Lecture 01 Introduction

Question 1

What is cell polarity?

Answer 1

cells have apical and basolateral membranes each with differential expression of proteins allowing net movement of ions across epithelium

Question 2

What are the two pathways that ions are dependent on?

Answer 2

transcellular (across cells) and paracellular (between cells)

Question 3

What are ‘leaky epithelium’?

Answer 3

epithelium that allows lots of ionic transport and ‘leak-back’

Question 4

What are ‘tight epithelium’?

Answer 4

epithelium that allows very little ionic transport or ‘leak-back’

Question 5

What is the transepithelial resistance (Rte) of leaky and tight epithelia?

Answer 5

leaky <200ohm.cm^2

tight >2000ohm.cm^2

Question 6

Give examples of leaky epithelial cells

Answer 6

proximal tubule
gallbladder
small intestine
choroid plexus

Question 7

Give examples of tight epithelial cells

Answer 7

distal tubule
stomach
frog skin

Question 8

What is the importance of frog skin?

Answer 8

important model of sodium reabsorption

Question 9

What determines tightness of epithelium?

Answer 9

cell-to-cell interactions - tight junctions
mediate ion transport between the epithelia
some proteins present that facilitate ion transport

Question 10

Compare tight and leaky epithelia in terms of ‘tightness’

Answer 10

if the quantity of ions transported are the same in both tight and leaky epithelia, the leaky epithelia will have much smaller proximal resistance and larger gaps in the tight junctions allowing greater ion ‘leak-back’ compared to the tight epithelia

Question 11

Compare the properties of leaky and tight epithelia

Answer 11

leaky
transepithelial potential ~0mV
flux large and isosmotic
high water permeability

tight
transepithelial potential ~50mV
flux small
low water permeability

Question 12

Define: Transepithelial Potential

Answer 12

the sum of the individual membrane potentials

Question 13

How is the Transepithelial Potentials measured?

Answer 13

place reference electrode one side of the cells (0mV) and the recording electrode on the other side
the difference between the two gives the Vte

Question 14

Can you measure the transepithelial potential for leaky epithelia?

Answer 14

no leaky cells cannot sustain a positive or negative Vte due to ion movement back between cells

Question 15

Give an example of how a negative Vte might be generated in tight epithelia?

Answer 15

potassium recycles across the basolateral membrane

sodium has net movement across the cell decreasing the number of cations on the apical side producing a negative Vte

Question 16

Give an example of how a positive Vte might be generated in tight epithelia?

Answer 16

thick ascending limb epithelial cells
NKCC2 cotransporter in the apical membrane each turnover produces 1Na+:1K+:2Cl-
potassium recycles across apical and basolateral membranes
sodium and chloride move across the cell and across the basolateral membrane
provides a net gain of one negative charge to the basolateral side therefore less anion on the apical side producing a positive charge

Question 17

What type of models can you use?

Answer 17

fresh tissues or cells - isolated structure or whole organ
cultured cells - sample, biopsy or stock
whole animal model organism

Question 18

What types of genetic modifications can you use?

Answer 18

wildtype
mutants (specific interest)
knock-out
overexpression

Question 19

What is the problem with over expressing a protein?

Answer 19

over expressing the channel doesn’t mean you have overexpressed the other factors as well

Question 20

What is an alternative to making genetic modifications?

Answer 20

pharmacological agents to activate or inhibit channels

Question 21

What different methodologies could you use? What information will you gain from them?

Answer 21

PCR - presence of mRNA
western blotting - presence of a protein or protein expression levels
immunostaining - location of the protein
flux radioactive compounds - transport function
electrophysiology - transport function
intracellular microelectrodes - IC potential (Vm)
patch clamp - single channel or whole cell currents
two-electrode voltage clamp - cell current
Ussing chamber - Vte, Rte, SCC

Question 22

Define: Short Circuit Current (SCC)

Answer 22

an indirect measure of the net ion flux across the membrane
the larger the SCC the bigger the movement of ions
the smaller the SCC the smaller the movement of ions

Question 23

Give an example of when intracellular electrodes might be used

Answer 23

calculating the resting membrane potential and the dominant or active ion channels present
example cells has high sodium and low potassium outside and low sodium and hight potassium inside
the resting membrane potential is sitting close to the Nernst potential for potassium
add amiloride the Vm becomes more negative (-40mV to -60mV) shows that sodium channels are present but potassium channels are dominant

Question 24

Why would you add amiloride?

Answer 24

it is a epithelial sodium channel blocker
check or prevent ENaC from working
i.e. to not interfere with an experiment

Question 25

What is signified if the resting membrane potential stays the same after amiloride has been added?

Answer 25

sodium are not active, present or important in that system

Question 26

What does the Patch Clamp technique allow us to measure?

Answer 26

measure the net movement of ions and therefore current

Question 27

Give an example of when the Patch Clamp technique can be used

Answer 27

measure the potassium currents in cortical collecting ducts of renal principal cells to observe the effects of regulation or mutations

Question 28

What would a smaller current in response to the addition of barium signify?

Answer 28

the channel is barium-sensitive

Question 29

How can the selectivity of the channel be calculated?

Answer 29

current-voltage curves

Question 30

What is the Vrev or Reversal Potential?

Answer 30

where there is no net movement of ions | the current is 0mV

Question 31

How does the Ussing chamber work?

Answer 31

four electrodes two on either side of the epithelia top two measure the transepithelial potential bottom two electrodes inject a specific voltage a epithelia sheet can be cut from a sample or cultured with a special insert the electrodes inject a known current and the resultant shift in potential in Vte is measured

Question 32

Give examples of cells that can be used in the epithelia sheet of the Ussing chamber

Answer 32

frog skin gut bladder

Question 33

What technique can be combined with the Ussing chamber technique?

Answer 33

tracer studies (radioactive compounds)

Question 34

What is Ohms law?

Answer 34

V = I x R

Question 35

How is Transepithelial Resistance calculated?

Answer 35

Rte = (change in voltage) / (current injected)

Question 36

How is Short Circuit Current (SCC) calculated?

Answer 36

SCC = Vte / Rte

Question 37

What does resistance of the epithelium mean?

Answer 37

high resistance = increase shift potential | low resistance = decrease shift potential

Question 38

What do Vte Traces show? What can they be used for?

Answer 38

show deflections with each individually representing the change in Vte in response to an injection of current can use this information to calculate the resistance

Question 39

What is the action of Lubiprostone (Lub)?

Answer 39

a prostaglandin that activates specific chloride channels