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1 doi:.3/nture93 d 5 Rttlesnke DRG (reds) Rttlesnke TG (reds) c 3 TRPV1 other TRPs Non-pit snke TG (reds) SFig other TRPs TRPV Non-pit snke DRG (reds) 5 Antomy of the pit orgn nd comprison of gene expression in snke sensory gngli Longitudinl projection showing trigeminl innervtion of rttlesnke pit orgn. (dpted from Tershim nd Ling, J Neurophysiol, 3-3 (1991)). Python hed showing loction of nostril nd lil pits (lck nd red rrows, respectively)(from Wikimedi Commons). (c - d) Numer of mrna-seq reds from snke gngli tht lign to the chicken proteome. nd TRPV1 re highlighted, s re other TRP fmily chnnels. The lue line indictes the expected numer of sequencing reds for genes with similr expression levels in the two smples sed on the totl numer of ligned reds SFig. 1 otined in ech. Signls elow reds re within the sttisticl noise of the RNA-seq protocol nd therefore considered s non-expressed sequences. Rttlesnke refers to C. trox nd non-pit refers to comintion of Texs Rt (Elphe osolete lindheimerii) nd Western Cochwhip (Msticophis flgellum testceus) snkes. 1
2 doi:.3/nture93 Rttlesnke Rt snke * * * Rttlesnke Humn * * *
3 doi:.3/nture93 SFig. Sequence lignment of orthologues Alignment of deduced protein sequences for rttlesnke versus rt snke chnnels showing 1% identity. Alignment of deduced protein sequences for rttlesnke versus humn chnnels showing 3% identity. Predicted trnsmemrne regions re denoted y lck rs over relevnt sequences; cysteine residues criticl for ctivtion of humn y re denoted with sterisks; N- terminl region contining nkyrin repets is indicted y green r (top pnel only). 3
4 doi:.3/nture rttlesnke F 3 /F rt snke Temperture (ºC) Rttlesnke 1 1 5⁰C - 7 C Temperture ( o C) ⁰C - Rt snke - 37 C Temperture ( o C) 5⁰C ⁰C -
5 doi:.3/nture93 SFig. 3 Het sensitivity of cloned snke chnnels Temperture-response profile for trnsfected HEK93 cells expressing cloned rttlesnke nd rt snke chnnels s determined y rtiometric clcium imging. Dt collected from 1 cells for rttlesnke nd 31 cells for rt snke chnnels; threshold =. ± 1.1 nd 3.3 ±.3ºC, respectively; P <.15. Representtive current-voltge nd temperture-response reltionships for rttlesnke nd rt snke chnnels expressed in oocytes (left nd middle). Current-voltge reltionships showing reltive responses to het or (1 mm) (right). 5
6 doi:.3/nture93 RT Het Humn Zerfish Zerfish SFig. Het nd sensitivity of humn nd zerfish HEK93 cells trnsfected with humn or zerfish or cdnas were sujected to live cell rtiometric clcium imging to mesure responses to het (5 C mximl stimulus) or ( µm). Note lck of het-evoked responses in ll cses. SFig.
7 doi:.3/nture93 RT Het c 5 C Normlized current C Temperture ( C) SFig. 5 Therml nd chemicl sensitivity of fly chnnel Therml nd chemicl sensitivity of Drosophil (d) s mesured y live-cell rtiometric clcium imging. Cloned receptors were trnsiently expressed in HEK93 cells nd sujected to temperture rmp, followed y ppliction of mustrd oil (; µm) t C. Current-voltge reltion determined y two-electrode voltge clmp recording from d-expressing oocyte in response to het or (1 mm). c Normlized curve showing het response profile for d chnnel in oocytes. Response t ny given temperture ws normlized to the mximl het-evoked current otined t 5⁰C. 7
8 doi:.3/nture93 Python log rtio TG/DRG Rttlesnke log rtio TG/DRG - 3 C 5⁰C ⁰C c - 3 C ⁰C ⁰C SFig. Anlysis of python chnnel Comprison of rttlesnke nd python trnscriptomes showing reltive expression levels in TG versus DRG sed on numer of ligned sequencing reds. Currentvoltge reltionships from oocytes expressing python in response to het or (1 mm). c Current-voltge reltionships from oocytes expressing o in response to het or (1 mm). SFig.
9 doi:.3/nture93 % of totl cells % of totl cells 5 3 Python Rt snke Python Rt snke Rt snke TRPV Cell dimeter (μm) c d Het F 3 /F na Temperture (ºC) s SFig. 7 Antomy nd physiology of python nd rt snke neurons Quntittive nlysis of or TRPV1 mrna expression in python nd rt snke TG s determined from histologicl sections shown in Fig. 5 (men ± s.d.; n = 7 neurons from 1 sections for python ; 795 neurons from sections for rt snke, nd 117 neurons from independent sections for rt snke TRPV1). Quntifiction of neuronl cell size from histologicl sections of python (grey) or rt snke (lck) TG (n cells from 5 sections ech). c Averge temperture-response profile for cpsicin-sensitive trigeminl neuron from rt snke s determined y clcium imging (n = 11 neurons). Therml response threshold = 3.7 ± 1.ºC (n = neurons). d Representtive ptch-clmp recording from cultured rt snke neuron shows het-evoked current with threshold of 35. ± 1.ºC (n = 3). 9
10 doi:.3/nture93 3C 35C C Cpsicin Untrnsfected cells Rttlesnke TRPV1 Current (μa) Cp, Het 5C Cp C SFig. Chrcteriztion of cloned rttlesnke TRPV1 chnnel Therml nd chemicl sensitivity of rttlesnke TRPV1 s mesured y live-cell rtiometric clcium imging. Cloned receptors were trnsiently expressed in HEK93 cells nd sujected to temperture rmp, followed y ppliction of cpsicin (Cp; 1 µm) t C. Current-voltge reltion determined y two-electrode voltge clmp recording from rttlesnke TRPV1-expessing oocyte in response to het or Cp (1 µm).
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