rtx  (Alomone Labs)


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    Structured Review

    Alomone Labs rtx
    <t>RTx-dependent</t> long-range conformational changes <t>in</t> <t>TRPV1.</t> a Cylinder representation of TRPV1 in turquoise (one subunit) and gray (the rest of the channel). The approximate distances from the RTx binding site (reference residue Y511) to subdomains are shown. b The cryo-EM densities (surface) and respective models (sticks) depicting close-up views of the vanilloid binding sites in TRPV1 C, RTx (skyblue), thresholding 0.19, TRPV1 IO, RTx (yellow), thresholding 0.04, and TRPV1 O, RTx (pink), thresholding 0.033. c The cryo-EM densities (surface) and respective models (sticks) depicting close-up views of the selectivity filter in TRPV1 C,RTx (skyblue), thresholding 0.19, TRPV1 IC,RTx (green), thresholding 0.04, TRPV1 IO,RTx (yellow), thresholding 0.1, and TRPV1 O,RTx (pink), thresholding 0.033. d – e Close-up view of the overlays of TRPV1 C, RTx (skyblue), TRPV1 IO, RTx (yellow), and TRPV1 O, RTx (pink) regarding the cytoplasmic domain, and S6 gate e , respectively.
    Rtx, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis"

    Article Title: Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis

    Journal: Nature Communications

    doi: 10.1038/s41467-022-30602-2

    RTx-dependent long-range conformational changes in TRPV1. a Cylinder representation of TRPV1 in turquoise (one subunit) and gray (the rest of the channel). The approximate distances from the RTx binding site (reference residue Y511) to subdomains are shown. b The cryo-EM densities (surface) and respective models (sticks) depicting close-up views of the vanilloid binding sites in TRPV1 C, RTx (skyblue), thresholding 0.19, TRPV1 IO, RTx (yellow), thresholding 0.04, and TRPV1 O, RTx (pink), thresholding 0.033. c The cryo-EM densities (surface) and respective models (sticks) depicting close-up views of the selectivity filter in TRPV1 C,RTx (skyblue), thresholding 0.19, TRPV1 IC,RTx (green), thresholding 0.04, TRPV1 IO,RTx (yellow), thresholding 0.1, and TRPV1 O,RTx (pink), thresholding 0.033. d – e Close-up view of the overlays of TRPV1 C, RTx (skyblue), TRPV1 IO, RTx (yellow), and TRPV1 O, RTx (pink) regarding the cytoplasmic domain, and S6 gate e , respectively.
    Figure Legend Snippet: RTx-dependent long-range conformational changes in TRPV1. a Cylinder representation of TRPV1 in turquoise (one subunit) and gray (the rest of the channel). The approximate distances from the RTx binding site (reference residue Y511) to subdomains are shown. b The cryo-EM densities (surface) and respective models (sticks) depicting close-up views of the vanilloid binding sites in TRPV1 C, RTx (skyblue), thresholding 0.19, TRPV1 IO, RTx (yellow), thresholding 0.04, and TRPV1 O, RTx (pink), thresholding 0.033. c The cryo-EM densities (surface) and respective models (sticks) depicting close-up views of the selectivity filter in TRPV1 C,RTx (skyblue), thresholding 0.19, TRPV1 IC,RTx (green), thresholding 0.04, TRPV1 IO,RTx (yellow), thresholding 0.1, and TRPV1 O,RTx (pink), thresholding 0.033. d – e Close-up view of the overlays of TRPV1 C, RTx (skyblue), TRPV1 IO, RTx (yellow), and TRPV1 O, RTx (pink) regarding the cytoplasmic domain, and S6 gate e , respectively.

