1. TRP channels
10/19/09

2. Plan Lecture on Wed - Richard Payne One week to gather sequences
The problem
Brainstorm a plan
One week to gather sequences
Each of you will do a different species
I am away Wed pm to Fri am. I’ll be in my office all afternoon and all Monday - bring questions to class!
We will then compile all the data
One week to analyze data
Present results to Dr. Payne

3. Drosophila mutant screen
Forward genetics
Screen for mutants which have visual defect
Find gene causing defect
Piece together parts of visual pathway

4. Screen by electroretinograms, ERGs
Send light pulse to eye
Wire outside of retina to record electrical signal coming from whole eye response
Sum of all neural responses in eye

5. Light coincident receptor potential

6. Light coincident receptor potential
Drosophila mutants
Norp - no receptor potential (phospholipase C)
Trp - transient receptor potential

7. Ion channel mutants
Trp = Transient receptor potential - channels close sooner

8. Trp is …
…an ion channel

9. Textbook example - nompC
Figure 3.5 Drosophila mutants identified as being very clumsy and uncoordinated Found mutation in gene NompC = No mechanoreceptor potential C Turned out to be different mutations in same gene NompC
So identified just like Shaker channel from Shaker mutants

12. 7 families of TRPs Trp NompC Montell 2005 Science STKE
Also TRPy which exist in yeast so these channels are very old!

13. TRP families TRPC Classic - like Drosophila Ca+2 DAG Light, pheremone
TRPV
Vanilloid receptors
Heat, cold, capsaicin
TRPM
Melastatin receptor
Taste, menthol, cool
TRPN
NOMPC
Mechanosensation
(none in mammals)
TRPA
ANK repeats
Cold, hearing
Melastatin is expressed in melanocytes and may be a tumor suppressor

14. How is Trp activated - DAG?

15. How is Trp activated - IP3 release of Ca+2?

16. Mammalian TRPCs Store operated means IP3 releases calcium stores
DAG means DAG activates channel directly
EPSC = excitatory post synaptic current

17. TRP domain - highly conserved
Occurs in TRPC, M and N though doesn’t occur in NOMPC???

18. Seems to be highly conserved within a family, particularly in TRPCs
TRP domain
Seems to be highly conserved within a family, particularly in TRPCs
Not in drosophila NOMPC
Highly conserved but don’t know its function. Does it have a function? What do you think?

19. #s of copies
Like to know if any of these TRP channels function in hearing. TRPN would be obvious candidate as it is mechanosensor, but mammals have 0 TRPNs!! So not sure what is hearing channel.

20. TRPC and TRPN most closely related
C. elegans has TRP3 which is like TRPC

21. Dr. Payne is world expert on limulus vision!
Response to IP3 Causes depolarization
Causes release of Ca2+
Adaptation
Can’t respond immediately - takes recovery time to rebuild Ca stores
Payne et al 1990

22. Vertebrate divergence times
The time scales show the times to most recent common ancestor.
Cartilagenous fish = sharks, rays
Agnatha = jawless fish = lamprey, hagfish
Vertebrates are MY old
Mammals are 100 MY
Tetrapods are 400 MY
Kumar and Hedges 1998

23. Comparative genomics Dr. Payne is interested in invertebrates

24. Placozoa are simplest animals with just 4 kinds of cells and few thousand cells total

25. Molting animals Trochozoa Lophophorata
Trochozoa
Lophophorata

27. Genome projects for inverts
Model systems
C. elegans, Drosophila
Sea urchins, amphioxus

28. Genome projects for inverts
Vectors for disease
Mosquitos, tick, louse, leech

29. Other insects
Bee, pea aphids, flour beetle, wasp

30. Miscellaneous
Polychaete worm
Limpet
Daphnia

31. BLAST-able invertebrate genomes
Deuterostomes
Craniate
Cephalochodates
Tunicate
Echinoderm
Human
Amphioxus
Sea squirt
Sea urchin
Protostome - annelid
Polychaete
Clitellate
Capitella
Leech
Protostome - Mollusc
Gastropod
Limpet
Protostome - Arthropod
Crustacean
Water flea
Insect - Coleoptera
- Diptera
- Hymenoptera
- Hemiptera
- Phthiraptera
Flour beetle
Fruit fly (1 of 12)
Mosquito (3)
Honey bee, wasp
Pea aphid
Louse

33. Horseshoe crab is closest to tick!!
Horseshoe crab is closest to tick!!

34. How do we get sequences from these genomes
Can’t use ortholog links in Ensembl
Haven’t made links for inverts!
We have to BLAST our way in

