Lecture 1

Question 1

What is the difference between transcellular and Paracellular ?

Answer 1

Transcellular = transport across the cell
Paracellular= Between the cells

Question 2

What makes a cell polarised ?

Answer 2

apical and basolateral membrane with different proteins on each membrane – net movement across the cell

Question 3

What does Transcellular transport require ?

Answer 3

Transcellular requires differential expression of transport membrane proteins

Question 4

What does the direction of movement depend on ?

Answer 4

Depends on the driving force

Question 5

What are the two classifications of epithelial cells ?

Answer 5

Leaky or tight

Question 6

Examples of Leaky epithelial cells ?

Answer 6

proximal tubule
gallbladder
small intestine
choroid plexus

Question 7

Examples of Tight epithelial cells ?

Answer 7

distal tubule
stomach
frog skin- important model of Na absorption

Question 8

What is the Transepithelial resistance (Rte) for leaky ?

Answer 8

Leaky < 200 .cm2

Question 9

What is the Transepithelial resistance (Rte) for tight ?

Answer 9

Tight > 2000 .cm2

Question 10

If you have high resistance what hapens to transport ??

Answer 10

it becomes smaller

Question 11

What junctions are found between epithelia?

Answer 11

Tight Junctions

Question 12

What determines the amount of transport?

Answer 12

Gaps between cells mediated by tight junction determines amount of transport
Tight- smaller holes/gaps less transport

Question 13

Leaky show what permeability to water ?

Answer 13

High

Question 14

Tight show what permeability to water ?

Answer 14

Low

Question 15

Tight epithelia transport

Answer 15

Tend to be across the cell and not between

Question 16

Definiton of Isosmotic -

Answer 16

having the same osmotic pressure.

In leaky- Flux is large and isosmotic

Question 17

Model 1 for how Vt is generated ?

Answer 17

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

Question 18

Model 2 for how Vt is generated ? - see lecture diagram

Answer 18

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

Question 19

What is the Vte for Leaky ?

Answer 19

0mV

Question 20

What is the Vte for Tight ?

Answer 20

approx 50mv

Question 21

-ve Vte

Answer 21

More anions or les cations

Question 22

+ve Vte

Answer 22

Less anions or more cations

Question 23

Types of tissues used

Answer 23

Fresh tissue
cultured cells
whole animals

Question 24

Types of preparations of tissues

Answer 24

Wildtype
mutant
Ko

Question 25

Over expression proteins in cells

Answer 25

prepared with Pharmacological agents

Question 26

what is the issue with overexpression data ?

Answer 26

may not be an absolute reflection/not in normal env

Question 27

What is PCR used for ?

Answer 27

mRNA presence

Question 28

Western blotting

Answer 28

Protein presence

Question 29

immunostaining

Answer 29

protein location

Question 30

flux radioactive compounds

Answer 30

Transport Function

Question 31

Electrophysiology

Answer 31

Transport Function

Question 32

electrophysiological techniques

Answer 32

• Intracellular microelectrode
• patch clamp
• two electrode voltage clamp
• Ussing chamber s

Question 33

Intracellular Microelctrode

Answer 33

IC potenial in Vm

Question 34

Patch clamp

Answer 34

-Single channel/cell current
-clamp to potential you decide-
Whole cell recording
Measure net current flow- set by ion channels open/how many/ selectivity

Question 35

two electrode voltage clamp

Answer 35

Cell current

Question 36

Ussing Chambers

Answer 36

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

Question 37

Amiloride

Answer 37

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

Question 38

Barium

Answer 38

potassium channel blocker

Question 39

Reversal potential for k currents

Answer 39

Reversal potential for k currents is at the Nernst potential – if 100% selective to K

Question 40

ussing chamber- How does it work ?

Answer 40

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.

Question 41

Rte

Answer 41

= total V/ I injected

Question 42

Isc

Answer 42

= Vte/Rte

Question 43

High resistance epithelium

Answer 43

Big shift in potential

Question 44

Low resistance epithelium

Answer 44

Small shift in potential

Question 45

Vte Traces

Answer 45

Downward deflection- injection of current

• Vte
• Vte in response to I injection to calculate resitance

Question 46

ohms law

Answer 46

V= I*R

Question 47

Negative Vte Trace

Answer 47

• 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

Question 48

Lub

Answer 48

Stimulates CFTR