Structure and Function nACh receptor

Question 1

how can the different modes of the patch-clamp technique can be
exploited to study various aspects of receptor/ion channel function?

Answer 1

This technique allows resolution at real time single receptors going through the process of opening and closing.

A pipette was used to suck at high pressure, to form a high resistance seal (Gigahom seal) which electrically and mechanically tight.

Different paths can be taken; can put whole cell with open pore into salt solution and record receptor population

Can snap the receptor so its upside down and measure its activity by changing the concentration at the back.

Can pull electrode, pipette away from cell so it snaps off, giving access to an intracellular membrane.

Question 2

What are the distinct gene families of transmitter-gated

ion channel subunits.

Answer 2

CYS-loop (pentamer)

Gluatmate (tetramer)

P2X (trimer)

Question 3

What has cloning of receptor subunits led to?

Answer 3

The discovery of numerous receptor isoforms, which exhibit distinct
physiological properties showing they belong to the Cys-loop transmitter-gated ion channel family.

Multiple subunits means targets for developing novel therapeutics.

Question 4

How fast can ions travel?

Answer 4

Ions flow selectively at a typical rate of 10,000,000 (10million) ions per second!!

Question 5

What kind of receptor is nAChR?

Answer 5

CATION-CONDUCTING (non selective to positive substrates)

Cation non-selective channel: usually primarily conducting
Na+ Ca2+ in to the cell & K+ out of the cell.

Question 6

What are the different modes of the patch- clamp technique?

Answer 6

1) Cell attached patch
2) Whole cell voltage clamp (removed)
3) Outside-out patch (reversed)
4) Inside- out patch (removed but not reversed)

Question 7

What are the pros and cons of the Cell attached patch?

Answer 7

Advantages:

1) The intracellular milieu is maintained (no biochemical wash-out).
2) Control of the extracellular solution.

Disadvantages:

1) The agonist always present, ACh cannot be washed off and so receptor desensitization.
2) No control of the intracellular content.

Question 8

What are the pros and cons of the Whole cell voltage patch?

Answer 8

Advantages:

1) Control of the intracellular & extracellular mileu.
2) Can record from a population of receptors/ion channels.
3) Can introduce biochemical modulators intracellularly via the pipette.

Disadvantages:

1) Biochemical “washout” of the intracellular mileu.
2) POOR Resolution- can rarely observe SINGLLE receptor activity

Question 9

What are the pros and cons of the Outside-out patch?

Answer 9

Advantages:

1) Control of the intracellular & extracellular mileu.
2) Can change/control extracellular drug (agonist) concentrations.

Disadvantages:

1) Biochemical “washout” of the intracellular mileu.
2) Cannot change intracellular messengers eg. c-AMP

Question 10

What are the pros and cons of the Inside-out patch?

Answer 10

Advantages:

1) Control of the intracellular & extracellular mileu.
2) Can introduce biochemical modulators eg. c-AMP to the intracellular face.

Disadvantages:
1) CANNOT change the concentration of agonist

Question 11

Explain in what way the nAChR isopening is complex.

Answer 11

It binds to TWO ligands; when it is just bound o one ligand it only opens briefly, however when it binds to two it is open for longer.

Double binding makes opening more stable.

Question 12

In nature, where were different nAChR found

Answer 12

In electric rays; electric organs need lots of ACh to discharge like a battery.

This gives evidence that similar subunits have developed in common ancestors.

Question 13

Describe Cys-loop transmitter-gated ion channel family basic structure.

Answer 13

The family members are nAChR, GABAA, glycine & 5HT3 receptors.

All have a PENTAMERIC assembly of the subunits with binding sites found between the subunit interfaces.

Question 14

Describe some of the different nAChR subunits?

Answer 14

2-9alpha, 2-4beta are neuronal

1alpha and 1beta are in muscle electric organ.

Question 15

What are PAMs?

Answer 15

positive allosteric modulators that are new drugs being made (as well as receptors antagonists,
agonists)

PAM amplifies ACh effect (on their own with no ACh there is no effect)

Different subtypes means SPECIFIC BINDING.

Question 16

What are some examples of disorders that have been linked to mutated receptors believed to be treated by drugs (PAM)?

Answer 16

Neuronal: 
Alzheimer’s 
Parkinson’s
Schizophrenia
Attention Deficit Disorder
Anxiety
Pain relief

Question 17

What is a alpha7-5HT3A chimera?

Answer 17

It is a hybrid synthetically made using 5HT3areceptor and A7-nAChreceptor

Question 18

What features does the chimera share with the original receptors?

Answer 18

It has the same agonist (ACh) as a7-nACh receptor (where 5-HT agonist is 5-HT)

It blocks a-bungarotoxin just like a7-nACh. (where 5-HT has no effect)

But it has a small channel conductance like 5-HT3areceptor

Question 19

What is a ACh BP?

Answer 19

ACh binding protein binds the the ALPHA SUBUNIT and its adjacent subunit

It is the same size of the extra cellular domain of the whole receptor

Crystallography has shown that exists as a HOMOPENTAMER (lacking the transmembrane components)

It is found in snail brains where it mops up EXCESS ACh.

Question 20

Describe the interaction between the AChBP and the agonist.

Answer 20

The agonist stabilises loop C of the protein in a contracted form, where open arms are shown to fold down so they no longer extend.

Question 21

How do amino acids influence the receptor? What amino acids are important in selectively?

Answer 21

Extracellular and intracellular rings of negatively charged
glutamate and aspartate residues influence cation conductance.

Cationic nAChR made anionic by mutating Glu to Arg.

Anionic α1-GlyR to cationic by mutating Arg to Glu

Question 22

Give an example of a variation seen in the 5-HT3 receptors. How does this compare with the nACh receptor?

Answer 22

Homomeric made up of the same subunit has a lower conductance

Heteromeric receptors have more than one type of subunits give a HIGHER CONDUCTANCE

However, homomeric nACh receptors have a HIGH conductance too

Question 23

Through chimeria experiments using the process of elimination, what was found out about the receptor structure and conductance?

Answer 23

the large intracellular loop of an important determinant of single channel conductance.

It has been narrowed down to 21 amino acids on the loop

Question 24

The receptor was Xrayed, what did this show?

Answer 24

There were 5 portals at the base for agonist exists like a watering can