Lecture 9

Question 1

What does it mean that nicotinic receptor is cation non-selective?

Answer 1

That is usually primarily conduct Na+ (and in some cases Ca2+) into the cell and K+ out of the cell.

Question 2

What are two advantages of the Cell-Attached patch?

Answer 2

The intracellular milieu is maintained (no biochemical washout)
2) Control of extracellular solution

Question 3

What are four disadvantages of the cell-attached patch?

Answer 3

1) Investigating different concentrations of drugs is complex.
2) The agonist is always present and this lead to receptor desensitization
3) No control of intracellular current
4) The resting membrane potential is not known.

Question 4

What are three advantages of whole-cell voltage clamp?

Answer 4

1) Control of the intra and extracellular milieu
2) Can record from a population of receptors/ion channels
3) Can introduce biochemical modulators (intracellularly) via the recording pipette

Question 5

What are two disadvantage of the whole-cell voltage patch?

Answer 5

1) Biochemical washout of the intracellular milieu

2) Resolution (can rarely observe single receptor activity)

Question 6

What are two advantages of Outside-out patch?

Answer 6

1) Control of the intracellular & extracellular milieu.

2) Can change/control extracellular drug concentrations

Question 7

What are two disadvantages of outside-out patch?

Answer 7

1) Biochemical washout of the intracellular milieu

2) Cannot change intracellular messengers.

Question 8

What are the two advantages if inside-out patch?

Answer 8

1) Control of the intracellular and extracellular milieu

2) Can introduce biochemical modulators to the intracellular face.

Question 9

What is the disadvantage of inside-out patch?

Answer 9

Cannot change the concentration of agonist.

Question 10

What are the four different types of patch-clamp recordings?

Answer 10

1) Cell-attached
2) Whole-cell
3) Outside-out
4) Inside-out.

Question 11

What are the two subtypes of nicotinic receptors revealed historically?

Answer 11

1) Ganglionic

2) Skeletal Muscle

Question 12

What are the two components that help obtaining a purified nAChR subunit protein?

Answer 12

1) α- Bungarotoxin obtained from Taiwan banded krait

2) nAChRs obtained from electric rays

Question 13

What did analysis of the cloning nAChR revealed?

Answer 13

nAChR is a membre of the Cys-loop transmitter gated ion channel family. All receptors are composed of pentameric assembly of subunits.

Question 14

What is nAChR closely related to?

Answer 14

GABAa
glycine
5HT3 receptors

Question 15

List the nicotinic receptors subunits normally found in muscle electric organ:

Answer 15

α1, β1, δ, γ, ε

Question 16

List the nicotinic receptor subunits normally found in an adult nmj:

Answer 16

2α, β, ε, δ

Question 17

List the nicotinic receptor subunits normmaly found in embryonic, extrajunctional, denervated receptors:

Answer 17

2α, β, γ, δ

Question 18

List the nicotinic receptor subunits normmaly found in neuronal nicotinic receptors:

Answer 18

α 2-9, β 2-4

Question 19

Where does the agonist ACh binds on the nicotinic receptor?

Answer 19

At the subunit interfaces.

Question 20

What has the discovery of nAChR subtypes encouraged?

Answer 20

It has encouraged drug discovery to identify novel receptors antagonists, agonists and postive allosteric modulators (PAMs)

Question 21

What does the α subunit provides together with the adjacent subunits?

Answer 21

The α subunit constitutes the principal component of the binding site, the adjacent subunit provide the complementary site.

Question 22

What is the peculiarity of ACh binding protein (AChBP)?

Answer 22

AChBP exists as a homopentamer and lacks the transmembrane components.

Question 23

Describe the action of the agonist with AChBP:

Answer 23

The agonist stabilises loop C (harbours 2 adjacent cysteines) in a contracted form (closed apposition to the adjacent subunits).

Question 24

What does loop C do when an agonist binds?

Answer 24

Loop C closes over the agonist binding pocket when the site is occupied by an agonist.

Question 25

What does loop C do when an antagonist binds?

Answer 25

Antagonist stabilise extended form and loop C is further away from the agonist binding pocket.

Question 26

Why are the small changes in conformation important in the AChBP?

Answer 26

Because the small changes in conformation of the ligand binding domain act to trigger the conformational change required for ion channel conduction.

Question 27

What happens if you mutate Glu or Asp to neutral or positive amino acids?

Answer 27

You have a decrease in conductance because extracellular and intracellular rings of negatively charged glutamate and aspartate influence cation conductance.

Question 28

What is the mutation that you should induced in nAChR α7 to make it anionic? What about α1-GlyR?

Answer 28

Cationic nAChR α7 to anionic by mutating Glu to Arg

Anionic α1-GlyR to cationic by mutating Arg to Glu.