TRP Channels

Question 1

1. to which divalent cation is TRPV5 and 6 highly selctive for?
2. which TRPV channel is predominantly expressed in the kidney and at lower levels in the small intestine?
3. which TRPV channel is predominantly expressed in the intestine, and at lower levels in the kidney?
4. to which divalent cation is TRPM6 and M7 selective for?

Answer 1

1. calcium
2. TRPV5
3. TRPV6
4. magnesium

Question 2

1. which condition implicates vitamin D3 in calcium homeostasis and why?
2. under what conditions are Parathyroid hormone (PTH) and VitD3 released?
3. what do PTH and VitD3 aim to do?
4. how does PTH act?
5. how does VitD3 act?

Answer 2

1. Rickets. Vitamin D deficiency. clinically characterised by hypocalcemia and skeletal abnormalities
2. hypocalcaemia
3. aim to increase calcium reabsorption from the kidney and intestine
4. PTH acts via the generation of cAMP (PTH receptor is a GPCR) to activate PKA - cAMP activates TRPV6 in intestine
5. VitD3 increases Ca reabsorption by inducing synthesis of calbindin and Ca channels

Question 3

1. describe the cell model for Ca handling in the small intestine
2. why is the driving force for calcium reabsorption high?
3. describe the role of calbindins.

Answer 3

1. TRPV6 (and some TRPV5) expressed on apical membrane. Ca ATPase and Ca/Na exchanger on the BL membrane
2. intracellular [Ca] is low (large concentration gradient for absorption)
3. to prevent calcium from acting as a signalling molecule as it is transported across epithelia. Binds to calcium, preventing it from being free. A localised low [Ca] at the BL membrane promotes dissociation of calbindins so Ca can be transported across the BL membrane.

Question 4

1. Describe the phenotype of TRPV6 KO mice (6 characteristics)
2. what was used to examine intestinal Ca handling?
3. describe Ca uptake in WT and KO mice under normal dietary calcium
4. describe Ca uptake in WT and KO mice on a low calcium diet.
5. why is it unexpected to see a large impact of V6 KO in the kidney?
6. describe the effects of KO on renal Ca handling
7. describe the role of the Ca sensing receptor and how it explains increased urinary volume in KOs
8. What can be used to explain the this larger than expected result?
9. describe one problem with the renal handling study.

Answer 4

1. normal lifespan, decreased body weight, impaired fertility, higher PTH and VitD3 and a lack of response to these hormones, alopecia (particularly if mother was KO, as Ca is not secreted into milk)
2. radioactive calcium, to look at the level of radioactivity in the plasma (oral gavage technique)
3. plasma Ca was lower in KO but not significant
4. Ca uptake was increased by 7.5 in WT and 9.6 in KO. However, because of the lower Ca uptake in the KO, levels were significantly lower in KO following this increase
5. becayse TRPV5 is the main Ca channel in the kidney
6. urinary Ca was double in KO; urinary volume was increased, urine osmolality was reduced (due to increased volume)
7. they are found in the A membrane, and respond to high Urinary calcium, to prevent the formation of kidney stones. They inhibit AQP2, to prevent the reabsorption of water to lower the calcium concentration of the urine. In KO, because urine Ca is high (due to a lack of reabsorption), the Ca sensing receptor will be inhibiting AQP2 to lower this concentration
8. age of animal. V6 is thought to be important pre-weaning. Mice used may have been at the age where their dependency is in transition from V6 to V5
9. they did not get evidence from western/northern blotting to confirm the change in V6/V5 levels.

Question 5

1. what are some cases of familial kidney stones caused by?
2. how did researchers investigate the cause of idopathic familial kidney stones?
3. what polymorphisms were identified?
4. what are the 3 possible genotypes of individuals?
5. which genotype confers a higher incidence of kidney stones?
6. when expressed in xenopus oocytes, which polymorphic TRP channel had enhanced Ca uptake?
7. What have these polymorphic functional differences been linked to?

Answer 5

1. mutations in the VitD3 receptor or the calcium sensing receptor
2. genetically screened 170 individuals with idiopathic familial kidney stones for mutations/polymorphisms
3. 3 synonymous (closely associated) polymorphisms in TRPV6 - CMM (derived) or RVT (ancestral)
4. homozygous derived (CC + MM + MM), heterozygous (CR + MV + MT) or homoygous ancestral (RR + VV + TT)
5. ancestral
6. ancestral - it has an enhanced function to absorb Ca from intestine
7. aggricultural revolution - derived polymorphism appeared as our calcium intake increased; prior to this, our Ca intake was lower therefore having an enhanced TRPV6 function was beneficial to absorb more Ca from our diets.