35. NCBI has links to all genome projects - Not all projects are BLASTable

36. Bee genome page with BLAST link

37. What kind of blast should we use?
MegaBLAST - highly similar
BLASTN - nucleotide
BLASTP - protein

38. Can blast to different databases

39. Ab initio = known + predicted genes

40. Use Drosophila Trp gene as bait

41. BLAST interim screen

42. Blast result

43. Download sequence

44. Seems to be highly conserved within a family, particularly in TRPCs
TRP domain
Seems to be highly conserved within a family, particularly in TRPCs
Not in drosophila NOMPC

45. Alignment = find Trp box

46. TRP domain - highly conserved
Occurs in TRPC, M and N though doesn’t occur in NOMPC???

47. Lots of variation before ion channel - we may need to deal just with channel part
ANK ANK
nompC1,2 and 3 have premature stops, nompC4 has missense
29 ANK repeats - maybe these bind to cytoskeleton??

48. Sometimes gene is poorly predicted
Note: Sequences which are NP are hand annotated and have very high confidence. Sequences which are XP are predicted from whole genome sequence.

49. Human_rho MNGTEGPNFYVPFSNATGVVRSPFEYPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLY
Chimp_rho MNGTEGPNFYVPFSNATGVVRSPFEYPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLY
Dog_rho MNGTEGPNFYVPFSNKTGVVRSPFEYPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLY
Mouse_rho MNGTEGPNFYVPFSNVTGVVRSPFEQPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLY
Rat_rho MNGTEGPNFYVPFSNITGVVRSPFEQPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLY
Cow_rho MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSMLAAYMFLLIMLGFPINFLTLY
Zfish_rho MNGTEGPAFYVPMSNATGVVRSPYEYPQYYLVAPWAYGFVAAYMFFLIITGFPVNFLTLY
Chick_rho MNGTEGINFYVPMSNKTGVVRSPFEYPQYYLAEPWKYRLVCCYIFFLISTGLPINLLTLL
****** ****:** *******:* *****. ** : ::..*:*:** *:*:*:*** 23 variable
Human_rho VTVQHKKLRTPLNYILLNLAVADLFMVLGGFTSTLYTSLHGYFVFGPTGCNLEGFFATLG
Chimp_rho VTVQHKKLRTPLNYILLNLAVADLFMVLGGFTSTLYTSLHGYFVFGPTGCNLEGFFATLG
Dog_rho VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNVEGFFATLG
Mouse_rho VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLG
Rat_rho VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLG
Cow_rho VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLG
Zfish_rho VTIEHKKLRTPLNYILLNLAIADLFMVFGGFTTTMYTSLHGYFVFGRLGCNLEGFFATLG
Chick_rho VTFKHKKLRQPLNYILVNLAVADLFMACFGFTVTFYTAWNGYFVFGPVGCAVEGFFATLG
**.:***** ******:***:*****. *** *:**: :****** ** :******** 17
Human_rho GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWS----
Chimp_rho GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWS----
Dog_rho GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWSSLLS
Mouse_rho GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVVFTWIMALACAAPPLVGWS----
Rat_rho GEIGLWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLVGWS----
Cow_rho GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLVGWS----
Zfish_rho GEMGLKSLVVLAIERWMVVCKPVSNFRFGENHAIMGVAFTWVMACSCAVPPLVGWS----
Chick_rho GQVALWSLVVLAIERYIVVCKPMGNFRFSATHAMMGIAFTWVMAFSCAAPPLFGWS----
*::.* *********::*****:.****. .**:**:.***:** :**.*** ***
Human_rho RYIPEGLQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIIIFFCYGQLVFTV
Chimp_rho RYIPEGLQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIIIFFCYGQLVFTV
Dog_rho HSPLVLRYIPEGMQCSCGIDYYTLKPEINNESFVIYMFVVHFAIPMIVIFFCYGQLVFTV
Mouse_rho RYIPEGMQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIVIFFCYGQLVFTV
Rat_rho RYIPEGMQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIVIFFCYGQLVFTV
Cow_rho RYIPEGMQCSCGIDYYTPHEETNNESFVIYMFVVHFIIPLIVIFFCYGQLVFTV
Zfish_rho RYIPEGMQCSCGVDYYTRTPGVNNESFVIYMFIVHFFIPLIVIFFCYGRLVCTV
Chick_rho RYMPEGMQCSCGPDYYTHNPDYHNESYVLYMFVIHFIIPVVVIFFSYGRLICKV
**:***:***** **** :***:*:***::** **:::***.**:*: .* 28

50. Wise2 - can predict mRNA from gDNA using related protein

51. Predict dog RH using cow RH AA
Cow AA
Dog seq

52. Finds introns so know where they are

53. Fixes intron prediction to get rid of extra stuff