dic8 pip2  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences dic8 pip2
    PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue <t>diC8-PIP</t> 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with <t>diC8-PIP2</t> had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P
    Dic8 Pip2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 40 article reviews
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    93/100 stars

    Images

    1) Product Images from "UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes"

    Article Title: UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201311094

    PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P
    Figure Legend Snippet: PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P

    Techniques Used: Transferring

    2) Product Images from "Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane"

    Article Title: Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200609576

    PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.
    Figure Legend Snippet: PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.

    Techniques Used: Transfection, Inhibition

    3) Product Images from "Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane"

    Article Title: Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200609576

    PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.
    Figure Legend Snippet: PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.

    Techniques Used: Transfection, Inhibition

    4) Product Images from "Phosphoinositide binding by the SNX27 FERM domain regulates its localization at the immune synapse of activated T-cells"

    Article Title: Phosphoinositide binding by the SNX27 FERM domain regulates its localization at the immune synapse of activated T-cells

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.158204

    SNX27 binds PtdIns P  lipids through its FERM domain.  The binding of SNX27 or the SNX27 FERM domain to water-soluble PtdIns P  species was measured by ITC. The full-length SNX27 protein is able to bind to all PtdIns P s tested, whereas the FERM domain binds
    Figure Legend Snippet: SNX27 binds PtdIns P lipids through its FERM domain. The binding of SNX27 or the SNX27 FERM domain to water-soluble PtdIns P species was measured by ITC. The full-length SNX27 protein is able to bind to all PtdIns P s tested, whereas the FERM domain binds

    Techniques Used: Binding Assay

    5) Product Images from "ORP5 and ORP8 bind phosphatidylinositol-4, 5-biphosphate (PtdIns(4,5)P2) and regulate its level at the plasma membrane"

    Article Title: ORP5 and ORP8 bind phosphatidylinositol-4, 5-biphosphate (PtdIns(4,5)P2) and regulate its level at the plasma membrane

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00861-5

    ORD8 can extract and transport PtdIns(4,5) P 2 . a Bar graph showing the percentage of PtdIns P extracted from liposomes by Osh6p, ORD8, and ORD8 (H514A, H515A) mutant. Error bars indicate s.d.; n = 3. b Schematic of the assay employed to examine lipid transport by ORPs. See text for details. c , d Brain PtdIns(4) P c and PtdIns(4,5) P 2 d transport assay. Donor liposomes (L A ) were incubated with NBD–PH FAPP followed by addition of acceptor liposomes (L B ) doped or not with PtdSer. After 3 min, the protein was injected. The broken line signifies NBD–PH FAPP signal upon complete PtdIns(4) P and PtdIns(4,5) P 2 equilibration between liposomes. e Plot of initial brain PtdIns(4) P and PtdIns(4,5) P 2 transport rates by Osh6p and ORP8 ORD. Error bars indicate s.d.; n = 3. f PtdIns(4,5) P 2 detected by PH-PLC–GFP in HeLa cells overexpressing ORP5A or ORP5AΔPH. g Quantitation of intensity in f , including the ratio of GFP fluorescence of the PM vs. cytosol, and the ratio of GFP signals detected by the TIRF microscopy vs. total epifluorescence (mean + s.d.; *** P
    Figure Legend Snippet: ORD8 can extract and transport PtdIns(4,5) P 2 . a Bar graph showing the percentage of PtdIns P extracted from liposomes by Osh6p, ORD8, and ORD8 (H514A, H515A) mutant. Error bars indicate s.d.; n = 3. b Schematic of the assay employed to examine lipid transport by ORPs. See text for details. c , d Brain PtdIns(4) P c and PtdIns(4,5) P 2 d transport assay. Donor liposomes (L A ) were incubated with NBD–PH FAPP followed by addition of acceptor liposomes (L B ) doped or not with PtdSer. After 3 min, the protein was injected. The broken line signifies NBD–PH FAPP signal upon complete PtdIns(4) P and PtdIns(4,5) P 2 equilibration between liposomes. e Plot of initial brain PtdIns(4) P and PtdIns(4,5) P 2 transport rates by Osh6p and ORP8 ORD. Error bars indicate s.d.; n = 3. f PtdIns(4,5) P 2 detected by PH-PLC–GFP in HeLa cells overexpressing ORP5A or ORP5AΔPH. g Quantitation of intensity in f , including the ratio of GFP fluorescence of the PM vs. cytosol, and the ratio of GFP signals detected by the TIRF microscopy vs. total epifluorescence (mean + s.d.; *** P