Question 6

1. describe the cell model for Mg absorption
2. why is there a strong driving force for for Mg reabsorption?
3. what does immunofluorescence show about M6 expression?
4. what does analysis of M6 and M7 by PCR in the kidney?

Answer 6

1. TRPM6 and TRPM7 are expressed in the apical membraneBL transporter yet to be determinedparacellular Mg transport also occurs in the intestine.
2. despite a Vm of -70 which would inhibit Mg absorption, in combo with the concentration gradient and overall Vte, the electrochemical driving force is for absorption
3. shows that TRPM6 is expressed in the A membrane of the early distal tubule (co-localises with NCC) and in the duodenum
4. M6 and M7 are expressed throughout the nephron, with the strongest bands being in the early distal tubule, the site of regulated Ca and Mg handling.

Question 7

1. in what TRP channel are mutations found that cause hypomagnesemia with secondary hypocalcaemia?
2. in what mendelian pattern are these mutations inherited?
3. name 4 characteristics of this disease

Answer 7

1. M6
2. recessive
3. seizures and tetany (involuntary contraction of muscles), neurological damage, poor intestinal Mg reabsorption and enhanced renal loss of Mg (normally, Mg is only seen in the urine when 1.5mg/dL is filtered; Mg is seen in the urine in these patients when less is filtered)

Question 8

describe the evidence that M6 and M7 function as heteromultimers which enhances their function.

Answer 8

when M6 is expressed alone in xenopus oocytes, currents are small. Currents are slightly bigger when M7 is expressed alone. When expressed together, the currents are greater than the sum of the currents when M6 and M7 are expressed alone

Fura-2 fluorescence and Mn uptake were also used to show the same thing:

• M6 and M7 are also permeable to Mn. Mn quences the fluorescence of Fura2
• the fluorescence was quenced to a greater extent when M6 and M7 are expressed together than the sum of when they were expressd separately.

Question 9

1. name the mutation responsible for hypomagnesemia and secondary hypocalcemia
2. what type of defect does this mutation cause?
3. what happened when this mutant was expressed alone in xenopus oocytes (Mn entry)
4. what happened when this mutant was expressed with M7? (Mn entry)
5. where is WT M6-YFP found?
6. where is WT M6-YFP found when co-expressed with M7?
7. where is mutant M6-YFP found?
8. what do these results suggest?

Answer 9

1. S141L
2. trafficking defect
3. rate of Mn entry was same as control cells (i.e. when no TRPM channel expressed)
4. rate of Mn entry was the same as when M7 was expressed alone (loss of enhancement)
5. to some extent at the cell surface
6. more is found at the cell surface
7. cytoplasm (not at cell surgace)
8. Impaired M6 trafficking, and loss of enhancement by the formation of heteromultimers means that there is less Mg (and Ca) absorption.

Question 10

1. what are the side effects of FK506 and Dexamethazone, in terms of Ca and Mg handling?
2. How does FK506 act as an immunosupressant?
3. how does dexamethazone act as an immunosupressant?
4. what were the effects of FK506 and Dex on urinary levels of Ca and Mg?
5. what were the effects of FK506 and Dex on Na excretion?
6. what were the effects of FK506 and Dex gamma-GT? What is this a marker for?
7. what were the effects of FK506 and Dex on Mg serum levels?
8. what were the effects of FK506 and Dex on V5 and M6 mRNA levels in the kidney?
9. How is FK506 thought to act?
10. How is Dex thought to act?

Answer 10

1. hypomagensemia, hypocalcemia and increased bone turnover
2. binds to FK506 binding proteins. resulting complex inhibits calcineurin in T lymphocytes, and as a result, a nuclear factor isnt dephosphorylated, thus remains in the cytosol. Hence IL-2 production isn’t activated
3. glutocorticoid
4. both cause calciurea and magnesurea
5. Dex causes Na excretion. FK506 has no effect
6. Dex causes the appearance of gamma-YT. This is a marker for renal tubule damage
7. FK506 causes a decrease in serum Mg
8. FK506 downregulates V5 and M6Dex upregulates V5 and M6
9. reduces renal expression of V5 and M6 leading to reduced Mg/Ca reabsorption
10. damages tubules (hence appearance of gamma-YT). V5 and M6 are upregulated as a compensatory mechanism. Na reabsorption from the prox tubule is reduced, hence increased excretion. paracellular transport of Mg and Ca are driven by Na reabsorption, hence why Mg and Ca reabsorption are reduced.