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