trp channels
DESCRIPTION
TRP channels. 10/19/09. 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! - PowerPoint PPT PresentationTRANSCRIPT
TRP channelsTRP channels
10/19/09
PlanPlan
Lecture on Wed - Richard PayneThe problemBrainstorm a plan
One week to gather sequencesEach 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
Drosophila Drosophila mutant screenmutant screen
Forward geneticsScreen for mutants which have visual
defectFind gene causing defectPiece together parts of visual pathway
Screen by Screen by electroretinograms, ERGselectroretinograms, 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
Light coincident receptor Light coincident receptor potentialpotential
Light coincident receptor Light coincident receptor potentialpotential
Norp - no receptor potential (phospholipase C)
Trp - transient receptor potential
Drosophila mutants
Ion channel mutantsIon channel mutants
Trp = Transient receptor potential - channels close sooner
Trp is …Trp is …
…an ion channel
Textbook example - nompCTextbook example - nompC
7 families of TRPs7 families of TRPsNompCTrp
TRP familiesTRP familiesTRPC
Classic - like Drosophila
Ca+2
DAGLight, pheremone
TRPV Vanilloid receptors
Heat, cold, capsaicin
TRPM
Melastatin receptor
Ca+2 Taste, menthol, cool
TRPN
NOMPC Ca+2 Mechanosensation(none in mammals)
TRPA ANK repeats
Cold, hearing
How is Trp activated - DAG?How is Trp activated - DAG?
How is Trp activated - How is Trp activated - IPIP3 3 release of Carelease of Ca+2+2??
Mammalian TRPCsMammalian TRPCs
TRP domain - highly TRP domain - highly conservedconserved
TRP domain
Seems to be highly conserved within a family, particularly in TRPCs
#s of copies#s of copies
TRPC and TRPN most closely related
C. elegans has TRP3 which is like TRPC
Dr. Payne is world expert on Dr. Payne is world expert on limulus vision!limulus vision!
Response to IP3 Causes depolarization
Causes release of Ca2+
AdaptationCan’t respond immediately - takes recovery time to rebuild Ca stores
Payne et al 1990
Vertebrate divergence timesVertebrate divergence timesHuman
Cow
Mouse
Rat
Reptiles
Birds
Amphibians
Tilapia
Fugu
Zebrafish
Cartilagenous fish
Agnatha
100200300400500600 0
Bonyfish
Time (Myr)
92
4171
276
310
360
250
120200
528
564
Kumar and Hedges 1998
Comparative genomicsComparative genomics
Dr. Payne is interested in invertebrates
Invertebrates
Trochozoa
Lophophorata
Molting animals
Genome projects for invertsGenome projects for inverts
Model systemsC. elegans, Drosophila
Sea urchins, amphioxus
Genome projects for invertsGenome projects for inverts
Vectors for diseaseMosquitos, tick, louse, leech
Other insectsOther insects
Bee, pea aphids, flour beetle, wasp
MiscellaneousMiscellaneous
Polychaete worm
Limpet
Daphnia
Deuterostomes CraniateCephalochodatesTunicateEchinoderm
HumanAmphioxusSea squirtSea urchin
Protostome - annelid PolychaeteClitellate
CapitellaLeech
Protostome - Mollusc Gastropod Limpet
Protostome - Arthropod
Crustacean Water flea
Protostome - Arthropod
Insect - Coleoptera - Diptera
- Hymenoptera - Hemiptera - Phthiraptera
Flour beetleFruit fly (1 of 12)Mosquito (3)Honey bee, waspPea aphidLouse
BLAST-able invertebrate genomes
Horseshoe crab is closest to tick!!
How do we get sequences How do we get sequences from these genomesfrom these genomes
Can’t use ortholog links in EnsemblHaven’t made links for inverts!
We have to BLAST our way in
NCBI has links to all genome NCBI has links to all genome projects - Not all projects are projects - Not all projects are
BLASTable BLASTable
Bee genome page with BLAST Bee genome page with BLAST linklink
What kind of blast should we What kind of blast should we use?use?
