MCDB 126B Midterm 2

Tetanus toxin

inhibits glycine release, muscle rigidity. continual excitatory discharge of the motor and autonomic nervous system. Spores enter via disrupted skin barrier

Direct transmitter action

transmembrane ion channels open/close in response to binding of a ligand

What are nicotinic acetylcholine receptors mainly involved in?

fast synaptic transmission

Do nicotinic acetylcholine receptors generate excitatory or inhibitory postsynaptic responses?

excitatory postsynaptic responses

T/F Nicotinic acetylcholine receptors are cationic channels

True

Where is ACh stored?

vesicular ACh transporter VACht, uses H+ gradient

presynaptic action potential releases ACh into synaptic cleft via

Ca2_ dependent exocytosis

Where is choline taken up into?

cholinergic neurons by choline transporter ChT

Choline + Acetyl CoA form

Acetylcholine

What does Choline Acetyltransferase (ChAT) do?

catalyzes transfer of an acetyl group from the coenzyme acetyl-CoA to choline

What does ACh binding to receptor in post synaptic memebrane cause

opening of AChR channel: Na+ flows into cell, K+ out synaptic cleft - depolarization

What depolarizes the membrane

excitatory postsynaptic potential

What is ACh metabolized into and by?

into choline and free acetate by acetylcholinesterase AChE

Main region of nicotinic ACh receptors in central nervous system

cortex and hippocampus

Alpha latrotoxin

Found in black widow spider venom. Promotes massive release of ACh at neuromuscular junction by binding to presynaptic proteins, causing presynaptic calcium increases that trigger neurotransmitter release

Nicotinic AChR agonists (nicotine)

in CNS cause neural excitation, increase heart rate and BO, causes cardiovascular problems, addicitive

Nicotinic AChR antagonists (alpha-bungarotoxin)

cobra/krait toxin irreversible binding

Goals of smoking cessation theraputics

1. Block alpha4-beta2 nAChRs
2. Nicotine replacement therapy

Block alpha4-beta2 nAChRs

decrease nicotine reinforcement of reward

Varenicline

alpha4-beta2 nAChR partial agonists

Bupropion

Inhibit reuptake of dopamine in CNS reward pathway and weak alpha4-beta2 nAChR antagonist

What does nicotine do?

cross blood-brain barrier to interact with specific nAChRs in brain which stimulates release of dopamine in nucleus accumbens (reward region)

How many agonist are needed to activate nicotinic AChR?

2 agonist molecules (positive cooperativity)

Patch clamp method

removes a tiny circle of plasma membrane and measures the electrical current in one or a few ion channels

What is used to predict transmembrane segments of receptors?

amino acid hydrophobicity

How many amino acids are required for a polypeptide to cross a membrane as an alpha helix?

20

how many subunits make up nAChR?

4 transmembrane domains which are alpha-helices

What are the unifying properties of Cys-loop superfamily

4 hydrophobic transmembrane segments and a conserved disulfide in N-terminal region

What does understanding the structure of nAChRs provide?

- How agonists and antagonists bind
- How nAChRs control ion flow
- How various protein domains affect function
- At the cellular level, interactions with other cellular components
- How to design ligands for therapeutic indications

Methods to determine molecular structure

-X-ray crystallography: 2Å resolution
- Cryo EM
- Molecular modeling

CyroElectron Micrscopy

- Spray acetylcholine on membranes with nAChR as sample is rapidly frozen in

TCurare (Tycocurarine)

nondepolarizing muscle relaxant alpha-bungarotoxin

What provides the molecular basis for cation selectivity of nAChR?

rings of negatively charged amino acids (Glu & Asp)

Conformational Changes in opening and closing of nACh receptor channel

Movement of TM2 transmembrane segment opens the pore like an iris opening, involving rotation/twisting/tilting of TM2 domain

Where is the narrowest part of the nACh receptor channel?

At leucine

Where are the agonist binding pockets on nACh receptor?

At interface between alpha and an adjacent subunit

Describe the binding site of ACh on nAChR

-rich in aromatic residues (Tryp & Tyr)
-pi electron cloud is above aromatic ring

Gly and Pro in nAChR

located between TM2 and TM3. Often seen where there is a bind in an amino side chain

nAChR pores

The M2 helixes from each of the 5 subunits form the pore
• Rings of negatively charged amino acid side chains from M2 line the pore: cation selective channel
• Pore opens like an iris when M2 transmembrane segments move
slightly & reposition the hydrophobic leucines that form the gate

Ligand binding site AChR cyrstal structure shows

- 2 ACh's bind at interfaces of a and adjacent subunits
- ACh binding pocket surrounded by aromatic tyrosine & tryptophans: p electrons stabilize + charge on acetylcholine

How does ligand binding open the pores?

A network of amino acids links the extracellular ligand binding domain to the transmembrane pore domain's M2-M3 linker, and conveys the conformational change of ligand binding into channel opening

Where does GABA occur?

mainly in brain tissue

What does GABA stand for

gamma-aminobutyric acid

GABA functions as an

inhibitory transmitter in many different CNS pathways

GABAa receptors is a

ligand gated Cl- channel that serves as a transmitter in 30-40% of all synapses in the CNS

GABAb receptor is a

GPCR (Gi)

What enzyme is involved in synthesis of GABA

GAD

What is the cofactor for GABA?

