TRP Channels

Question 1

how is the presence of pain inferred in model organisms?

Answer 1

through physical and behavioural interractions?

Question 2

1. What is the phenotype of trp mutant photoreceptors in Drosophila?
2. What does TRP couple to in Drosophila receptors
3. Which 3 other proteins does TRP form a supermolecular complex with?

Answer 2

1. transient receptor potentials
2. rhodopsin
3. PLC, PKC and INAD (scaffold protein)

Question 3

1. how many TM domains do TRP proteins have?
2. how many proteins come together to form a functional channel?
3. Which structures form the gating and selectivity filter of the channel?
4. Which domain do all TRP channels contain? Is it found in the C or N terminus?
5. Name 3 other domains that may be found in TRP channels
6. what is the shape of the IV curve

Answer 3

1. 6
2. 4
3. S5, S6 and the extracellular loop between them
4. TRP box in the c terminus
5. ankryn repeats, CIRB domains, and PDX domains
6. rectification

Question 4

Name the 2 models which give an explanation as to why upon increaseing sensitivity, innocuous stimuli produce a noxious sensation

Answer 4

1. Intensity Code - same receptors respond to both innocuous and noxious stimuli; the increase in intensity causes a shift in sensation
2. Specificity Code - specific neurons detect noxious stimuli but not innocuous stimuli.

Question 5

1. What is the difference between the nerve endings of mechanosensory neurons and nociceptors?
2. what is the difference in size and myelination of axons involved in touch/proprioception and pain
3. what do large, unmyelinated neurons express?
4. what do peptidergic neurons express?
5. What do non-peptidergic neurons express?
6. What type of neurons is TRPA1 a feature of? What is the evidence for this?
7. Name 2 TRP channels which are often expressed together?
8. Which TRP channel is often expressed on its own?

Answer 5

1. Mechanosensory neurons have specialised nerve endings such as merkel cells and ruffini corpuscles; nociceptors have unspecialised bare nerve endings
2. neurons associated with proprioception and touch are LARGE and MYELINATEDneurons associated with nociception are SMALL and UNMYELINATED
3. NF200
4. Substance P, CGRP and TRPV1
5. FRAP, IB4 and TRPV1
6. peptidergic. It co-localises with CGRP but not NF200
7. TRPV1 and TRPA1
8. TRPM8

Question 6

Name 5 problems associated with studying TRP channels

Name 3 ways in which these problems are addressed

Answer 6

1. there are a large number of subtypes with broad expression patterns and overlapping properties
2. their expression is not exclusive to the DRG
3. they tend to form heteromultimers which can affect functionality
4. lack of specific blockers
5. poorly understood mechanism of activation
6. genetically altered animals
7. heterologous expression patterns (using cDNA to exogenously express TRP channels)
8. generation of specific pharmacological blockers.

Question 7

1. How was capsacin used to identify TRPV1
2. describe how capsaicin can cause depolarisation
3. What was performed to show that TRPV1 is a multimodal receptor?
4. What has also been found to activate TRPV1 and what does this explain?

Answer 7

1. Responsiveness to capsaicin was used as a screening readout. A cDNA library was continuously fractioned until only a single clone responded to capsaicin. This clone expressed TRPV1.
2. capsaicin binding to receptor > opening of TRPV1 > Ca and Na influx > depolarisation
3. TRPV1 was expressed in xenopus oocytes. The addition of capsaicin and high temperatures evoked an inward current
4. inflammatory mediators. Explains the burning sensation in inflamed tissue.

Question 8

1. What does prolonged exposure to capsaicin lead to?
2. give 2 reasons for the long term response to prolonged exposure to capsaicin

Answer 8

1. sensitisation. Nociceptors become insensitive to capsaicin and less sensitive to high temperatures
2. death of nociceptor; destruction of peripheral terminals

Question 9

1. what is the Phenotype of TRPV1 KOs?
2. what were used to test mechanosensitivity? what did they find?
3. What else was abolished in TRPV1 KOs?
4. Why were KOs still thermosensitive?
5. At what temperatures are TRPV1 channels activated?
6. At what temperatures are TRPV2 channels activated?
7. At what temperatures are TRPV3 channels activated?

Answer 9

1. completely insensitive to capsaicin and less sensitive to high temperatures (higher threshold of innocuous heat)
2. von Frey fillaments. they found that snesitivity to mechanical pain is normal
3. thermal hypersensitivity caused by secondary hyperalgesia was abolished
4. because of the expression of other TRP channels such as TRPV2
5. 40^oC
6. 50^oC
7. 30^oC

Question 10

1. What was used to identify TRPM8?
2. What was the phenotype of TRPM8 knockouts?
3. How was this phenotype shown?

Answer 10

1. fractioning of a cDNA library until only one clone responded to cold/menthol
2. reduced threshold to respond to cold, but they were still thermosensitive.
3. KO mice only moved to a warm chamber at a colder temperature than WT mice, but at extremely cold temperatures, they moved to the chamber

Question 11

1. what do TRPA1 channels respond to?
2. What is the phenotype of TRPA1 KOs?

Answer 11

1. extreme cold and horseradish
2. defects in cold and mechanical sensitivity and sensitivity to bradykinin.

Question 12

Give 3 possible mechanisms for temperature dependent TRP gating

provide evidence to disprove any of these mechanisms

* how does temperature alter channel activity in terms of voltage dependence?

Answer 12

1. production of chemicals at certain temperatures which bind to specific receptors. Disproven for TRPM8 and TRPV1 in cell free patches. Potential mechanism for TRPV4 activation
2. undergo temperature sensitive structural rearrangements
3. sense changes in membrane tension due to temperatre dependent lipid bilayer rearrangements.

* teperature changes cause voltage dependent changes of the channel meaning that the channel is likely to be active at more physiological temperatures.

Question 13

1. Which TRP channels are responsible for making the skin temperature sensitive. In which cell type are they expressed?
2. which channel contributes to hearing an equilibrium. How?
3. How do inflammatory mediators acting on TRPV1 cause hyperalgesia? (2)
4. How do the 2 TRPV1 splice varients regulate each other
5. How do the 2 TRPM8 splice varients regulate each other
6. what happens to TRPV1 expression following axonal injury. What does this lead to?

Answer 13

1. TRPV3 and TRPV4 in keratinocytes
2. TRPA1. They enable mechanosensation in vertebrate hair cells
3. they lower the threshold for heat pain from 42^oC towards body temperature. They also promote the phosphorylation of TRPV1, which causes it to have increased activity.
4. TRPV1 beta acts as a dominant negative channel, reducing TRPV1 alpha activity
5. the short isoform lacks a TM domain therefore can’t act as a functional channel. However it blocks the trafficking of the long, functional isoform
6. it is downregulated in the injured axon, but upregulated in the neighbouring uninjured axons, including novel expression in low threshold A fibres. This contributes to neuropathic pain.

Question 14

1. What does the general approach of studying TRP channels involve?
2. What evidence supports the use of dominant negative mutants
3. Give 2 issues regarding the use of siRNA

Answer 14

1. generally involves peturbing/neutralising the protein of interest
2. because TRP channels function as tetramers. Incorporation of a dominant negative protein into the tetramer will declare the channel unfunctional
3. data obtained is succeptable to off target effects of the siRNA, which can lead to false positive results

TRP channels have long turnover times, and effective KD can take many days. This can give cells enough time to adapt and readjust their need for a particualr TRP channel by upregulating redundant machinery, which can give false negative results.