Ligand Gated Channels and NeuroTransmission: Module 2.6-2.7

Question 1

What are the three families of ligand-gated channels (ionotropics)?

Answer 1

1. Cys-loop, Pentamers (Multiple Subunits)
2. Tetramers (2 Subunits)
3. Trimers

There is one class of Channels missing in this image

Question 2

How many members are part of the cys-loop family of receptors?

Answer 2

4

All of them are pentrameric and ionotropic have extracellular cys-loop,

Question 3

What are the cationic members of the cysloop family?

Answer 3

1. Acetylcholine Nicotinic Receptor (nAChR)
2. Serotonin (5-HT3 Receptor)

Question 4

What are the structural characteristics of the Cys Loop Family?

Answer 4

Characteristic Loop formed by disulfide bond between 2 cysteine residues 13 amino acids apart near the N-terminal domain of the alpha subunit.

M2 is where the selectivity filter is.

Question 5

What are the anionic members of the cysloop family?

Answer 5

1. Glycine (GlyR)
2. GABA (GABAa Receptor)

Question 6

What type of receptor are nicotinic acetylcholine receptors; what agonist are they responsive to and what is its type?

Answer 6

Ionotropic

Acetylcholine

Endogenous Agonist

Endogenous = Naturally Occuring substance within the body.

Question 7

nAChRs got nicotinic name due to response to nicotine

What type of agonist is nicotine?

Answer 7

Exogenous Agonist

Question 8

Who discovered nAChr?

Answer 8

• 1914: muscle receptor by Henry H. Dale and Otto Loewi
• 1986: nicotinic ACh receptors were found in neuron

Question 9

Explain the structure of nAChRs in muscle.

Answer 9

Each Subunit -> 4 Transmembrane Domains Need 5 Subunits for functional Receptor alpha subunit = ligand binding domain M2 segments form Pore Walls M1-M2 and M3-M4 Linkers (Strings things between subunits) are sites of modulation. (Phosphorylation, Oxidation, Ca Binding) Positive Charges Can go through.

Question 10

Explain the nAChR composition and distribution.

Answer 10

Subfamilies 1 and 2 -> 5 Alpha Subunits - A7 Found in CNS (Limbic/Cortex) - Fast Desensitization Subfamily 3 -> 2 alpha and 3 Beta subunits - [A3B4 -> Found in PNS - Slow Densitizaiton][A4B2 Found in CNS (Thalanus/Cortex) - Fast Desensitization] SubFamily 4 -> 2 alpha1, 1 beta1, 1 Gamma or Epsilon, 1 delta - Found in Skeletal Muscle

Question 11

What is a-Bungarotoxin(a-BTX or a-BgT)?

Answer 11

Snake Venom - Neurotoxin

Competitive Irreversible Antagonist for nAchRs (muscle a1, a7, a9) at the Neromuscular junction.

Causes paralysis, respitory failure and death.

Question 12

What does the IV plot for a Neuronal nAChR (2 alpha, 3 beta) look like and what causes this?

Answer 12

Inward Rectification

Due to Polyamine-mediated Block: Polyamine has alot of positive charges - depolarized memebrane potentials and the ring of negative cysteine residues pushes/attracts polyamine outside the cell through nAChRs however these pores are too small and cause a block.

At negative potentials polyamine is released and channel is unblocked

Question 13

Why do muscle nAchR show ohmic behavior?

Answer 13

Receptors are much larger and polyamine can go through causing receptors to not become plugged.

Question 14

When nAChRs are bound by. Ach

What ions does it let pass?

Answer 14

It allows the non selective flow of all cations.

Causes flow of Na, K, Ca; sometimes show permeability for a certain ion.

Question 15

What experiment can be used to find nAchR reversal potential and what is it?

Answer 15

0 mV

Voltage Clamp of Post-Synaptic Cell

Question 16

What are the adult and fetal subunits of nAChRs and what is their difference in conductance and open probability?

Answer 16

Adult Subunit = Epsilon - Higher Conductance and Less open Probability.

Fetal Subunit = Gamma - Less Conductance and More Open Probability.

Though to reflect different roles in synaptic transmission and synapse formation for developement .

Question 17

,

What is a-BTX used to study?

Answer 17

Used to study nACh funciton and cell surface Expression.

Green: Motor nerve labelled with antibodies against neurofilament Red-Orange: Tetramethylrhodamine conjugate of a-BTX

Question 18

What is Curare (d-tubocuranine)?

Answer 18

Curare is a general term for a group of plant-derived alkaloid toxins traditionally used as arrow poisons by Indigenous peoples in South America. These toxins are known for their ability to cause muscle paralysis by interfering with the transmission of nerve impulses to muscles.

Competitive Reversible Antagonist for nAChRs at the neuromuscular junction.

Question 19

What neurotransmitters mediate alot of the synaptic inhibition in th CNS?