    Techniques Used: Binding Assay, Cryo-EM Sample Prep

    PH-S5-S6 triad hydrogen bond network in TRPV1 for RTx gating. a The cryo-EM maps (surface) and respective models (sticks) depicting the tripartite hydrogen bond network of PH-S5-S6 in TRPV1 C, RTx (skyblue), thresholding 0.15, TRPV1 IC, RTx (yellow), thresholding 0.045, TRPV1 IO, RTx (gold), thresholding 0.1, and TRPV1 O, RTx (pink), thresholding 0.04. The black dotted-lines indicate hydrogen bonds. The red dotted-lines indicate distance measurements between atoms where hydrogen bonds are broken. b – e TRPV1 Y584F and T641A reduce large cation permeabilty (YO-PRO-1, M.W. 376 Da) in the presence of RTx. Representative inside-out current traces of TRPV1 WT b , TRPV1 Y584F c , and TRPV1 T641A d . Current traces for basal, RTx (200 nM) activation (red trace) and intracellular application of 10 μM YO-PRO-1 (blue trace). e Summary of current inhibition by YO-PRO-1 (10 µM) of TRPV1 WT, TRPV1 Y584F and TRPV1 T641A after application of a saturating concentration of RTx (200 nM). Data are presented as mean ± s.e.m.; P
    Figure Legend Snippet: PH-S5-S6 triad hydrogen bond network in TRPV1 for RTx gating. a The cryo-EM maps (surface) and respective models (sticks) depicting the tripartite hydrogen bond network of PH-S5-S6 in TRPV1 C, RTx (skyblue), thresholding 0.15, TRPV1 IC, RTx (yellow), thresholding 0.045, TRPV1 IO, RTx (gold), thresholding 0.1, and TRPV1 O, RTx (pink), thresholding 0.04. The black dotted-lines indicate hydrogen bonds. The red dotted-lines indicate distance measurements between atoms where hydrogen bonds are broken. b – e TRPV1 Y584F and T641A reduce large cation permeabilty (YO-PRO-1, M.W. 376 Da) in the presence of RTx. Representative inside-out current traces of TRPV1 WT b , TRPV1 Y584F c , and TRPV1 T641A d . Current traces for basal, RTx (200 nM) activation (red trace) and intracellular application of 10 μM YO-PRO-1 (blue trace). e Summary of current inhibition by YO-PRO-1 (10 µM) of TRPV1 WT, TRPV1 Y584F and TRPV1 T641A after application of a saturating concentration of RTx (200 nM). Data are presented as mean ± s.e.m.; P

    Techniques Used: Cryo-EM Sample Prep, Activation Assay, Inhibition, Concentration Assay

    RTx-mediated TRPV1 gating mechanism. In the unstimulated apo state, the channel is closed both at the selectivity filter and S6 gate. 1 Initially, RTx binds with no significant conformational changes. 2 RTx binding induces S6 gate dilation. 3 The M644 sidechain flips outward, and the CD moves towards the channel core. 4 Finally, rearrangement of the PL, PH, TJ results in further dilation of the S6 gate.
    Figure Legend Snippet: RTx-mediated TRPV1 gating mechanism. In the unstimulated apo state, the channel is closed both at the selectivity filter and S6 gate. 1 Initially, RTx binds with no significant conformational changes. 2 RTx binding induces S6 gate dilation. 3 The M644 sidechain flips outward, and the CD moves towards the channel core. 4 Finally, rearrangement of the PL, PH, TJ results in further dilation of the S6 gate.

