Na+,Choline - symporter. - dependent on Na and Cl. - normal [Choline] plasma ---> 10mM - km > 10mm so in normal tissues LOW affinity - This transport, Km is 1-5mM. THUS HIGH AFFINITY

Done by ---> Choline Acetyltransferase. - cytoplasmic - NOT rate limiting - used for immunohistochemical mapping of cholinergic neurons.

Cholinergic Neurotransmission Flashcards by Naufel Atia

Where do we find Cholinergic Neurons?

Neuromuscular junction
Autonomic preganglionic Neurons
Parasympathetic postganglionic fibres (heart,glands,smooth muscle)
CNS

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Steps in Cholinergic Nerve Axon Terminal

High affinity uptake of Choline
ACh synthesis
Uptake of ACh into vesicle (storage)
Exocytosis of ACh
ACh inactivation

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High Affinity uptake

Na+,Choline - symporter.

dependent on Na and Cl.
normal [Choline] plasma —> 10mM
km > 10mm so in normal tissues LOW affinity
This transport, Km is 1-5mM. THUS HIGH AFFINITY

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What is the Choline uptaker inhibited by?

-Hemicholinium

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synthesis of ACh

Done by —> Choline Acetyltransferase.

cytoplasmic
NOT rate limiting
used for immunohistochemical mapping of cholinergic neurons.

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Uptake of ACh into vesicles?

Done by —> Vesicular ACh Transporter (VAChT)

-
-H+,ACh antiporter.

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What is VAChT inhibitted by?

-Vesamicol

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What triggers the exocytosis of ACh vesicles?

AP -> Depolarisation of Neurons -> Ca2+ entry via VDCC –> exocytosis of vesicles.

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Steps in Exocytosis of Synaptic Vesicles

uptake of neurotransmitter into vesicle.
formation of reserve pool - clumping of vesicles.
Docking of vesicles onto active zone.
Priming - vesicle becomes competent for Ca++ signal.
Ca++ signal - fusion pore opens.
Vesicle recyling:
- kiss and stay : ->refilling w/o undocking
- Kiss and run : ->vesicle releases NT and returns
- full endocytosis

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Docking

SNARE and Synaptotagmin not involved.

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Priming

SNARE complex forms on vesicle.

Synaptotagmin become associated with complex.

Vesicle and plasma are in close proximity - forming unstable intermediate.

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Fusion

Ca++ signal -> Synaptotagmin C2 domain inserts into phospholipid membrane.

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What are SNARE’s receptors for?

NSF (N-ethylmaleide Sensitive Factor) and NSF soluble factors.

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SNARE in Synaptic Vesicle

VAMP (vesicular Associated membrane protein)

- Synaptobrevin

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SNARE in plasma membrane

Syntaxin 1 A/B

- SNAP-25 (synaptosomal-associated protein)

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Energy for fusion of vesicles

-Done by the sponatenous formation of a-helices in the cytosol by SNARE motiffs.

-These a-helices become supercoiled and parallel coiled structures.
This provides the energy

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How many AA in SNARE proteins

70 AA homolog sequence (SNARE motiff)

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Fusion-core complex:

parallel helices are formed with the SNARE sequences that span into the cytosol (four sequences from three proteins).

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What is the Ca++ sensor?

Synaptotagmin!

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Regulatory proteins involved in Exocytosis

Munc18-1
Complexis
Synaptophysins (vesicular)
Synapsins (vesicular)

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v-snares

Synaptobrevin

- VAMP

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T-snare

Syntaxin 1 A/B

- SNAP-25

formation of fusion pore after Ca2+ signal

T-snare + V-snare bind.
They begin to zip from the N-terminal. Bringing membranes close to one another.

2.Zipping causes ->lateral tension on PM
curving of PM
hemifusion of the outer layers

Hemifusion of the inner layers
complete fusion causes pore complex
contents emptied out

Structure of Botulinum Toxins

Heavy Chain and Light chain

How is Botulinum toxins taken up into the Cholinergic Neurons

-Acceptor mediated Endocytosis.

