INTRO LEC

Question 1

Why are cells polarised and what does this mean?

Answer 1

Means that different proteins are expressed at the apical and basolateral membranes of the cells.
This allows net movement of ions across cells

Question 2

What does transcellular and paracellular transport of ions mean?

Answer 2

Paracellular - between cells

Transcellular - across cells

Question 3

What is the difference between tight and leaky epithelium and what are their different transepithelial resistances?

Answer 3

Leaky epithelia allow lots of transport through them (large driving force for the movement of ions) and the trans epithelial resistance is < 200ohms/cm2. This is due to the large number of tight junctions (gaps/holes) that mediate the amount of transport

Tight epithelia allow less/hardly any transport across cells and have a transepithelial resistance of > 2000ohms/cm2

Question 4

Where can tight and leaky epithelia be found?

Answer 4

TIGHT - distal tubule, stomach

LEAKY - prox tubule, gall bladder, small intestine, choroid plexus

Question 5

Properties of leaky epithelia

Answer 5

Rte < 200ohms/cm2
Large flux
High h20 perm
Vte = around 0mV

Question 6

Properties of tight epithelia

Answer 6

Rte > 2000ohms/cm2
Small flux
Low h20 perm
Vte = around 50mV (no back leak of ions so positive value generated!)

Question 7

what is the Vte?

Answer 7

Transepithelial potential - the potential generated across the epithelium by the movement of ions

Question 8

Generating Vte - what generates a -ve and +ve potential?

Answer 8

-Ve = more anions move and less cations move 
\+Ve = more cations move and less anions

Question 9

What preparations are commonly used in measuring movement of ions across epithelia?

Answer 9

• cultured cells
• whole animals
• epithelial sheets
• fresh tissues and cells

Question 10

How does the activity of the Na+/K+ ATPase leave a net Negative- charge behind?

Answer 10

Na+/K+ ATPase maintains a constant low intracellular Na+ concentration, creating a driving force for the movement of Na+ out of the cell
This means that Na+ (positive) moves out, leaving a negative Vte/potential behind

Question 11

How does the activity of the Na+/K+/2Cl- cotransporter leave behind a net +ve charge?

Answer 11

2Cl- ions move across and out of the cell for only one Na+ –> a net positive charge is left behind

Question 12

What methods are involved (with proteins) in investigating electrophysiology and why are they used?

Answer 12

PCR –> detects the present of RNA
western blot –> tests for expression of proteins
immuno staining –> tests for localisation of proteins
Flux of radioactive compounds –> tests for functional transport of ions

Question 13

What are common methods used to measure ion movement in electrophysiology?

Answer 13

Patch clamp - measure single channel or whole cell conductance
2 electrode voltage clamp - clamps potential so that whole cell current can be measured
Ussing chamber - measures Vte, SSC (net flux of ions across epithelia) and resistance

Question 14

What are the nerst potentials for Na+ and K+ channels and what happens when one of these channels opens?

Answer 14

When an ion channel opens, the membrane drives the membrane potential closer to the nerst potential for that ion

K+ = -90mV
Na+ = +60mV

Question 15

How does Amiloride work and impact the nerst potential and membrane potential

Answer 15

Amiloride is an Na+ channel blocker so when added to cells blocks Na+ channels.
This prevents the activity of these channels from driving the membrane potential towards the potential for Na+, so the Vm shifts towards the nerst for K+ (as K+ channels are now dominant)

Question 16

the Ussing chamber –> how does it work and what calculations does it allow to be made?

Answer 16

The ussing chamber uses an epithelial sheet.
You inject a known amount of current into the chamber and measure the shift in potential upon injection
You / this shift in potential by the current to calculate resistance
S.S.C can be calculated by doing Vte (transepi potential) / Rte (resistance)

Question 17

Examples of epithelial sheets used in ussing chamber?

Answer 17

Frog skin, bladder, cultured cells, gut epithelium

Question 18

How does the patch clamp / 2 electrode voltage clamp method work and how does adding Ba2+ impact experimental data?

Answer 18

1) clamp membrane at particular potential
2) measure net current flow at this potential

By changing the potential you are changing the driving force for the movement of ions in that channel.
If Vm is close to the nerst potential for that channel, that channel is dominant in generating potential

Ba2+ is a Na+ channel blocker so can add and smaller currents will result!

Question 19

What does a downward deflection on a Vte trace represent?

Answer 19

The amount the potential has changed by when current has been injected

Question 20

Why does adding amiloride cause an initial increase in Vte?

Answer 20

Have blocked the movement of Na+ therefore there is more positive current left in the cell causing an initial increase in Vte

Question 21

Why does Amiloride send the Vte closer to 0 (more +ve?)

Answer 21

Less Na+ channels open = less +ve potential lost!!

Question 22

How does adding CFTR inhibitor impact the effect of Prostoglandins

Answer 22

Prostoglandins mimic release of Cl- –> by inhibiting Cl- channel (CFTR) can reverse secretion of Cl-, leaving a more negative Vte!