    Techniques Used: Binding Assay

    Thermal titration cryo-EM experiment and the effect of RTx on TRPV1 heat sensitivity. a A representative macroscopic current time-course (top panel) recorded from a HEK-293T cell expressing rat TRPV1 in response to the temperature ramp (10–50 °C) at a membrane potential of −60 mV and then followed by a saturating concentration of RTx (50 nM) and 20 µM ruthenium red (RR). The dashed line indicates zero current. The recorded temperature is shown in the middle panel. The Arrhenius plot for the temperature activation was shown in the bottom panel. Fitted Q 10 values for high (blue line) and low (red line) temperature ranges are shown. b A representative time-course recording for RTx-bound TRPV1 temperature sensitivity. First the channel was challenged by 10 nM RTx for ~20 s followed by a temperature ramp (10–48 °C), then a saturating concentration of RTx (50 nM) was introduced, and finally RR (20 µM) was applied to completely block the channel. The dashed line indicates zero current. The recorded temperature is shown in the middle panel and the Arrhenius plot for the temperature activation is shown in the bottom panel. Fitted Q 10 values for high and low temperature (T) ranges are shown. c Q 10 values as a function of I/I 50nM RTx for low and high temperature ranges. Each experiment was conducted as shown in a and b . The low T range Q 10 value is steady at 1.7, while the high T range Q 10 rapidly collapses from ~38 to ~3. Each pair of high and low temperature sensitivity data points represents independent time-course recordings from individual cells ( n = 17 cells). Source data are provided as a Source Data file. d Representative micrographs of TRPV1 recorded in the presence of 50 μM RTx at 4 °C, 25 °C and 48 °C, respectively. Cryo-EM maps of RTx-TRPV1 determined at 4 °C (class I, class II, and class III), 25 °C (class A and class B), and 48 °C (class α). Note the differences between central pore sizes amongst different classes at 4 °C. The classes not found in each dataset are shown as transparent. The pie charts depict particle distributions among classes for each dataset along with representative micrographs. Each pie chart represents an average value for four independent data processes (Supplementary Fig. 2a , b ).
    Figure Legend Snippet: Thermal titration cryo-EM experiment and the effect of RTx on TRPV1 heat sensitivity. a A representative macroscopic current time-course (top panel) recorded from a HEK-293T cell expressing rat TRPV1 in response to the temperature ramp (10–50 °C) at a membrane potential of −60 mV and then followed by a saturating concentration of RTx (50 nM) and 20 µM ruthenium red (RR). The dashed line indicates zero current. The recorded temperature is shown in the middle panel. The Arrhenius plot for the temperature activation was shown in the bottom panel. Fitted Q 10 values for high (blue line) and low (red line) temperature ranges are shown. b A representative time-course recording for RTx-bound TRPV1 temperature sensitivity. First the channel was challenged by 10 nM RTx for ~20 s followed by a temperature ramp (10–48 °C), then a saturating concentration of RTx (50 nM) was introduced, and finally RR (20 µM) was applied to completely block the channel. The dashed line indicates zero current. The recorded temperature is shown in the middle panel and the Arrhenius plot for the temperature activation is shown in the bottom panel. Fitted Q 10 values for high and low temperature (T) ranges are shown. c Q 10 values as a function of I/I 50nM RTx for low and high temperature ranges. Each experiment was conducted as shown in a and b . The low T range Q 10 value is steady at 1.7, while the high T range Q 10 rapidly collapses from ~38 to ~3. Each pair of high and low temperature sensitivity data points represents independent time-course recordings from individual cells ( n = 17 cells). Source data are provided as a Source Data file. d Representative micrographs of TRPV1 recorded in the presence of 50 μM RTx at 4 °C, 25 °C and 48 °C, respectively. Cryo-EM maps of RTx-TRPV1 determined at 4 °C (class I, class II, and class III), 25 °C (class A and class B), and 48 °C (class α). Note the differences between central pore sizes amongst different classes at 4 °C. The classes not found in each dataset are shown as transparent. The pie charts depict particle distributions among classes for each dataset along with representative micrographs. Each pie chart represents an average value for four independent data processes (Supplementary Fig. 2a , b ).

    Techniques Used: Titration, Cryo-EM Sample Prep, Expressing, Concentration Assay, Activation Assay, Blocking Assay

    Pore comparison across TRPV1 C, RTx , TRPV1 IC, RTx , TRPV1 IO, RTx , and TRPV1 O, RTx . The cryo-EM densities (grey surface) and respective models (cartoon) depicting bottom-up views of the S6 gate (top), top-down views of the selectivity filter (middle), top-down views of the monomeric outer pore (bottom), and local estimated resolutions for TRPV1 C, RTx a , blue, thresholding 0.12); TRPV1 IC, RTx b , cyan, thresholding 0.035); TRPV1 IO, RTx c , orange, thresholding 0.09); TRPV1 O, RTx,4 °C d , green, thresholding 0.1); TRPV1 O, RTx,25 °C e , brown, thresholding 0.08); and TRPV1 O, RTx,48 °C f , red, thresholding 0.033).
    Figure Legend Snippet: Pore comparison across TRPV1 C, RTx , TRPV1 IC, RTx , TRPV1 IO, RTx , and TRPV1 O, RTx . The cryo-EM densities (grey surface) and respective models (cartoon) depicting bottom-up views of the S6 gate (top), top-down views of the selectivity filter (middle), top-down views of the monomeric outer pore (bottom), and local estimated resolutions for TRPV1 C, RTx a , blue, thresholding 0.12); TRPV1 IC, RTx b , cyan, thresholding 0.035); TRPV1 IO, RTx c , orange, thresholding 0.09); TRPV1 O, RTx,4 °C d , green, thresholding 0.1); TRPV1 O, RTx,25 °C e , brown, thresholding 0.08); and TRPV1 O, RTx,48 °C f , red, thresholding 0.033).