How does Botulinum affect Cholinergic Neurons

-Inhibits Exocytosis with LC (Catalytic domain). - -> Zn2+ - dependent endproteases - -> has specificity for sites in the SNARE Protein

affect of Botulinum Toxins on SNARE complex stability

Botulinum either increases or decreases the stability of SNARE.

Types of Cholinergic Receptors

1) Muscarinic | 2) Nictonic

Where do we find Muscuranic receptors

Organs innervated by: - > Parasympathetic postganglionic - > CNS

Where do we find Nicotonic receptors?

Neuromuscular junction

Inhibitor of Muscuranic receptor

Atropine | is a competitive inhibitor

Inhibitor of Nictonic receptor

Crurae

Molecular mechanism of Muscuranic receptors?

1. PLC activation 2. [Ca]i Increase 3) inhibition of Adenyl Cyclase 4) Activation of K+ channels

Molecular mechanism of Nictonic receptors

Na+ and K+ channels

M1 receptor

- 7 transmembrane protein - Gq coupled -PLC ---> PIP2 Hydrolysis --> IP3 ---> Ca signal

M2 receptor location

M2 receptors are found in the heart

Which G-protein is M2 receptors coupled with?

Gi - K+ channel activation---> HYPERPOLARIZATION --> BRADYCARDIA - inhbition of cAMP - inhibition of Ca++ signal

M3 receptor location

Gastro-intestinal tract

M3 receptor coupled G-protein

M3 receptor effects

- Smooth muscle contraction | - secretion stimulation

Muscle Type Nictonic Receptor

- In striatal muscle - has Na+ and K+ permeability. - causes membrane depolarisation which leads to Ca++ release from SR.

What is muscle type nictonic receptor inhibitted by?

D-Tubocuranine (competitive inhinb)

How many subunits in Nictonic receptors

5. - A x 2 (binding site of ACh) - B - D - G

Inhibitors of Nictonic receptors

- >>>D-tubocruranine: - comp inhibitor ->>>a-bungarotoxin: - from a indian - SPECIFIC for muscular Nictonic receptors

Events following the stimulation of nicotinic receptors in skeletal muscle

1. Neurotransmitter released diffuses across the synapse and binds to the Nictonic receptors on the sarcolemmma. 2. Causes depolarisation and AP propagates down the T-tubules. 3. Causes release of Ca++ from cisternae of SR 4. Ca++ binds to Trop-C causes change of shape, which pulls Tropomyosin off the actin. etc etc

What is Malignant Hyperthermia

-rare complication during inhalation of anaesthia due to genetic defect in ryanodine receptors.

What happens in Malignant Hyperthermia

1. Mutated Ryanodine receptor. 2. Altered kinetics of Ca++ channels. 3. Persistent large increase in [Ca++]i causes: - --> HYPER METABOLISM (fever) - --> Persistent activation of the muscle (muscle ridgity)

Acetylcholine Esterase - facts

- Located in the synapse. - Specific for ACh - It is a serine protease

Mechanism of Acetylcholine esterase

1. ACh binds to active site of enzyme. 2. Choline released and we have Acetyl-Enzyme complex. 3. Hydrolysis of the complex to form -> ACETATE , H+ and Acetylcholine Esterase.

Pseudocholinesterase

- found in Plasma and Liver. - function is UNKNOWN BUT we think it involved in: ->hydrolysis of other esters (acting as short-term muscle relaxant).

effects of: --> suxamethonium

- It is a Nic ACh receptor agonist. | - causes muscle relaxation and temporary paralysis.

Reversible inhibitors of Acetylcholine esterase

- have a pharmalogical use. | examples: 1) Physostigmin 2) Neostigmin

Disease which is treated with reverisble acetylcholine esterase

- Myasthenia Gravis - we use short lasting drug to test. - physostigmin and neostigmin are long loasting. - eye drops used for glaucoma.