Vitamin B6

Stiff Person Syndrome

Antibodies against GAD

Origin of the neuronal membrane potential and synaptic integration

1. Depolarization: positive ion influx and negative ion efflux
2. Hyperpolarization: positive ion efflux and negative ion influx

excitation + inhibition on different branch vs same branch

same branch: only excitation
different branch: excitation and inhibition

Immature neurons

-cotransporter: Na+, K+, Cl- (into cell)
-high [Cl-] inside cells, GABA receptor has depolarizing response

Mature neurons

Cotransporter: K+, Cl- (outside cell)
-Low [Cl-] inside cell, GABAr has hyperpolarizing response

GABAa receptor

-pentamer consisting of combo of 19 subunit
-member of the Cys-loop superfamily

subunit on GABAa receptor

-4 transmembrane segments
-both amino and carboxyl termini extracellular
-extracellular segment form the binding sites for GABA and for allosteric modulators

GABAa receptor phasic inhibition

at synapse, fast transient inhibitory postsynaptic currents due to synaptically released GABA

GABAa receptor tonic inhibition

at extrasynaptic sites, responds to background GABA concentrations

GABA

endogenous agonist

Muscimol

derived from hallucinogenic mushroom, resembles GABA chemically, powerful GABAA receptor agonist

Gaboxadol

synthetic analogue, partial agonist

Biscuculline

naturally occurring convulsant compound; a specific competitive antagonist that blocks fast inhibitory synaptic potential in most CNS synapses

Gabazine

synthetic GABA analogue, competitive antagonist

Picrotoxin

convulsant noncompetitive antagonist, acts by blocking Cl- channel pore of the GABAA receptor, thus blocking the postsynaptic inhibitory effect of GABA

Benzodiazepines, barbiturates, steroid metabolites, alcohol

Allosteric modulators, potentiates GABA action

Where do agonists and competitive antagonists act on GABAa receptor?

at alpha-beta interface

Where do noncompetitive antagonists act on GABAa receptor?

block Cl- channel pore

Where do allosteric modulators act on GABAa receptor?

alpha-gamma interface

neutral allosteric ligand

No change in affinity or efficacy, unaltered signaling

negative allosteric modulator

activity decreases when modulator binds

positive allosteric modulator

activity increases when modulator binds

Competitive antagonists

increase EC50 (shift right), no change in maximum response

noncompetitive antagonist

No change in EC50, decrease maximum response, negative effect on response cannot be overcome by adding more agonist (shift down)

Allosteric affinity modulator

increase potency, decrease EC50 (shift left)

Allosteric efficacy modulator

increase efficacy/max response, EC50 same (shift up)

Benzodiazepine

- Selectively potentiate the effects of GABA on GABAA receptors
- Bind with high affinity to a site on GABAA receptor that is different from the agonist binding site (a - g interface)
- Binding of GABA is facilitated, thereby its agonist effect is enhanced.

Benzodiazepine agonist example

Diazepam, sedative effect

Benzodiazepine antagonist example

flumazenil, Neutral reverses benzo. overdose

Benzodiazepine inverse agonists example

beta-carbolines, proconvulsive effect

Barbiturates

-Pentobarbital, Phenobarbital
- Anesthesia, epilepsy treatment
- Potentiate GABA affinity as well as efficacy

Neurosteroids

allosterically enhance activation of GABAA R, as well as activating at conventional intracellular steroid receptors

IV anesthetic for fast acting induction of anesthesia

propofol, etomidate, potentiate the effect of GABA

Benzodiazepine inverse agonists \____ GABA affinity

decrease (shift right)

Benzodiazepine agonists \____ GABA affinity

increase (shift left)

Benzodiazepine agonists increase GABA response by

increasing the frequency of channel openings (increase affinity)

Barbiturates increase GABA response by

causing channels to stay open longer (increase efficacy)

GABAa receptor is a \___-mer and consist of a combo of \___ subunits

pentamer, 19 subunits

Side effects of benzodiazepine agonists

block memory and addictive

Anxiolytic action of diazepam is selectively mediated by which subunit?

alpha2

Glycine receptors mediate major inhibitory actions in

spinal cord and brainstem

Glycine R agonists, glycine and alanine are released by

inhibitory interneurons involved in spinal reflexes

Glycine receptor structure

Cys-loop family; pentamers with 3α:2β configuration

What are glycine receptors anchored to

postsynaptic cytoskeleton by gephyrin

What does low level of gephyrin cause?

hyperexcitable neuron (epilepsy)

Strychnine

competitive antagonist - causes spinal convulsions

Glycine receptors are essential for

crossed extensor reflect

crossed extensor reflex

flexion of one limb withdrawn from pain stimulus followed by extension of opposite limb for maintaining balance

Stretch reflex

the contraction of a muscle in response to stretch of that muscle

glycine in crossed extensor and stretch reflex

Will inhibit the signal causing muscle to relax

Tetaus cause

- Lockjaw, opisthotonus (arched back), muscle rigidity/spasms, suffocation
- Respiratory or cardiac arrest and severe BP changes due to uncontrollable muscle spasms

How does tetanus toxin work?

-Retrogradely transported through the axons of the periphery neurons and reach the inhibitory interneurons in the spinal cord
-Toxin cleaves the proteins (synaptobrevin) involved in vesicle fusion, thereby blocking neurotransmitter release (glycine)