Lecture 01 Introduction Flashcards
What is cell polarity?
cells have apical and basolateral membranes each with differential expression of proteins allowing net movement of ions across epithelium
What are the two pathways that ions are dependent on?
transcellular (across cells) and paracellular (between cells)
What are ‘leaky epithelium’?
epithelium that allows lots of ionic transport and ‘leak-back’
What are ‘tight epithelium’?
epithelium that allows very little ionic transport or ‘leak-back’
What is the transepithelial resistance (Rte) of leaky and tight epithelia?
leaky <200ohm.cm^2
tight >2000ohm.cm^2
Give examples of leaky epithelial cells
proximal tubule
gallbladder
small intestine
choroid plexus
Give examples of tight epithelial cells
distal tubule
stomach
frog skin
What is the importance of frog skin?
important model of sodium reabsorption
What determines tightness of epithelium?
cell-to-cell interactions - tight junctions
mediate ion transport between the epithelia
some proteins present that facilitate ion transport
Compare tight and leaky epithelia in terms of ‘tightness’
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
Compare the properties of leaky and tight epithelia
leaky
transepithelial potential ~0mV
flux large and isosmotic
high water permeability
tight
transepithelial potential ~50mV
flux small
low water permeability
Define: Transepithelial Potential
the sum of the individual membrane potentials
How is the Transepithelial Potentials measured?
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
Can you measure the transepithelial potential for leaky epithelia?
no leaky cells cannot sustain a positive or negative Vte due to ion movement back between cells
Give an example of how a negative Vte might be generated in tight epithelia?
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
Give an example of how a positive Vte might be generated in tight epithelia?
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
What type of models can you use?
fresh tissues or cells - isolated structure or whole organ
cultured cells - sample, biopsy or stock
whole animal model organism
What types of genetic modifications can you use?
wildtype
mutants (specific interest)
knock-out
overexpression
What is the problem with over expressing a protein?
over expressing the channel doesn’t mean you have overexpressed the other factors as well
What is an alternative to making genetic modifications?
pharmacological agents to activate or inhibit channels
What different methodologies could you use? What information will you gain from them?
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
Define: Short Circuit Current (SCC)
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
Give an example of when intracellular electrodes might be used
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
Why would you add amiloride?
it is a epithelial sodium channel blocker
check or prevent ENaC from working
i.e. to not interfere with an experiment
What is signified if the resting membrane potential stays the same after amiloride has been added?
sodium are not active, present or important in that system
What does the Patch Clamp technique allow us to measure?
measure the net movement of ions and therefore current
Give an example of when the Patch Clamp technique can be used
measure the potassium currents in cortical collecting ducts of renal principal cells to observe the effects of regulation or mutations
What would a smaller current in response to the addition of barium signify?
the channel is barium-sensitive
How can the selectivity of the channel be calculated?
current-voltage curves
What is the Vrev or Reversal Potential?
where there is no net movement of ions
the current is 0mV
How does the Ussing chamber work?
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
Give examples of cells that can be used in the epithelia sheet of the Ussing chamber
frog skin
gut
bladder
What technique can be combined with the Ussing chamber technique?
tracer studies (radioactive compounds)
What is Ohms law?
V = I x R
How is Transepithelial Resistance calculated?
Rte = (change in voltage) / (current injected)
How is Short Circuit Current (SCC) calculated?
SCC = Vte / Rte
What does resistance of the epithelium mean?
high resistance = increase shift potential
low resistance = decrease shift potential
What do Vte Traces show? What can they be used for?
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
What is the action of Lubiprostone (Lub)?
a prostaglandin that activates specific chloride channels
