Lecture 1 Flashcards
What is the difference between transcellular and Paracellular ?
Transcellular = transport across the cell Paracellular= Between the cells
What makes a cell polarised ?
apical and basolateral membrane with different proteins on each membrane – net movement across the cell
What does Transcellular transport require ?
Transcellular requires differential expression of transport membrane proteins
What does the direction of movement depend on ?
Depends on the driving force
What are the two classifications of epithelial cells ?
Leaky or tight
Examples of Leaky epithelial cells ?
proximal tubule
gallbladder
small intestine
choroid plexus
Examples of Tight epithelial cells ?
distal tubule
stomach
frog skin- important model of Na absorption
What is the Transepithelial resistance (Rte) for leaky ?
Leaky < 200 .cm2
What is the Transepithelial resistance (Rte) for tight ?
Tight > 2000 .cm2
If you have high resistance what hapens to transport ??
it becomes smaller
What junctions are found between epithelia?
Tight Junctions
What determines the amount of transport?
Gaps between cells mediated by tight junction determines amount of transport
Tight- smaller holes/gaps less transport
Leaky show what permeability to water ?
High
Tight show what permeability to water ?
Low
Tight epithelia transport
Tend to be across the cell and not between
Definiton of Isosmotic -
having the same osmotic pressure.
In leaky- Flux is large and isosmotic
Model 1 for how Vt is generated ?
Negative mem potential and low NA – influx of positive charge and leaves across basolateral membrane- net movement
Tight epithelium – Na cant leak back – net loss of positive charge therefore negative potential
Model 2 for how Vt is generated ? - see lecture diagram
Na/ K transport protein on apical membrane
Na lost- apical to baso
K in and leaking back across apical mem- recycled
2 CL but one positive ion moving- apical to basolateral
Overall loss of one negative charge- leaves positive potential
Large positive transepithelial potential
What is the Vte for Leaky ?
0mV
What is the Vte for Tight ?
approx 50mv
-ve Vte
More anions or les cations
+ve Vte
Less anions or more cations
Types of tissues used
Fresh tissue
cultured cells
whole animals
Types of preparations of tissues
Wildtype
mutant
Ko
Over expression proteins in cells
prepared with Pharmacological agents
what is the issue with overexpression data ?
may not be an absolute reflection/not in normal env
What is PCR used for ?
mRNA presence
Western blotting
Protein presence
immunostaining
protein location
flux radioactive compounds
Transport Function
Electrophysiology
Transport Function
electrophysiological techniques
- Intracellular microelectrode
- patch clamp
- two electrode voltage clamp
- Ussing chamber s
Intracellular Microelctrode
IC potenial in Vm
Patch clamp
-Single channel/cell current
-clamp to potential you decide-
Whole cell recording
Measure net current flow- set by ion channels open/how many/ selectivity
two electrode voltage clamp
Cell current
Ussing Chambers
Transepithelial potential
- transepithelial resistnace
- Short circuit current
- Mem potential of a cell determined by which ion channels open and there selectivity
- K channels – drive mem potential towards Nernst potential for K
- Lots of K channels open- very negative mem potential
- Na- opposite
- Closer to Ena- more Na open
- Closer to EK – more K open
Amiloride
Blocks ENac – shifts mem potential (-40 to -60) Na no longer contributing- k channels dominate
Epithelial sodium channel is there at rest and maintains mem potential -active
Barium
potassium channel blocker
Reversal potential for k currents
Reversal potential for k currents is at the Nernst potential – if 100% selective to K
ussing chamber- How does it work ?
Vte, Rte and Isc
- Epithelial sheet in middle of chamber separating either side
- 4 electrodes
- 2 measure transepithelial potential either side
- 2 inject currents
- see diagram
1. Current injected
2. Resulting shift in Vte measured.
Rte
= total V/ I injected
Isc
= Vte/Rte
High resistance epithelium
Big shift in potential
Low resistance epithelium
Small shift in potential
Vte Traces
Downward deflection- injection of current
- Vte
- Vte in response to I injection to calculate resitance
ohms law
V= I*R
Negative Vte Trace
- More anions or less cations
- amiloride sends Vte to zero
- Blocks Na channels
- Less transport
- Reduced loss of positive charge
- vte due to loss of Na
Lub
Stimulates CFTR