    Techniques Used: Mutagenesis, Transport Assay, Incubation, Injection, Planar Chromatography, Quantitation Assay, Fluorescence, Microscopy

    A PtdIn P s gradient is required for PtdSer transport by ORD8. a Schematic of the assay employed to study PtdSer transport by ORD8 under a PtdIns P gradient. b PtdSer transport assay. Donor liposomes (L A ) were incubated with NBD-C2 Lact followed by addition of acceptor liposomes (L B ) doped with or without PtdIns P s. After 3 min, the transfer protein was injected. The broken line signifies NBD-C2 Lact signal upon complete PtdSer equilibration between liposomes. PtdIns(3) P and PtdIns(3,4,5) P 3 are 18:1/18:1. PtdIns(4) P and PtdIns(4,5) P 2 are from brain. c Plot of initial PtdSer transport rates demonstrates ORP8 transports PtdSer more efficiently under a PtdIns(4,5) P 2 gradient. Error bars indicate s.d.; n = 3. d Distribution of PtdSer as detected by Lact-C2–GFP in HeLa cells deficient in both ORP5 and ORP8. e The ratio of GFP signals detected by the TIRF microscopy vs. total epifluorescence (mean + s.d.; ** P
    Figure Legend Snippet: A PtdIn P s gradient is required for PtdSer transport by ORD8. a Schematic of the assay employed to study PtdSer transport by ORD8 under a PtdIns P gradient. b PtdSer transport assay. Donor liposomes (L A ) were incubated with NBD-C2 Lact followed by addition of acceptor liposomes (L B ) doped with or without PtdIns P s. After 3 min, the transfer protein was injected. The broken line signifies NBD-C2 Lact signal upon complete PtdSer equilibration between liposomes. PtdIns(3) P and PtdIns(3,4,5) P 3 are 18:1/18:1. PtdIns(4) P and PtdIns(4,5) P 2 are from brain. c Plot of initial PtdSer transport rates demonstrates ORP8 transports PtdSer more efficiently under a PtdIns(4,5) P 2 gradient. Error bars indicate s.d.; n = 3. d Distribution of PtdSer as detected by Lact-C2–GFP in HeLa cells deficient in both ORP5 and ORP8. e The ratio of GFP signals detected by the TIRF microscopy vs. total epifluorescence (mean + s.d.; ** P