    Techniques Used: Cryo-EM Sample Prep

    RTx-dependent conformational trajectory of TRPV1. a Comparison of the pore domain structures, only two subunits are shown for clarity, with the S6 gate (S6b), selectivity filter (SF), pore loop (PL) and pore helix (PH) as indicated. The pore profiles are shown as surfaces (gray). The red arrows indicate direction of movement. b Comparison of TRPV1 C,RTx (gray) and TRPV1 IC,RTx (green) structures (left) and close-up view of TRPV1 C,RTx and TRPV1 IC,RTx pore region (right). c The cryo-EM densities and the models for M644 in TRPV1 IC,RTx (green) and TRPV1 IO,RTx (gold). The cryo-EM map thresholdings are 0.03, and 0.04, respectively. d Comparison of TRPV1 IO, RTx (gold) and TRPV1 O, RTx (pink) outer pore region. Representative residues showing large motions are shown as sticks. TJ, turret junction. Phospholipids are shown as sticks and cryo-EM densities, with thresholding at 0.035 and 0.029, respectively.
    Figure Legend Snippet: RTx-dependent conformational trajectory of TRPV1. a Comparison of the pore domain structures, only two subunits are shown for clarity, with the S6 gate (S6b), selectivity filter (SF), pore loop (PL) and pore helix (PH) as indicated. The pore profiles are shown as surfaces (gray). The red arrows indicate direction of movement. b Comparison of TRPV1 C,RTx (gray) and TRPV1 IC,RTx (green) structures (left) and close-up view of TRPV1 C,RTx and TRPV1 IC,RTx pore region (right). c The cryo-EM densities and the models for M644 in TRPV1 IC,RTx (green) and TRPV1 IO,RTx (gold). The cryo-EM map thresholdings are 0.03, and 0.04, respectively. d Comparison of TRPV1 IO, RTx (gold) and TRPV1 O, RTx (pink) outer pore region. Representative residues showing large motions are shown as sticks. TJ, turret junction. Phospholipids are shown as sticks and cryo-EM densities, with thresholding at 0.035 and 0.029, respectively.

    Techniques Used: Cryo-EM Sample Prep

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    Alomone Labs s c
    Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. On day 19, αPD-L1 potentiated the nociceptor ablation mediated reduction in B16F10-OVA tumour volume. ( i–k ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into a cohort of nociceptor neuron-ablated mice 3 days prior to the injection given to nociceptor intact mice. Mice from each group with similar tumour size (~85mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~47%) in nociceptor-ablated mice than was observed in nociceptor-intact mice (~32%; i–j ). In addition, nociceptor ablation increased the proportion of intratumoral tumour-specific ( k ; defined as H-2Kb + ) CD8 + T cells. These differences were further enhanced by αPD-L1 treatment ( i–k ). ( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected <t>with</t> <t>RTX</t> <t>(s.c.,</t> 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). Experiments were independently repeated two ( c–g ), three ( h–q ) or six ( a , b ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( b–f , k , q ), or two-way ANOVA post-hoc Bonferroni ( h–j , l–p ). Source Data
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    Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. On day 19, αPD-L1 potentiated the nociceptor ablation mediated reduction in B16F10-OVA tumour volume. ( i–k ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into a cohort of nociceptor neuron-ablated mice 3 days prior to the injection given to nociceptor intact mice. Mice from each group with similar tumour size (~85mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~47%) in nociceptor-ablated mice than was observed in nociceptor-intact mice (~32%; i–j ). In addition, nociceptor ablation increased the proportion of intratumoral tumour-specific ( k ; defined as H-2Kb + ) CD8 + T cells. These differences were further enhanced by αPD-L1 treatment ( i–k ). ( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected <t>with</t> <t>RTX</t> <t>(s.c.,</t> 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). Experiments were independently repeated two ( c–g ), three ( h–q ) or six ( a , b ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( b–f , k , q ), or two-way ANOVA post-hoc Bonferroni ( h–j , l–p ). Source Data
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    Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. On day 19, αPD-L1 potentiated the nociceptor ablation mediated reduction in B16F10-OVA tumour volume. ( i–k ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into a cohort of nociceptor neuron-ablated mice 3 days prior to the injection given to nociceptor intact mice. Mice from each group with similar tumour size (~85mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~47%) in nociceptor-ablated mice than was observed in nociceptor-intact mice (~32%; i–j ). In addition, nociceptor ablation increased the proportion of intratumoral tumour-specific ( k ; defined as H-2Kb + ) CD8 + T cells. These differences were further enhanced by αPD-L1 treatment ( i–k ). ( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected with RTX (s.c., 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). Experiments were independently repeated two ( c–g ), three ( h–q ) or six ( a , b ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( b–f , k , q ), or two-way ANOVA post-hoc Bonferroni ( h–j , l–p ). Source Data

