Lecture 2 Flashcards
Frog skin epithelium
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
Short circuit current technique using an ussing chamber
- see diagram
- Shift in transepithelia potential to calculate resistance
- If looking at sodium uptake use the same solution - Standard Kreb solution
In frog skin and SCC if you lose Na from apical to basolateral
Expect negative transepithelial current
- use radioactive Na measure difference either side of chamber
- calibration curve show Na transport per unit time in both directions
In an ussing chamber for passively transported ion flux is what ?
0
In an ussing chamber for actively transported ion what is the equation for ion flux ??
Isc= JNET
Na outflux tends to show what ??
Leak back
To measure flux what is needed ??
An active component
- If tight epithelium has net transport across you can then measure flux.
NA/K atpase is the active component which allows ?
Flux measurement and allows net reabsorption of Na
Frog skin
very little net movement of chloride- cant test flux
Change extracellular sodium-
Shows apical membrane has Na transporter
Effect of amiloride on epithelial Na transport
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
why is there Small transepithelial potential in human colonic biopsy compared to frog ?
not as tight as frog skin and mainly Na in frog skin not the case in the colon
Human nasal biopsy
- Less invasive way of obtaining airway epithelium
- Vm is cell potential
- Na entry depolarises the cell
- add amiloride and the cell hyperpolarises
Human nasal Biopsy Mall et al.
- 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
Patch clamp analysis of ENac
- Directly look at currents and how they are impacted
- Direct demonstration of NA channel
patch clamp recording - what does it show ??
Each flickering is one channel opening – higher conc size of deflection
Expression cloning of ENac
- 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
Mutations in ENac - two main syndromes
Liddles or Pseudohypoaldosteronism
Liddles Syndrome
GOF
Hypertension
High ENac
Nedd4- Channel retrieval
PHA
LOF
Hypotension
ENac Intact
- lose salt and vol depleted because water follows the salt
Functional Expression Screen
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
Na channel
3 gene seq that code for 3 separate subunits – all 3 needed for normal function
Na channel currents - pure clone vs Poly A
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
Alpha subunit
Alpha subunit can make a channel but other subunits needed for full activity
If all subunits were required for ENac
ENac wouldnt have been cloned
