Lecture 2

Question 1

Frog skin epithelium

Answer 1

tight epithelium model 
•	Apical surface outside and inside surface basolateral 
•	Absorb sodium from external env 
•	Same mechanism we use in upper airway 
•	Robust/easy to use/lots of it

Question 2

Short circuit current technique using an ussing chamber

Answer 2

• see diagram
• Shift in transepithelia potential to calculate resistance
• If looking at sodium uptake use the same solution - Standard Kreb solution

Question 3

In frog skin and SCC if you lose Na from apical to basolateral

Answer 3

Expect negative transepithelial current

• use radioactive Na measure difference either side of chamber
• calibration curve show Na transport per unit time in both directions

Question 4

In an ussing chamber for passively transported ion flux is what ?

Answer 4

0

Question 5

In an ussing chamber for actively transported ion what is the equation for ion flux ??

Answer 5

Isc= JNET

Question 6

Na outflux tends to show what ??

Answer 6

Leak back

Question 7

To measure flux what is needed ??

Answer 7

An active component

- If tight epithelium has net transport across you can then measure flux.

Question 8

NA/K atpase is the active component which allows ?

Answer 8

Flux measurement and allows net reabsorption of Na

Question 9

Frog skin

Answer 9

very little net movement of chloride- cant test flux

Question 10

Change extracellular sodium-

Answer 10

Shows apical membrane has Na transporter

Question 11

Effect of amiloride on epithelial Na transport

Answer 11

Use Human colonic Biopsy

• Na Channel in apical membrane of colon
• Amiloride blocks Na on the epithelium
• Amiloride is low in the lumen -Ve voltage due to low Na absorption

Question 12

why is there Small transepithelial potential in human colonic biopsy compared to frog ?

Answer 12

not as tight as frog skin and mainly Na in frog skin not the case in the colon

Question 13

Human nasal biopsy

Answer 13

• Less invasive way of obtaining airway epithelium
• Vm is cell potential
• Na entry depolarises the cell
• add amiloride and the cell hyperpolarises

Question 14

Human nasal Biopsy Mall et al.

Answer 14

• Lots of NA reabsorption and CL secretion
• Add amiloride- hyperpolarization moving mem potential away from Nernst potential for sodium evidence for na selective channels in apical membrane of the nasal epithelium
• Predict Enac on apical

Question 15

Patch clamp analysis of ENac

Answer 15

• Directly look at currents and how they are impacted

- Direct demonstration of NA channel

Question 16

patch clamp recording - what does it show ??

Answer 16

Each flickering is one channel opening – higher conc size of deflection

Question 17

Expression cloning of ENac

Answer 17

• conventional cloning > isolate protein and sequence e.g using amiloride affinity column
• Protein abundnace too low in Native cells
• Native channel properties well defined > highly Na selective

Question 18

Mutations in ENac - two main syndromes

Answer 18

Liddles or Pseudohypoaldosteronism

Question 19

Liddles Syndrome

Answer 19

GOF
Hypertension
High ENac
Nedd4- Channel retrieval

Question 20

PHA

Answer 20

LOF
Hypotension
ENac Intact
- lose salt and vol depleted because water follows the salt

Question 21

Functional Expression Screen

Answer 21

Take mRNA instead of proteins – from kidney or colons and chopped into pools and inject into oocyte and identified which mRNA section made sodium selective amiloride ihibitable currents- functional expression

• mRNA from salt depleted rats
• aldosterone upregs mRNA for epithelial Na channel to maximise likelihood of getting mRNA for Na channel

Question 22

Na channel

Answer 22

3 gene seq that code for 3 separate subunits – all 3 needed for normal function

Question 23

Na channel currents - pure clone vs Poly A

Answer 23

Thought with pure clone they’d get large Na current
Bigger deflection the bigger the function- not pure clone bigger current WHY? – because of 3 subunits
Alpha can make a functional sodium channel
Size of deflection indicates function
Massive enhancement of function

Question 24

Alpha subunit

Answer 24

Alpha subunit can make a channel but other subunits needed for full activity

Question 25

If all subunits were required for ENac

Answer 25

ENac wouldnt have been cloned