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend Snippet: Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. On day 19, αPD-L1 potentiated the nociceptor ablation mediated reduction in B16F10-OVA tumour volume. ( i–k ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into a cohort of nociceptor neuron-ablated mice 3 days prior to the injection given to nociceptor intact mice. Mice from each group with similar tumour size (~85mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~47%) in nociceptor-ablated mice than was observed in nociceptor-intact mice (~32%; i–j ). In addition, nociceptor ablation increased the proportion of intratumoral tumour-specific ( k ; defined as H-2Kb + ) CD8 + T cells. These differences were further enhanced by αPD-L1 treatment ( i–k ). ( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected with RTX (s.c., 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). Experiments were independently repeated two ( c–g ), three ( h–q ) or six ( a , b ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( b–f , k , q ), or two-way ANOVA post-hoc Bonferroni ( h–j , l–p ). Source Data

    Article Snippet: RTX (ref. ; Alomone Labs, R-400) was injected (subcutaneously; s.c.) in three dosages (30, 70 and 100 μg kg−1 ) into the right flank of Rag1 −/ − and C57BL/6J mice of around three weeks of age.

    Techniques: Injection, Mouse Assay, Activation Assay, FACS, Whisker Assay

    RTx-dependent long-range conformational changes in TRPV1. a Cylinder representation of TRPV1 in turquoise (one subunit) and gray (the rest of the channel). The approximate distances from the RTx binding site (reference residue Y511) to subdomains are shown. b The cryo-EM densities (surface) and respective models (sticks) depicting close-up views of the vanilloid binding sites in TRPV1 C, RTx (skyblue), thresholding 0.19, TRPV1 IO, RTx (yellow), thresholding 0.04, and TRPV1 O, RTx (pink), thresholding 0.033. c The cryo-EM densities (surface) and respective models (sticks) depicting close-up views of the selectivity filter in TRPV1 C,RTx (skyblue), thresholding 0.19, TRPV1 IC,RTx (green), thresholding 0.04, TRPV1 IO,RTx (yellow), thresholding 0.1, and TRPV1 O,RTx (pink), thresholding 0.033. d – e Close-up view of the overlays of TRPV1 C, RTx (skyblue), TRPV1 IO, RTx (yellow), and TRPV1 O, RTx (pink) regarding the cytoplasmic domain, and S6 gate e , respectively.

    Journal: Nature Communications

    Article Title: Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis

    doi: 10.1038/s41467-022-30602-2

    Figure Lengend Snippet: RTx-dependent long-range conformational changes in TRPV1. a Cylinder representation of TRPV1 in turquoise (one subunit) and gray (the rest of the channel). The approximate distances from the RTx binding site (reference residue Y511) to subdomains are shown. b The cryo-EM densities (surface) and respective models (sticks) depicting close-up views of the vanilloid binding sites in TRPV1 C, RTx (skyblue), thresholding 0.19, TRPV1 IO, RTx (yellow), thresholding 0.04, and TRPV1 O, RTx (pink), thresholding 0.033. c The cryo-EM densities (surface) and respective models (sticks) depicting close-up views of the selectivity filter in TRPV1 C,RTx (skyblue), thresholding 0.19, TRPV1 IC,RTx (green), thresholding 0.04, TRPV1 IO,RTx (yellow), thresholding 0.1, and TRPV1 O,RTx (pink), thresholding 0.033. d – e Close-up view of the overlays of TRPV1 C, RTx (skyblue), TRPV1 IO, RTx (yellow), and TRPV1 O, RTx (pink) regarding the cytoplasmic domain, and S6 gate e , respectively.

    Article Snippet: All cryo-EM samples in this study were prepared on freshly glow-discharged UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil), using a Leica EM GP2 to plunge freeze in LN2-cooled liquid ethane. (i) For TRPV14C, RTx , the TRPV1 sample was mixed with 50 µM RTx (Alomone, dissolved in DMSO) for 30 min before applying to the grid.