    Techniques Used: Transport Assay, Incubation, Injection, Microscopy

    Non-canonical PH domain–PtdIns P association is indispensable for translocation of ORP5 to ER–PM junctions. a Cartoon representation of the crystal structure of ORP8 PH domain. b Ribbon representation of the superposition of ORP8 PH ( brown ), Osh3p PH ( cyan ; PDB id: 4IAP) 20 , Cert PH ( yellow ; PDB id: 2RSG) 20 , and ARHGAP9 PH ( green ; PDB id: 2P0D) 20 . c Model of ORP8 PH domain constructed by superimposing PH domains of ARHGAP9 20 , Cert 20 , and ARNO 20 , highlights the putative PtdIns P -binding site (between β1–β2 and β5–β6). The electrostatic surface representation shows the presence of a positively charged cleft presented by the β1–β2 and β5–β6 loops for PtdIns P binding. Electrostatic potential rendered surface was computed in ccp4mg 45 , negatively charged surfaces are shown in red , whereas positively charged surfaces are blue in color, colors are contoured from −0.5 V to +0.5. d A combined sequence alignment and secondary structure comparison of the PH domain of ORP5, ORP8, Osh3p, OSBP, and Cert. Secondary structure elements for ORP8 and Cert PH derived from the crystal structure are indicated above and below the alignment, respectively. Alignments were made with ESPript 2.2 (http://espript.ibcp.fr/ESPript/ESPript/) 46 . Red inverted triangles indicate the positively charged amino acids constituting the basic patch on the PH module of ORP8 and ORP5, which are absent in the other PH domains. Green triangles indicate the amino acids mediating PtdIns(4) P binding on the PH domains of Osh3p, Cert, and OSBP, which are absent in ORP5 and ORP8 PH domains. e The binding of ORP5 and ORP8 PH domain to PtdIns P s was measured by ITC. See Table 1 for a complete list of results. The binding of ORP5 R136Q, R179Q, ORP8 R158Q, R201Q, and WT ORP5 and ORP8 PH domains are shown in red , green , and black , respectively. Experiments were performed at 25 °C using 25 μM protein and 500 μM PtdIns P s. Top panels show raw data and bottom panels show integrated normalized data. f Co-localization of GFP and mCherry-tagged WT ORP5A and mutants (R136Q, R179Q) with MAPPER and DsRed-ER in HeLa cells. The GFP-fused ORP5A mutants (R136Q, R179Q) mainly overlap with DsRed-ER, but not MAPPER, suggesting that these mutants lose cortical ER localization. Bar = 10 μm
    Figure Legend Snippet: Non-canonical PH domain–PtdIns P association is indispensable for translocation of ORP5 to ER–PM junctions. a Cartoon representation of the crystal structure of ORP8 PH domain. b Ribbon representation of the superposition of ORP8 PH ( brown ), Osh3p PH ( cyan ; PDB id: 4IAP) 20 , Cert PH ( yellow ; PDB id: 2RSG) 20 , and ARHGAP9 PH ( green ; PDB id: 2P0D) 20 . c Model of ORP8 PH domain constructed by superimposing PH domains of ARHGAP9 20 , Cert 20 , and ARNO 20 , highlights the putative PtdIns P -binding site (between β1–β2 and β5–β6). The electrostatic surface representation shows the presence of a positively charged cleft presented by the β1–β2 and β5–β6 loops for PtdIns P binding. Electrostatic potential rendered surface was computed in ccp4mg 45 , negatively charged surfaces are shown in red , whereas positively charged surfaces are blue in color, colors are contoured from −0.5 V to +0.5. d A combined sequence alignment and secondary structure comparison of the PH domain of ORP5, ORP8, Osh3p, OSBP, and Cert. Secondary structure elements for ORP8 and Cert PH derived from the crystal structure are indicated above and below the alignment, respectively. Alignments were made with ESPript 2.2 (http://espript.ibcp.fr/ESPript/ESPript/) 46 . Red inverted triangles indicate the positively charged amino acids constituting the basic patch on the PH module of ORP8 and ORP5, which are absent in the other PH domains. Green triangles indicate the amino acids mediating PtdIns(4) P binding on the PH domains of Osh3p, Cert, and OSBP, which are absent in ORP5 and ORP8 PH domains. e The binding of ORP5 and ORP8 PH domain to PtdIns P s was measured by ITC. See Table 1 for a complete list of results. The binding of ORP5 R136Q, R179Q, ORP8 R158Q, R201Q, and WT ORP5 and ORP8 PH domains are shown in red , green , and black , respectively. Experiments were performed at 25 °C using 25 μM protein and 500 μM PtdIns P s. Top panels show raw data and bottom panels show integrated normalized data. f Co-localization of GFP and mCherry-tagged WT ORP5A and mutants (R136Q, R179Q) with MAPPER and DsRed-ER in HeLa cells. The GFP-fused ORP5A mutants (R136Q, R179Q) mainly overlap with DsRed-ER, but not MAPPER, suggesting that these mutants lose cortical ER localization. Bar = 10 μm

    Techniques Used: Translocation Assay, Construct, Binding Assay, Sequencing, Derivative Assay

