Lab 4: ENaC

Question 1

Where is the ROMK1 channel found?

Answer 1

This is in the apical membrane for both the kidney thick ascending limb epithelial cells and the principle cells of the collecting duct. K+ flows through this channel so K+ is secreted from the epithelial cells to the developing urine

Question 2

What do restriction enzymes do?

Answer 2

They cut DNA into smaller strands, you can study the effect of length differences in individuals

Question 3

What is the name of the mutation in ENaC? Where is it located?

Answer 3

R566X

This is close to the COOH terminus

Question 4

What is the name of the mutation in the ROMK gene? Where is it located?

Answer 4

Y60X which is closer to the NH2 terminus

Question 5

What is the purpose of the this lab?

Answer 5

We are trying to work out which one is ENaC and which one is ROMK. We use APE to try and find cut sites for specific enzymes in each transporter. We incubate them with a restriction enzyme in agarose gel which separates DNA based on their base pair length

Question 6

What is the relationship between the number of basepairs and the distance traveled?

Answer 6

As you increase the number of base pairs, you decrease the distance traveled.

Question 7

The cutting site for the enzymes used for βENaC was located ___________ from the PstI site, meaning that PstI ended up at the _______ end of the βENaC gene

Answer 7

upstream

5`

Question 8

The cutting site for the enzymes used for ROMK1 was located ___________ from the PstI site, meaning that PstI ended up at the _______ end of the ROMK1 gene

Answer 8

downstrean

3`

Question 9

Where does the ROMK mutation affect?

Answer 9

This is the ROMK1 channel on the apical membrane of the thick ascending limb

Question 10

Is Barter’s syndrome a loss of gain of function mutation? Is it dominant or recessive?

Answer 10

This is a recessive mutation which means that it is a loss of function mutation

Question 11

What happens in Barter’s syndrome?

Answer 11

• type 1 there is a mutation in NKCC2
• type 2 there is a Y60X mutation in ROMK1
• type 3 there is a mutation in the Cl- channel (ClCNKB)

Question 12

What happens in Liddle’s syndrome?d

Answer 12

There is a R566X mutation which means that arginine 566 is changed to a stop codon which means that there is no binding site for E3 and so it can’t be ubiquitinated so it can’t be endocytose

Question 13

Is Liddle’s syndrome a loss of gain of function mutation? Is it dominant or recessive?

Answer 13

It is a dominant mutation which means that it is gain of function