    Techniques: Binding Assay, Cryo-EM Sample Prep

    PH-S5-S6 triad hydrogen bond network in TRPV1 for RTx gating. a The cryo-EM maps (surface) and respective models (sticks) depicting the tripartite hydrogen bond network of PH-S5-S6 in TRPV1 C, RTx (skyblue), thresholding 0.15, TRPV1 IC, RTx (yellow), thresholding 0.045, TRPV1 IO, RTx (gold), thresholding 0.1, and TRPV1 O, RTx (pink), thresholding 0.04. The black dotted-lines indicate hydrogen bonds. The red dotted-lines indicate distance measurements between atoms where hydrogen bonds are broken. b – e TRPV1 Y584F and T641A reduce large cation permeabilty (YO-PRO-1, M.W. 376 Da) in the presence of RTx. Representative inside-out current traces of TRPV1 WT b , TRPV1 Y584F c , and TRPV1 T641A d . Current traces for basal, RTx (200 nM) activation (red trace) and intracellular application of 10 μM YO-PRO-1 (blue trace). e Summary of current inhibition by YO-PRO-1 (10 µM) of TRPV1 WT, TRPV1 Y584F and TRPV1 T641A after application of a saturating concentration of RTx (200 nM). Data are presented as mean ± s.e.m.; P

    Journal: Nature Communications

    Article Title: Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis

    doi: 10.1038/s41467-022-30602-2

    Figure Lengend Snippet: PH-S5-S6 triad hydrogen bond network in TRPV1 for RTx gating. a The cryo-EM maps (surface) and respective models (sticks) depicting the tripartite hydrogen bond network of PH-S5-S6 in TRPV1 C, RTx (skyblue), thresholding 0.15, TRPV1 IC, RTx (yellow), thresholding 0.045, TRPV1 IO, RTx (gold), thresholding 0.1, and TRPV1 O, RTx (pink), thresholding 0.04. The black dotted-lines indicate hydrogen bonds. The red dotted-lines indicate distance measurements between atoms where hydrogen bonds are broken. b – e TRPV1 Y584F and T641A reduce large cation permeabilty (YO-PRO-1, M.W. 376 Da) in the presence of RTx. Representative inside-out current traces of TRPV1 WT b , TRPV1 Y584F c , and TRPV1 T641A d . Current traces for basal, RTx (200 nM) activation (red trace) and intracellular application of 10 μM YO-PRO-1 (blue trace). e Summary of current inhibition by YO-PRO-1 (10 µM) of TRPV1 WT, TRPV1 Y584F and TRPV1 T641A after application of a saturating concentration of RTx (200 nM). Data are presented as mean ± s.e.m.; P

    Article Snippet: All cryo-EM samples in this study were prepared on freshly glow-discharged UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil), using a Leica EM GP2 to plunge freeze in LN2-cooled liquid ethane. (i) For TRPV14C, RTx , the TRPV1 sample was mixed with 50 µM RTx (Alomone, dissolved in DMSO) for 30 min before applying to the grid.

    Techniques: Cryo-EM Sample Prep, Activation Assay, Inhibition, Concentration Assay

    RTx-mediated TRPV1 gating mechanism. In the unstimulated apo state, the channel is closed both at the selectivity filter and S6 gate. 1 Initially, RTx binds with no significant conformational changes. 2 RTx binding induces S6 gate dilation. 3 The M644 sidechain flips outward, and the CD moves towards the channel core. 4 Finally, rearrangement of the PL, PH, TJ results in further dilation of the S6 gate.

    Journal: Nature Communications

    Article Title: Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis

    doi: 10.1038/s41467-022-30602-2

    Figure Lengend Snippet: RTx-mediated TRPV1 gating mechanism. In the unstimulated apo state, the channel is closed both at the selectivity filter and S6 gate. 1 Initially, RTx binds with no significant conformational changes. 2 RTx binding induces S6 gate dilation. 3 The M644 sidechain flips outward, and the CD moves towards the channel core. 4 Finally, rearrangement of the PL, PH, TJ results in further dilation of the S6 gate.

    Article Snippet: All cryo-EM samples in this study were prepared on freshly glow-discharged UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil), using a Leica EM GP2 to plunge freeze in LN2-cooled liquid ethane. (i) For TRPV14C, RTx , the TRPV1 sample was mixed with 50 µM RTx (Alomone, dissolved in DMSO) for 30 min before applying to the grid.

