Unit 3

Synapsin

“Molecular Cowboy”

• Sequesters/tethers vesicles in the reserve pool

• Mechanism: binds to the vesicle and actin cytoskeleton

RIM

“Rab3 Interacting Molecule”

• Scaffolding

  • Mechanism: binds to P/Q and N-Type voltage gated calcium channels and Rab3

RIM-BP

“Rab3 Interacting Molecule Binding Protein”

• Recruits calcium channels for the active zone

• Mechanism: binds to RIM and calcium channels

MUNC-13

The savior of syntaxin.

• Exists in either an inactive-homodimer conformation or active conformation when bound to RIM.

• The MUN domain helps kick MUNC-18 off of syntaxin, freeing the Habc domain and allowing syntaxin to become its active conformation.

Synaptotagmin

Calcium sensor

• Couples with calcium to then interact with complexin and allow the trans-SNARE domain become the cis-SNARE domain.

• C2 domains allow calcium binding (5 total sites)

Components of SNARE complex

1. Synaptobrevin (vesicular-SNARE)

2. Syntaxin (target-SNARE)

3. SNAP-25 (target-SNARE)

How is SNAP-25 associated with the plasma membrane?

Via lipid side chain interactions with the plasma membrane.

NSF

SNARE Complex disassembler; utilizes the energy of ATP hydrolysis plus adaptor proteins to take apart the complex.

Kiss and Run Model of Fusion

Phenomenon where some vesicles will not fully fuse with the membrane and instead form a transient pore which closes immediately following neurotransmitter release.

Clathrin

• Associated with receptor mediated endocytosis (recycling process)

• Honeycomb lattice structure

Clathrin Formation

1. Coat assembly and cargo selection

2. Bud formation

3. Vesicle formation

4. Uncoating

Triskelion

Unique structure of the honeycomb lattice

• Contains one heavy chain and one light chain

• Flexible

• Concave

• Connected to the membrane via adaptins

Endosomes

• Sorting center

• Allocates proteins for reloading, retrograde transport, etc.

Ba or Sr Release

Both barium and strontium CAN cause neurotransmitter release, however it is desynchronized and therefore does not produce a response.

Model Synapses

• Calyx of Held (in the brainstem)

• Giant squid synapse

• Neuromuscular junction

Neuromuscular Junction (NMJ)

• The junction between the nervous ad skeletomuscular systems.

• The most commonly used synapse in experimentation.

• Highly excitable

Motor End Plate

Where a nerve makes contact with muscle.

Junctional Fold

Invaginations beneath the presynaptic release sites. Increases the surface area of the postsynaptic cell which allows for a greater density of ion channels and receptors.

Basal Lamina

A structure in the synaptic cleft which holds enzymes that degrade acetylcholine (acetylcholinesterase) plus other molecules.

T-Tubule System

Network of invaginations in the muscle to help distribute action potentials everywhere.

Excitation-Contraction Coupling

1. Depolarization activates L-Type Ca2+ channels.

2. The L-Type Ca2+ channels are physically coupled to RyR (ryodine receptors, calcium channels on the smooth endoplasmic reticulum membrane).

3. The RyR opens

4. Calcium ions flow in and interact with the sarcomere.

Tubocurarine

Toxin used to keep the neuromuscular junction response below threshold which allows experimenters to measure the end plate potential.

End Plate Potential (EPP)

• Dependent on time constant and how far away from the NMJ

• Dependent on extracellular calcium concentration

k[CaX]^4

The theory that the binding of calcium to some unknown thing (X) to complete the complex.

• Depends on multiple complexes

Calcium Conductance

• Peaks during the afterhyperpolarization phase

• Rises slowly

Family Tree of Calcium Channels

• Voltage gated calcium channels (VGCCs) split into high voltage activated and low voltage activated channels.

• Low voltage channels are T-Type channels.

• High voltage activated channels split into L-Type or P/Q, N, or R-Type channels

ω Agatoxin

Binds to P/Q-Type calcium channels (Cav2.1)

ω Conotoxin

Binds to N-Type calcium channels

CD2

Toxin that blocks ALL calcium channels.

Most common calcium channel type?

P/Q-Type

Subunits of VGCCs

1. α1

2. α2

3. δ

4. γ

5. β

Transmembrane Subunits of VGCCs

1. α1

2. β

3. δ

4. γ

Intracellular Subunits of VGCCs

β

Extracellular Subunits of VGCCs

α2

Quantum Hypothesis

Bernard Katz; theory that postulated neurotransmitters were released in discrete packets called quanta.

• This theory was confirmed by electron microscopy

Prostigamine

A toxin that blocks acetylcholinesterase, thereby increasing the available acetylcholine in the synaptic cleft.

• Is used experimentally to look at the decay of minis.

How are mEPPs distributed and what does that mean?

Exponentially, that means they are random events. They require no stimulation and occur spontaneously.

What does changing the Ca/Mg Ratio Affect?

When lowers, it allows minis to be measured.

EPPs are _______ multiples of mEPPs

Integer

Residual Calcium Hypothesis

Katz proposed that calcium from an initial pulse may remain in the presynaptic terminal for some time even after the stimulus has diminished and the calcium of a second pulse will sum with the leftover calcium.

Quantal Analysis Assumptions

1. Enough receptors on the postsynaptic cell

2. Lots of release sites

3. Neurotransmitters are released in quanta

4. Independent probability of release

n

Quantal release sites

• Neuromuscular junctions have lots

• Central synapses have few

Pr

Release probability

• Independent factor

• Low when cell is at rest

• High when calcium concentration increases

Evoked Response

Occurs when the calcium concentration increases.

