Mariq Flashcards
Anatomy of AchR
4 transmembrane regions / subunit
2 alpha
1 beta
1 gamma
1 delta
Acetylcholine History
Nerve stimulation vs Muscle
* Stimulate motor nerve – Muscles
did Notcontract
* Stimulate muscle - Contraction
* Is it a general anesthetic? Motor
disruption – Sensation intact!
* Limit curare to nerve – Muscle did
contract
* Expose muscle to curare – Muscle
did Notcontract
* Cannulate vasculature to NMJ –
Muscles did Notcontract
Evidence for chemical synaptic transmission, 1921 (Otto Loewi)
physostigmine
a reversible cholinesterase inhibitor.
Why Chemical Neurotransmission?
*Sign Inversion, integration and modulation
*Chemical NT allows for amplification!
*Muscle is big cell (150 μm diameter)
*Nerve terminal tiny (1.5 μm)
*NMJ: AP in nerve –> AP in muscle
*Ohm’s Law: V = IR
*How much current needed to depolarize muscle?
*How much current can be supplied by the neuron
if purely electrical communication?
Estimating Receptor Properties
- Consider a channel that opens
and closes - I = Nipo ; I = mean current, N = #
of channels, i = current through
single channel, po= probability
of channel being open - Var = Ii(1- po); Var = variance
- Plot Variance against Mean
Conductivity of AchR
28-32 pS
Ways to separate proteins
Size exclusion chromatography (mass)
ion exchange chromatography (charge)
affinity chromatography (binding)
which subunit of AchR controls gating?
delta
which subunit of AchR is required for binding to alpha-bungalotoxin?
alpha
which region of AchR controls conductance?
M2
Isolation and Identification of
Proteins
- Need Tissues or Cells that Express Protein
- Need Method to Separate Complex Mixture
of Proteins by Physical Characteristics - Need Assay to Detect Protein (Purification)
– Biochemical association
– Physiological function
what ions does AChR conduct?
Na, K
What charateristic of AChR may change conductance?
(3) Ring(s) of charge around pore
Organization of AChR Channel Pore
*Chlorpromazine (CPZ) is channel
blocker
*UV flash causes reaction with protein
side chains
*Incubate AChR vesicles with [3H]-CPZ
and agonist. UV flash.
*Label found in every subunit
*All labeled amino acids in M2, at
relative positions 2’, 6’, 9’
Mutations in Pore Affect Blockers,
Desensitization, Conductance and Affinity
9’L: Part of Symmetrical Gate
a7/5HT3 Chimera: Ligand Binding
Ligand-gated Ion Channels
- Multimeric integral membrane glycoproteins
- Signal in two ways:
* Change in membrane voltage - most
common
* Ca2+ entry - critical for synaptic plasticity - Gating typically rapid (μsecs after ligand
binding) - Ion Selectivity:
* Cationic, often non-selective (Na+, K+, Ca2+)
* Cl- (GABA and Glycine receptors) - Subject to modulation in a variety of ways
AChR alpha7 ligand
ACh
AChR 5HT ligand
serotonin
GABA receptors
I.g-amino-butyric acid (GABA) is the principle inhibitory
transmitter in the brain.
A. GABA receptors are cys-loop receptors; thus, they are
pentamers – at least four different types of subunits.
B. 6 asubunits, 3 bsubunits, 3 gsubunits, d, e, p, and rsubunits.
C. In general, a, b, and gsubunits are all required for receptor
function.
D. Only a limited number of subunit combinations exist.
Glycine receptors
Glycine (Gly) is the predominant inhibitory transmitter in
the spinal cord.
A. Glycine receptors are pentamers of two different types of
subunits.
B. 4 asubunits, and 1 bsubunit
C. Stoichiometry is 3a:2b
GABA Receptor Subunit Composition
Two GABA Binding Sites at
a-bInterfaces
Benzodiazepine Site at
a-gInterface
How does diazapram effect GABA current
increases probability of channel opening
how does barbituate effect GABA current
increases length of time channel is open
Difference between cationic channels (ACh/Serotonin) and chloride channels (GABA/Glycine)?
cationic channels have acidic residues @ most intracellular spot, chloride channels have basic residues at that location
Cys-loop receptors vs. Glutamate
Receptors
criticisms of glutamate as NT
- Too abundant–10 mM soluble constituent of brain
- Involved in many biochemical/metabolic pathways
- Broad action: depolarized essentially all neurons
- No known mechanisms of activation or inactivation
Classification of iGluRs
*NMDA - N-methyl-D-aspartate
– Competitive antagonist: AP5,
APV
– Pore blocker: MK-801
*AMPA
– Competitive antagonist: CNQX
*Kainate
– Competitive antagonist:
LY382884
AMPA response is….
fast, short
NMDA response is…
slow, long
Structure of iGluRs
iGluRs are Tetramers
AMPARs are typically heteromers, but
can function as homomers
NMDARs are obligate heteromers, and
must include the GluN1 subunit
iGluR Relevance to Neurological
Disorders
*Excitotoxicity/Stroke/Ischemia
*Epilepsy
*Neurodegenerative Disorders
*Psychiatric Disorders
Receptor Cloning Strategies(Functional Expression)
`* Find tissue that expresses receptor
* Make total RNA
* Inject into Xenopus oocyte
* If receptor activity detected, make
cDNA library
* Make pools of cRNA
* Test in oocytes
* Fractionate pools, retest…
* Isolate single clone…
* Note: strategy dependent on
receptor that is single protein or
homo-oligomer
What is the topology of iGlurs?
- iGluRs ~ 1000 a.a.
- No Cys-Cys loop
- C-terminus phosphorylated
- RNA editing
- Alternative splicing: flip/flop region
proximal to TM region - Loop 3 most conserved
- Little sequence identity with AChRs,
etc. - Regions of sequence identity with
bacterial periplasmic glutamaine
binding proteins
________ Studies Reveal
Topology of iGluRs
Glycosylation
GluR3’s ligand
AMPA
GluR6’s ligand
Kainate
Where does glutamate bind?
S1/S2 regions
Topology of Ion Channels (AChR, iGluR, K)
Gating and Stoichiometry of iGluRs
Topology of glutamate receptors
NMDARs’ general properties
- voltage dependent
- permeant to Ca2+
- can function as coincidence detector
- gated by Mg2+
- Ligands
– Need 2 molecules of glutamate & glycine
– Glycine (or D-serine) is required co-agonist - Ionic permeability
– Channel permeable to Na+, K+, and Ca2+ - Voltage sensitivity/Mg2+block
– At resting membrane potential, channel blocked by
Mg2+ion.
– As neuron becomes depolarized, Mg2+block is
removed.
– Region of “negative slope conductance”
– “Coincidence detector” - Time course of NMDA currents
– Slower and more prolonged than AMPA/Kainate
receptor-mediated currents
_____ is a Co-Agonist for NMDARs
Glycine
Stargazin Protein
Founding Member of TARP
Family of Transmembrane
AMPA Receptor Regulatory
Proteins
3 roles:
* Obligate Chaperone
* Receptor Localization
* Receptor Function
