Structure of AMPA Glutamate R

Sobolevsky, Rosconi, Gouaux (2009) Nature 462, 745

Ligand Binding Domain:• 4 binding sites for

agonist/antagonist• one within each subunit

clamshell

Transmembrane domainForms the pore

Amino Terminal Domain:involved in receptor assembly, trafficking and modulation

• 4 subunits• Structure is a dimer of dimers

Pore region (shaped like an upside-down KV channel):Re-entrant loop “M2” forms the inner part of the poreM3 lines the outer part of the pore and forms the gate for channel opening

Critical site in the pore:

•NMDA R have an “N” (asparagine) at this site. This makes a channel that IS Calcium permeable, blocked by Mg2+

•AMPA R undergo mRNA editing. Their mRNA encodes a “Q” (glutamine). On the GluR2 subunit, this site is edited to encode “R” (arginine). QR editing introduces a positive charge that makes channels that are NOT Calcium permeable, not blocked by Mg2+

3 Classes of Glutamate Receptors:

AMPA R

Kainate R NMDA R

A combination of 4 homologous subunits forms each glutamate receptor.

Newer Nomenclature:

NMDA Receptor: Binding Sites

3 Types of Glutamate Receptors:•NMDA•AMPA•Kainate

NMDA receptors require glycine as a co-agonist

NMDA Receptor

Kainate ReceptorAMPA Receptor

NMDA Receptor

NMDA Receptor:•Glutamate binds to 2 of the subunits (GluN2) and glycine binds to the other two subunits (GluN1 or GluN3)•Physiological background [glycine] often sufficient to serve as the co-agonist

Non-competitive antagonists:Ketamine is a veterinary anesthetic, also used for children; drug of abuse; currently being evaluated for treatment of depression

Phencyclidine (PCP) is a drug of abuse

Both are dissociative anesthetics

How do non-competitive antagonists inhibit the NMDA R?

How can we learn how they work?

Non-Competitive NMDA Antagonists

Synaptic PhysiologyMost central excitatory synapses have both AMPA and NMDA receptors.

AMPA R• Fast excitatory response

NMDA R• Slightly slower excitatory response

Excitatory action:Both NMDA and AMPA produce inward Na currents, which will depolarize the cell membrane.

Mg2+ blocks NMDA Receptor at Negative Potentials

At negative membrane potentials, the electrical driving force drives Mg2+ into the pore and blocks it

At more positive voltages, Mg2+

is expelled from the channel, and ions can flow through the pore

Calcium

Excitotoxicity

NMDA receptors serve as a coincidence detector:They are only open when the post-synaptic cell is: a) depolarized and b) glutamate is present

NMDA receptors cause calcium influx, which is important for learning & memory, but also cause neuronal death

SummaryGlutamate: inhibitory for worm muscle; allosteric agonist for treatment of filarial infection (e.g. river blindness)Glutamate: major excitatory neurotransmitter in mammalian brain

• Structure: tetramer, cation channel, sites within pore loop determine Ca2+ permeability & Mg2+ block

• 3 classes of mammalian Glutamate Receptors:– NMDA R (agonist: glutamate, aspartate, NMDA; co-agonist: glycine;

antagonist AP-5; channel blocker: PCP, ketamine), slow EPSP, excitotoxicity, Na+, K+ & highly Ca2+ permeable, blocked by Mg2+, widely distributed

– AMPA R (agonist: glutamate, AMPA, Quisqualate; Antagonist: NBQX, CNQX) fast EPSP, Na+ & K+ permeable but low Ca2+

permeability, widely distributed– Kainate R (agonist: glutatmate, kainate) Fast EPSP, low Ca2+

permeability, limited distribution

• “Use-dependent block”: ketamine & PCP must gain access to the open pore to block the channel