ACh Release

Question 1

Describe how activation of the nAChR leads to opening of the associated cation-conducting ion channel

Answer 1

• agonist binding to the receptor induces a conformational change to open associated ion channel
• nAChR channel is a cation channel primarily conducting Na into the cell and K out of the cell
• signalling is extremely rapid

Question 2

How can we study the functional properties of the receptor-channels in real time

Answer 2

patch-clamp technique

Question 3

Describe the patch-clamp technique

Answer 3

• sharp electrodes penetrate into muscle fibre
• patch electrodes polish end and make it smooth
• patch electrode touches surface of neuron
• suck on syringe and hope that in membrane there is a receptor or two
• seal is mechanically tight and electrically tight
• ACh binds to receptor
• can see current that flows with sodium going through receptors

Question 4

How can we analyse results from the patch-clamp technique to study the activity of single receptors

Answer 4

• jump down on graph is channel opening -> sodium fluxes through
• can be used to explore effects of drugs on singular receptors

Question 5

Describe the inside-out formation of the receptor arising from the patch-clamp technique

Answer 5

receptor is located facing inside the glass electrode and is immediately pulled from the cell

Question 6

Describe the outside-out formation of the receptor arising from the patch-clamp technique

Answer 6

membrane snaps back in such a way that there is a piece of membrane at each end of patch electrode that fold to have receptor pointing out towards the back (facing the whole cell)

Question 7

Advantages of the patch-clamp technique

Answer 7

• can record the activity of single receptors
• the intracellular milieu is maintained
• control of the extracellular solution

Question 8

Disadvantages of the cell-attached patch-clamp technique

Answer 8

• investigating different concentrations of drugs e.g. agonists is complex
• the agonist is always present, meaning receptors can become desensitized and inactive
• no control of the intracellular content
• the resting membrane potential is not known

Question 9

Advantages of the whole-cell voltage clamp patch-clamp technique

Answer 9

• control of the intracellular and extracellular milieu
• can record from a population of receptors/ion channels
• can introduce biochemical modulators (intracellularly) via the recording pipette

Question 10

Disadvantages of the whole-cell voltage-clamp patch-clamp technique

Answer 10

• biochemical “washout” of intracellular milieu
• can rarely observe single receptor activity

Question 11

Advantages of the outside-out patch patch-clamp technique

Answer 11

• control of the intracellular and extracellular milieu
• can change/control extracellular drug (agonist) concentrations

Question 12

Disadvantages of the outside-out patch patch-clamp technique

Answer 12

• biochemical “washout” of the intracellular milieu
• cannot easily change intracellular messengers e.g. cAMP

Question 13

Advantage of the inside-out patch patch-clamp technique

Answer 13

• control of the intracellular and extracellular milieu
• can introduce biochemical modulators e.g. cAMP to the intracellular face

Question 14

Disadvantages of the inside-out patch patch-clamp technique

Answer 14

cannot easily change concentration of agonist

Question 15

What is the reversal potential

Answer 15

the potential at which there is no net current flow

Question 16

How can the reversal potential be used

Answer 16

• can be used as a fingerprint to identify which ions are being conducted through the channel
• can be coupled with molecular biology techniques to determine the part of the protein that controls the conduction of cations/anions

Question 17

High net current indicates outward flow of which ion

Answer 17

Potassium

Question 18

Low net current indicates inward flow of which ion

Answer 18

Sodium

Question 19

Where net current = 0 (reversal potential)

Answer 19

equal inward and outward flow of sodium and potassium (respectively)

Question 20

Therapeutic prospects of the neuronal nicotinic receptors

Answer 20

• Alzhimers - cognitive enhancement
• Parkinson’s
• Schizophrenia
• ADD
• Pain - analgesia
• Anxiety
• Smoking cessation

Question 21

How can we study functional properties of recombinant wild type and mutant receptors

Answer 21

• Xenopus oocyte expression system with voltage-clamp
• epithelial cell transfection system with whole-cell voltage-clamp and patch-clamp

Question 22

Describe the oocyte expression system

Answer 22

oocytes derived from xenopus laevis are injected with DNA/RNA and placed into a voltage-clamp recording and the dunction is studied using the two-electrode voltage-clamp technique

Question 23

Application of neurotransmitter to oocyte

Answer 23

receptor opens, allowing chloride ions to leave cell, resulting in transient inward current that is recorded and analysed by computer software