TRP channels

1. TRP channels 10/19/09

2. Plan • Lecture on Wed - Richard Payne • 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

12. 7 families of TRPs Trp NompC

13. TRP families

14. How is Trp activated - DAG?

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

16. Mammalian TRPCs

17. TRP domain - highly conserved

18. TRP domain Seems to be highly conserved within a family, particularly in TRPCs

19. #s of copies

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 Kumar and Hedges 1998

23. Comparative genomics • Dr. Payne is interested in invertebrates • Invertebrates

25. Molting animals 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

33. 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. TRP domain Seems to be highly conserved within a family, particularly in TRPCs

45. Alignment = find Trp box

46. TRP domain - highly conserved

47. Lots of variation before ion channel - we may need to deal just with channel part ANK ANK

48. Sometimes gene is poorly predicted

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---- *::.* *********::*****:.****. .**:**:.***:** :**.*** *** 23 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