Spontaneous Release

Occurs when the release probability is low, random event.

m

Quantal content

Quantal Content Equation

m=n*Pr

q

Quantal size

• Defined as the postsynaptic response to the release of quanta

• Dependent on the number of receptors and the number of neurotransmitters released (these are two separate factors but they are very difficult to differentiate)

Postsynaptic Potential Equation

PSP=q*n*Pr or m*q

Short Term Plasticity

Two stimuli delivered in rapid succession.

Different Types of Vesicle Pools and % Breakdown:

1. Reserve, 80-90%

2. Recycle, 10-15%

3. Readily releasable, 1%

Presynaptic Depression

Occurs when the readily releasable pool is depleted and there is not sufficient time to replenish it.

Glutamate Reuptake and Recycle Steps:

1. Glutamate is taken up from the synaptic cleft by astrocytes.

2. In astrocytes, glutamate is transformed into glutamine by glutamine synthetase.

3. Glutamine is transported BACK into the presynaptic cell.

4. In the presynaptic cell, glutamine is transformed back into glutamate by glutaminase.

Glutamate Receptor Family Tree

NMDA, AMPA, and Kinate.

AMPA Receptors Family Tree

GluR1, GluR2, GluR3, GluR4

• Differ in their biophysical properties.

NMDA Receptors Family Tree

NR1, NR2, NR3

• NR2 splits into NR2A, NR2B, NR2C, NR2D

Defining Features (topology) of AMPA Receptors:

• Tetrameric

  • Can homotetramerize by incorporating different GluR subunits

• Extracellular N-Terminus

• Intracellular C-Terminus

• Amino binding domain (NBD)

• Ligand binding domain (LBD)

• Transmembrane domain (TMD)

‘Line’ Structure of AMPA Receptor

N-Terminus ~ NBD ~ S1 ~ M1-3 ~ S2 ~ M4 ~ CBD ~ C-Terminus

• S1 and S2 make up the ligand binding domain (LBD)

• M1-3 and M4 make up the transmembrane domain (TMD)

AMPA I-V Plot

• Reversal potential = 0 mV

  • Monovalent cation selectivity (K+ and Na+)

• Slope = 8 pS

How does an AMPA receptor open?

1. In its closed conformation, the M3 helix occludes the pore.

2. When the ligand binding domain clamps down on glutamate, its linker rotates

3. This motion tugs on the M3 helix, causing it to open

Important structures of NMDA receptor?

• Tetrameric

  • Two NR1 subunits and two NR2 subunits

• Glycine is a co-agonist (required).

• Mg can bind and block NMDA receptors and is only displaced by depolarization.

NMDA I-V Plot(s)

• Reversal potential = 0 mV

  • Monovalent cation selectivity (K+, Na+, and Ca2+)

• Slope w/ NO extracellular Mg = 20-50 pS

• Slope w/ extracellular Mg = exponential

Defining difference between an EPSP and IPSP

The relationship between the reversal potential and threshold of the postsynaptic current.

• Reversal potential < Threshold = IPSP

• Reversal potential > Threshold = EPSP

EPSC of a Glutamatergic Cell

Defined by both the postsynaptic current of the AMPA and NMDA receptors.

• AMPA is the fast component

• NMDA is the slow component (voltage dependent)

Three Layers of Proteins in the Post Synaptic Density (PSD)

1. Neurotransmitter receptors

2. Primary scaffolds

3. Secondary scaffolds

Primary Scaffolding Protein for PSD

PSD-95

• Defined by the binding of PSD-95 to the C-Terminus of NR2

GABA Synthesis

Glutamic acid is transformed into γ-amino-butyric-acid (GABA) by glutamic acid decarboxylase (GAD).

• Synthesized in the presynaptic terminal

Where is GAD synthesized?

In the soma.

Two Types of GAD

67 and 65

• 67 is the main one

• 65 boosts GABA synthesis when needed

vGAT

Vesicular GABA transporter

• Utilizes the v-ATPase to pump GABA into a vesicle (remember v-ATPase uses the energy from ATP hydrolysis to pump hydrogen ions into the vesicle)

GABA Recycling

GABA in the synaptic cleft is taken BACK to the presynaptic terminal by either astrocytes or GABA transporters (GAT).

• It’s then converted into glutamate by GABA transaminase which produces glutamine.

What current mediates the IPSC?

Chloride

GABA Topology:

• Pentameric: two α₁ subunits, β₂ subunits, and δ or γ subunits

• Contains both a ligand binding domain (cys loop) and transmembrane domain (M1-4)

• Extracellular N and C Termini

Importance of M2 in GABA

Has residues for chloride selectivity; makes up the pore interface.

Where does GABA bind?

Between the N-Terminal ligand binding domain and a subunit.

How interneurons are differentiated?

1. Calcium protein binding domains (CB, CR, and PV)

2. Shape (Basket, chandelier, …)

3. Synapse region

How is the PSD of GABAergic cells and glutamatergic cells different?

Glutamatergic cells postsynaptic densities are asymmetrical while GABAergic cells postsynaptic densities are symmetrical and therefore have pleomorphic synaptic vesicles.

How do benzodiazepines work?

They increase the frequency of GABA binding.

GABA I-V Plot

• Reversal potential = 0 mV

• Slope = 30 pS

• Chloride selective