    ORP5 and ORP8 bind PtdIns P species through a conserved binding mechanism. a The binding of purified ORP5 and ORP8 ORD domain to water-soluble PtdIns P species was measured by ITC. The WT ORD domain of ORP5 and ORP8 binds to all the PtdIns P s (Table 1 and Supplementary Fig. 8 ), including PtdIns(4) P . No binding was observed with ORD5 (H478A, H479A) and ORD8 (H514A, H515A) suggesting a conserved cargo-binding mode. Experiments were performed at 25 °C using 25 µM protein in the cell and 500 µM PtdIns P s injected from the syringe. Top panels show raw data, and bottom panels show integrated normalized data. b The homology model of human ORD8 and ORD5 ( orange ) is superimposed on the Osh6p ( blue , PDB id: 4Ph7, 4B2Z) 14 , 16 demonstrating their similar overall architectures. The blow up cartoon representation of the putative cargo-binding site in the ORP8/5 ORD is constructed by superposition of the Osh6p–PtdIns(4) P complex on the ORD8 homology model. The amino acids that mediate cargo binding in the Osh6p structure are conserved in both ORD8 ( orange ) and ORD5 ( black )
    Figure Legend Snippet: ORP5 and ORP8 bind PtdIns P species through a conserved binding mechanism. a The binding of purified ORP5 and ORP8 ORD domain to water-soluble PtdIns P species was measured by ITC. The WT ORD domain of ORP5 and ORP8 binds to all the PtdIns P s (Table 1 and Supplementary Fig. 8 ), including PtdIns(4) P . No binding was observed with ORD5 (H478A, H479A) and ORD8 (H514A, H515A) suggesting a conserved cargo-binding mode. Experiments were performed at 25 °C using 25 µM protein in the cell and 500 µM PtdIns P s injected from the syringe. Top panels show raw data, and bottom panels show integrated normalized data. b The homology model of human ORD8 and ORD5 ( orange ) is superimposed on the Osh6p ( blue , PDB id: 4Ph7, 4B2Z) 14 , 16 demonstrating their similar overall architectures. The blow up cartoon representation of the putative cargo-binding site in the ORP8/5 ORD is constructed by superposition of the Osh6p–PtdIns(4) P complex on the ORD8 homology model. The amino acids that mediate cargo binding in the Osh6p structure are conserved in both ORD8 ( orange ) and ORD5 ( black )

    Techniques Used: Binding Assay, Purification, Injection, Construct

    6) Product Images from "Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane"

    Article Title: Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200609576

    PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.
    Figure Legend Snippet: PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.

    Techniques Used: Transfection, Inhibition

    7) Product Images from "Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P2 hydrolysis"

    Article Title: Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P2 hydrolysis

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200910388

    The TRP domain mutants R696A and K705A are insensitive to changes in PI(4,5)P 2 . Inside-out patches were excised from HM1 cells expressing TRPV3 R696A or K705A, and voltage was clamped at +80 mV. (A and D) Representative current records from inside-out patches exposed to mAb (1:200 dilution; black bars, left). Open boxes indicate the time segments used for analysis of single-channel properties ( a , before exposure to mAb; b , after exposure to mAb). Expanded current traces (right) during the time period shown in box a or b . (B and E) All-points histograms of raw data shown in A and D and similar recordings (see Fig. S3) in nontreated controls or patches treated with 0.5 U/ml PI-PLC and 30 µM diC8-PI(4,5)P 2 . (C and F) Average NP OPEN in patches expressing R696A or K705A either before (open bars) or after (black bars) the addition of mAb or PI-PLC, or in nontreated control recordings. Data represent means ± SEM from n = 3–5 patches.
    Figure Legend Snippet: The TRP domain mutants R696A and K705A are insensitive to changes in PI(4,5)P 2 . Inside-out patches were excised from HM1 cells expressing TRPV3 R696A or K705A, and voltage was clamped at +80 mV. (A and D) Representative current records from inside-out patches exposed to mAb (1:200 dilution; black bars, left). Open boxes indicate the time segments used for analysis of single-channel properties ( a , before exposure to mAb; b , after exposure to mAb). Expanded current traces (right) during the time period shown in box a or b . (B and E) All-points histograms of raw data shown in A and D and similar recordings (see Fig. S3) in nontreated controls or patches treated with 0.5 U/ml PI-PLC and 30 µM diC8-PI(4,5)P 2 . (C and F) Average NP OPEN in patches expressing R696A or K705A either before (open bars) or after (black bars) the addition of mAb or PI-PLC, or in nontreated control recordings. Data represent means ± SEM from n = 3–5 patches.