MegaBLAST - highly similar
BLASTN - nucleotide
BLASTP - protein
Can blast to different Can blast to different databasesdatabases
Ab initio = known + Ab initio = known + predicted genespredicted genes
Use Use Drosophila Drosophila Trp gene as Trp gene as baitbait
BLAST interim screenBLAST interim screen
Blast resultBlast result
Download sequenceDownload sequence
TRP domain
Seems to be highly conserved within a family, particularly in TRPCs
Alignment = find Trp boxAlignment = find Trp box
TRP domain - highly TRP domain - highly conservedconserved
Lots of variation before ion channel - we may need to deal just with channel part
ANK ANK
Sometimes gene is poorly Sometimes gene is poorly predictedpredicted
Human_rho MNGTEGPNFYVPFSNATGVVRSPFEYPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYChimp_rho MNGTEGPNFYVPFSNATGVVRSPFEYPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYDog_rho MNGTEGPNFYVPFSNKTGVVRSPFEYPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYMouse_rho MNGTEGPNFYVPFSNVTGVVRSPFEQPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYRat_rho MNGTEGPNFYVPFSNITGVVRSPFEQPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYCow_rho MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSMLAAYMFLLIMLGFPINFLTLYZfish_rho MNGTEGPAFYVPMSNATGVVRSPYEYPQYYLVAPWAYGFVAAYMFFLIITGFPVNFLTLYChick_rho MNGTEGINFYVPMSNKTGVVRSPFEYPQYYLAEPWKYRLVCCYIFFLISTGLPINLLTLL ****** ****:** *******:* *****. ** : ::..*:*:** *:*:*:*** 23 variable Human_rho VTVQHKKLRTPLNYILLNLAVADLFMVLGGFTSTLYTSLHGYFVFGPTGCNLEGFFATLGChimp_rho VTVQHKKLRTPLNYILLNLAVADLFMVLGGFTSTLYTSLHGYFVFGPTGCNLEGFFATLGDog_rho VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNVEGFFATLGMouse_rho VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLGRat_rho VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLGCow_rho VTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLGZfish_rho VTIEHKKLRTPLNYILLNLAIADLFMVFGGFTTTMYTSLHGYFVFGRLGCNLEGFFATLGChick_rho VTFKHKKLRQPLNYILVNLAVADLFMACFGFTVTFYTAWNGYFVFGPVGCAVEGFFATLG **.:***** ******:***:*****. *** *:**: :****** ** :******** 17
Human_rho GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWS----Chimp_rho GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWS----Dog_rho GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWSSLLSMouse_rho GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVVFTWIMALACAAPPLVGWS----Rat_rho GEIGLWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLVGWS----Cow_rho GEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLVGWS----Zfish_rho GEMGLKSLVVLAIERWMVVCKPVSNFRFGENHAIMGVAFTWVMACSCAVPPLVGWS----Chick_rho GQVALWSLVVLAIERYIVVCKPMGNFRFSATHAMMGIAFTWVMAFSCAAPPLFGWS---- *::.* *********::*****:.****. .**:**:.***:** :**.*** *** 23
Human_rho ------RYIPEGLQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIIIFFCYGQLVFTVChimp_rho ------RYIPEGLQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIIIFFCYGQLVFTVDog_rho HSPLVLRYIPEGMQCSCGIDYYTLKPEINNESFVIYMFVVHFAIPMIVIFFCYGQLVFTVMouse_rho ------RYIPEGMQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIVIFFCYGQLVFTVRat_rho ------RYIPEGMQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIVIFFCYGQLVFTVCow_rho ------RYIPEGMQCSCGIDYYTPHEETNNESFVIYMFVVHFIIPLIVIFFCYGQLVFTVZfish_rho ------RYIPEGMQCSCGVDYYTRTPGVNNESFVIYMFIVHFFIPLIVIFFCYGRLVCTVChick_rho ------RYMPEGMQCSCGPDYYTHNPDYHNESYVLYMFVIHFIIPVVVIFFSYGRLICKV **:***:***** **** :***:*:***::** **:::***.**:*: .* 28
Wise2 - can predict mRNA Wise2 - can predict mRNA from gDNA using related from gDNA using related
proteinprotein
Predict dog RH using cow Predict dog RH using cow RH AARH AA
Cow AA
Dog seq
Finds introns so know where Finds introns so know where they arethey are
Fixes intron prediction to get Fixes intron prediction to get rid of extra stuffrid of extra stuff