    Techniques: Binding Assay

    Thermal titration cryo-EM experiment and the effect of RTx on TRPV1 heat sensitivity. a A representative macroscopic current time-course (top panel) recorded from a HEK-293T cell expressing rat TRPV1 in response to the temperature ramp (10–50 °C) at a membrane potential of −60 mV and then followed by a saturating concentration of RTx (50 nM) and 20 µM ruthenium red (RR). The dashed line indicates zero current. The recorded temperature is shown in the middle panel. The Arrhenius plot for the temperature activation was shown in the bottom panel. Fitted Q 10 values for high (blue line) and low (red line) temperature ranges are shown. b A representative time-course recording for RTx-bound TRPV1 temperature sensitivity. First the channel was challenged by 10 nM RTx for ~20 s followed by a temperature ramp (10–48 °C), then a saturating concentration of RTx (50 nM) was introduced, and finally RR (20 µM) was applied to completely block the channel. The dashed line indicates zero current. The recorded temperature is shown in the middle panel and the Arrhenius plot for the temperature activation is shown in the bottom panel. Fitted Q 10 values for high and low temperature (T) ranges are shown. c Q 10 values as a function of I/I 50nM RTx for low and high temperature ranges. Each experiment was conducted as shown in a and b . The low T range Q 10 value is steady at 1.7, while the high T range Q 10 rapidly collapses from ~38 to ~3. Each pair of high and low temperature sensitivity data points represents independent time-course recordings from individual cells ( n = 17 cells). Source data are provided as a Source Data file. d Representative micrographs of TRPV1 recorded in the presence of 50 μM RTx at 4 °C, 25 °C and 48 °C, respectively. Cryo-EM maps of RTx-TRPV1 determined at 4 °C (class I, class II, and class III), 25 °C (class A and class B), and 48 °C (class α). Note the differences between central pore sizes amongst different classes at 4 °C. The classes not found in each dataset are shown as transparent. The pie charts depict particle distributions among classes for each dataset along with representative micrographs. Each pie chart represents an average value for four independent data processes (Supplementary Fig. 2a , b ).

    Journal: Nature Communications

    Article Title: Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis

    doi: 10.1038/s41467-022-30602-2

    Figure Lengend Snippet: Thermal titration cryo-EM experiment and the effect of RTx on TRPV1 heat sensitivity. a A representative macroscopic current time-course (top panel) recorded from a HEK-293T cell expressing rat TRPV1 in response to the temperature ramp (10–50 °C) at a membrane potential of −60 mV and then followed by a saturating concentration of RTx (50 nM) and 20 µM ruthenium red (RR). The dashed line indicates zero current. The recorded temperature is shown in the middle panel. The Arrhenius plot for the temperature activation was shown in the bottom panel. Fitted Q 10 values for high (blue line) and low (red line) temperature ranges are shown. b A representative time-course recording for RTx-bound TRPV1 temperature sensitivity. First the channel was challenged by 10 nM RTx for ~20 s followed by a temperature ramp (10–48 °C), then a saturating concentration of RTx (50 nM) was introduced, and finally RR (20 µM) was applied to completely block the channel. The dashed line indicates zero current. The recorded temperature is shown in the middle panel and the Arrhenius plot for the temperature activation is shown in the bottom panel. Fitted Q 10 values for high and low temperature (T) ranges are shown. c Q 10 values as a function of I/I 50nM RTx for low and high temperature ranges. Each experiment was conducted as shown in a and b . The low T range Q 10 value is steady at 1.7, while the high T range Q 10 rapidly collapses from ~38 to ~3. Each pair of high and low temperature sensitivity data points represents independent time-course recordings from individual cells ( n = 17 cells). Source data are provided as a Source Data file. d Representative micrographs of TRPV1 recorded in the presence of 50 μM RTx at 4 °C, 25 °C and 48 °C, respectively. Cryo-EM maps of RTx-TRPV1 determined at 4 °C (class I, class II, and class III), 25 °C (class A and class B), and 48 °C (class α). Note the differences between central pore sizes amongst different classes at 4 °C. The classes not found in each dataset are shown as transparent. The pie charts depict particle distributions among classes for each dataset along with representative micrographs. Each pie chart represents an average value for four independent data processes (Supplementary Fig. 2a , b ).

    Article Snippet: All cryo-EM samples in this study were prepared on freshly glow-discharged UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil), using a Leica EM GP2 to plunge freeze in LN2-cooled liquid ethane. (i) For TRPV14C, RTx , the TRPV1 sample was mixed with 50 µM RTx (Alomone, dissolved in DMSO) for 30 min before applying to the grid.