Answer 19

GABA = Majority

Glycine = Rest

Question 20

GABA type A and Glycine Receptors are ionotropic

What ion do they select for?

Answer 20

Cl-

Question 21

Why is synaptic inhibiton tightly regulated?

Answer 21

Too Much Inhibition: Loss of Consciousness and Coma

Too Little Inhibition: Siezure Activity

Question 22

What are 2 significant modulators of GABA type A and what is their effect?

Answer 22

Benzodiazepines (Tranquilizer Valium): Increases Frequency of channel opening aswell as can increase Cl conductance.

Bariturates (sedative phenobarbital, pentobarbital): Increase the duration of channel opening.

Each bind to own specific site on the receptor.

Question 23

What are some potential natural modulators of GABA type A receptor?

Answer 23

Metabolites of these steroid hormones: Progesterone, Corticosterone, Testosterone.

Question 24

Expain how GABA responses change in the CNS as we develop.

Answer 24

Immature CNS neurons: Express higher levels of NKCC1(Brings Cl inside) receptors than KCC2 (Brings Cl outside) leading to a **high intracellular Cl **concentration. When GABA channels open in this case -> Cl leaves the cell causing a depolarizing effect.

Mature CNS neurons: Express higher levels of KCC2 than NKCC1 leading to low intracellular Cl. When GABA channels open -> Cl influx which causes a hyperpolarizing effect.

Nerst Equation for Cl changes.

Thought to occur during Embryonic Developement - Need depolarization to form new synapses and switches to prevent siezure activity in adults.

In Embryo GABA is Excitatory NT

Question 25

What is the perforated patch configuration?

Answer 25

Modified version of whole-cell recording with little pores instead of big hole. Holes created by pore-forming antibiotics. Pores allow flow of small ions and prevent passing of larger ions and molecules.

Congifuration is still whole cell as you create an eletrical connection between the pipette and cytoplasm

Question 26

What are some different antibiotics used in the perforated patch technique and what are their functions?

Answer 26

Nystatin and Amphotericin-B: Allows Cation to pass but Preserves Intracellular Ca

Gramicidin: Allows everything through but Preserves intracellular Cl

ATP is also preserved.

Question 27

What are the advantages and disadvantages of the perforated patch technique?

Answer 27

Advantages
* Maintains integrity of Cytoplasmic Components including soluble second mesengers.
* Prevents channel “run-down”
* It reduces dialysis of cells

Disadvantages
* Higher access resistance, which increases noise and reduces signal
* Time
* The membrane perforated by the antibiotic is prone to rupture.

Question 28

Why do perforated currents have a smaller amplitude and why does the reversal potential shift to 0mV in whole cell.

Answer 28

1. Perforated currents have a smaller amplitude due to resistance at the tip of the pipette. The whole cell membrane break reduces this resistance.
2. Experimental Conditions use a symmetrical solution of pipette to bath, so that after you are done the perforated reversal potential reading; you can break the membrane to whole cell and make sure the reversal potential is 0mV meaning you got an accurate perforated reading of reversal potential(this would be the naturally occuring reversal potential).

Question 29

What is the main excitatory NT of the CNS?

Answer 29

Glutamate

Question 30

What are the 3 classes of ionotropic glutamate receptors?

Answer 30

AMPA, NMDA and Kainate

Question 31

Explain glutamate receptor structure?

Answer 31

Tetramer (4 sub-units), heteromultimers.

Heteromultimers are protein complexes made up of two or more different types of polypeptide chains

Question 32

What ions to ionotropic glutumate receptors let through?

Answer 32

They are Cation Non-Selective

Question 33

What are the different glutumate receptors gated or blocked by?

Answer 33

Question 34

What are the Antagonists of the different classes of ionotropic glutumate receptors?

Answer 34

Question 35

Explain the Mg Block of NMDA receptors.

Answer 35

At the resting membrane potential (−65 mV) extracellular Mg2+ binds tightly to a site in the pore of the channel, blocking ionic current. But when the membrane is depolarized (for example, by the opening of AMPA receptor channels), Mg2+ is expelled from the channel by electrostatic repulsion, allowing Na+ and Ca2+ to enter.

Important Figures.

In the presence of MG -> IV Plot is shaped as an outward rectifier.

Question 36

What glutamatergic synapses are open at negative values of postsynaptic Vm?

Answer 36

AMPA Channels can be activated by glutumate depolarizing the cell.

NMDA receptors have a Mg block at these potentials.

Question 37

What glutamatergic synapses are open at more positive values of postsynaptic membrane potential?

Answer 37

AMPA and NMDA

Mg detaches from NMDA

Question 38

What ions are AMPA channels most permeable too?

Answer 38

Na and K

Question 39

What ions are NMDA channels most permeable too?

Answer 39

Ca

Question 40

NMDA channels are ___ ___and ____ ____.

Answer 40

NMDA channels are ligand gated and voltage dependant.