    Techniques: Titration, Cryo-EM Sample Prep, Expressing, Concentration Assay, Activation Assay, Blocking Assay

    Pore comparison across TRPV1 C, RTx , TRPV1 IC, RTx , TRPV1 IO, RTx , and TRPV1 O, RTx . The cryo-EM densities (grey surface) and respective models (cartoon) depicting bottom-up views of the S6 gate (top), top-down views of the selectivity filter (middle), top-down views of the monomeric outer pore (bottom), and local estimated resolutions for TRPV1 C, RTx a , blue, thresholding 0.12); TRPV1 IC, RTx b , cyan, thresholding 0.035); TRPV1 IO, RTx c , orange, thresholding 0.09); TRPV1 O, RTx,4 °C d , green, thresholding 0.1); TRPV1 O, RTx,25 °C e , brown, thresholding 0.08); and TRPV1 O, RTx,48 °C f , red, thresholding 0.033).

    Journal: Nature Communications

    Article Title: Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis

    doi: 10.1038/s41467-022-30602-2

    Figure Lengend Snippet: Pore comparison across TRPV1 C, RTx , TRPV1 IC, RTx , TRPV1 IO, RTx , and TRPV1 O, RTx . The cryo-EM densities (grey surface) and respective models (cartoon) depicting bottom-up views of the S6 gate (top), top-down views of the selectivity filter (middle), top-down views of the monomeric outer pore (bottom), and local estimated resolutions for TRPV1 C, RTx a , blue, thresholding 0.12); TRPV1 IC, RTx b , cyan, thresholding 0.035); TRPV1 IO, RTx c , orange, thresholding 0.09); TRPV1 O, RTx,4 °C d , green, thresholding 0.1); TRPV1 O, RTx,25 °C e , brown, thresholding 0.08); and TRPV1 O, RTx,48 °C f , red, thresholding 0.033).

    Article Snippet: All cryo-EM samples in this study were prepared on freshly glow-discharged UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil), using a Leica EM GP2 to plunge freeze in LN2-cooled liquid ethane. (i) For TRPV14C, RTx , the TRPV1 sample was mixed with 50 µM RTx (Alomone, dissolved in DMSO) for 30 min before applying to the grid.

    Techniques: Cryo-EM Sample Prep

    RTx-dependent conformational trajectory of TRPV1. a Comparison of the pore domain structures, only two subunits are shown for clarity, with the S6 gate (S6b), selectivity filter (SF), pore loop (PL) and pore helix (PH) as indicated. The pore profiles are shown as surfaces (gray). The red arrows indicate direction of movement. b Comparison of TRPV1 C,RTx (gray) and TRPV1 IC,RTx (green) structures (left) and close-up view of TRPV1 C,RTx and TRPV1 IC,RTx pore region (right). c The cryo-EM densities and the models for M644 in TRPV1 IC,RTx (green) and TRPV1 IO,RTx (gold). The cryo-EM map thresholdings are 0.03, and 0.04, respectively. d Comparison of TRPV1 IO, RTx (gold) and TRPV1 O, RTx (pink) outer pore region. Representative residues showing large motions are shown as sticks. TJ, turret junction. Phospholipids are shown as sticks and cryo-EM densities, with thresholding at 0.035 and 0.029, respectively.

    Journal: Nature Communications

    Article Title: Vanilloid-dependent TRPV1 opening trajectory from cryoEM ensemble analysis

    doi: 10.1038/s41467-022-30602-2

    Figure Lengend Snippet: RTx-dependent conformational trajectory of TRPV1. a Comparison of the pore domain structures, only two subunits are shown for clarity, with the S6 gate (S6b), selectivity filter (SF), pore loop (PL) and pore helix (PH) as indicated. The pore profiles are shown as surfaces (gray). The red arrows indicate direction of movement. b Comparison of TRPV1 C,RTx (gray) and TRPV1 IC,RTx (green) structures (left) and close-up view of TRPV1 C,RTx and TRPV1 IC,RTx pore region (right). c The cryo-EM densities and the models for M644 in TRPV1 IC,RTx (green) and TRPV1 IO,RTx (gold). The cryo-EM map thresholdings are 0.03, and 0.04, respectively. d Comparison of TRPV1 IO, RTx (gold) and TRPV1 O, RTx (pink) outer pore region. Representative residues showing large motions are shown as sticks. TJ, turret junction. Phospholipids are shown as sticks and cryo-EM densities, with thresholding at 0.035 and 0.029, respectively.

    Article Snippet: All cryo-EM samples in this study were prepared on freshly glow-discharged UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil), using a Leica EM GP2 to plunge freeze in LN2-cooled liquid ethane. (i) For TRPV14C, RTx , the TRPV1 sample was mixed with 50 µM RTx (Alomone, dissolved in DMSO) for 30 min before applying to the grid.

    Techniques: Cryo-EM Sample Prep