    Techniques Used: Expressing, Planar Chromatography

    PI(4,5)P 2 decreases TRPV3 single-channel open probability. Inside-out patches excised from HM1 cells expressing WT TRPV3 (V m = +80 mV) were treated with 0.5 U/ml PI-PLC (open bar), followed by water-soluble diC8-phosphatidylinositol phosphate analogues. (A and B) Representative current records showing the [diC8-PI(4,5)P 2 ]–dependent decrease in TRPV3 channel activity (A) and lack of similar effect in vehicle-treated patches (B). Filled bars in B represent serial dilutions of vehicle that mimic those used to achieve the indicated [diC8-PI(4,5)P 2 ] shown in A. (C) A representative patch recording in which TRPV3 activity was stimulated by mAb and reversibly inhibited by 30 µM diC8-PI(4,5)P 2 . (D) The average diC8-PI(4,5)P 2 concentration–response relation (data represent means ± SEM; n = 5 patches). Solid line represents a Hill fit to the data for 1–60 µM diC8-PI(4,5)P 2 (IC 50 = 10.0 ± 4.5 µM). (E) Average change in NP OPEN induced by the addition of the indicated diC8-phosphatidylinositol phosphates. Data represent means ± SEM from n = 3–6 patches.
    Figure Legend Snippet: PI(4,5)P 2 decreases TRPV3 single-channel open probability. Inside-out patches excised from HM1 cells expressing WT TRPV3 (V m = +80 mV) were treated with 0.5 U/ml PI-PLC (open bar), followed by water-soluble diC8-phosphatidylinositol phosphate analogues. (A and B) Representative current records showing the [diC8-PI(4,5)P 2 ]–dependent decrease in TRPV3 channel activity (A) and lack of similar effect in vehicle-treated patches (B). Filled bars in B represent serial dilutions of vehicle that mimic those used to achieve the indicated [diC8-PI(4,5)P 2 ] shown in A. (C) A representative patch recording in which TRPV3 activity was stimulated by mAb and reversibly inhibited by 30 µM diC8-PI(4,5)P 2 . (D) The average diC8-PI(4,5)P 2 concentration–response relation (data represent means ± SEM; n = 5 patches). Solid line represents a Hill fit to the data for 1–60 µM diC8-PI(4,5)P 2 (IC 50 = 10.0 ± 4.5 µM). (E) Average change in NP OPEN induced by the addition of the indicated diC8-phosphatidylinositol phosphates. Data represent means ± SEM from n = 3–6 patches.

    Techniques Used: Expressing, Planar Chromatography, Activity Assay, Concentration Assay

    8) Product Images from "Direct inhibition of the cold-activated TRPM8 ion channel by G?q"

    Article Title: Direct inhibition of the cold-activated TRPM8 ion channel by G?q

    Journal: Nature cell biology

    doi: 10.1038/ncb2529

    Activated Gα q directly inhibits TRPM8 in excised patches . (a) Left: typical example of channel activity at +40mV in inside-out patches excised from HEK cells expressing TRPM8 after addition of 50nM Gα q * (Gα q pre-incubated with GTPγS) in the presence of 50μM DiC8-PIP 2 . Arrows indicate time of addition of Gα q *. Sections of traces are shown below at higher time resolution. Note that single-channel currents are smaller than in Fig. 3a , because membrane potential was lower. (b) Real time quantification of NP o in a. Red dashed lines give mean NP o over indicated time period. Inset shows silver stain of purified Gα q protein. Mean NP o before Gα q *, 0.151 ± 0.0088; after Gα q *, 0.013 ± 0.001; P
    Figure Legend Snippet: Activated Gα q directly inhibits TRPM8 in excised patches . (a) Left: typical example of channel activity at +40mV in inside-out patches excised from HEK cells expressing TRPM8 after addition of 50nM Gα q * (Gα q pre-incubated with GTPγS) in the presence of 50μM DiC8-PIP 2 . Arrows indicate time of addition of Gα q *. Sections of traces are shown below at higher time resolution. Note that single-channel currents are smaller than in Fig. 3a , because membrane potential was lower. (b) Real time quantification of NP o in a. Red dashed lines give mean NP o over indicated time period. Inset shows silver stain of purified Gα q protein. Mean NP o before Gα q *, 0.151 ± 0.0088; after Gα q *, 0.013 ± 0.001; P

    Techniques Used: Activity Assay, Expressing, Incubation, Silver Staining, Purification

    9) Product Images from "Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane"

    Article Title: Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200609576

    PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.
    Figure Legend Snippet: PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.

    Techniques Used: Transfection, Inhibition

    10) Product Images from "PIP2 regulation of TRPC5 channel activation and desensitization"

    Article Title: PIP2 regulation of TRPC5 channel activation and desensitization

    Journal: bioRxiv

    doi: 10.1101/2021.01.25.428089

    Regulation by OAG or PIP 2 does not alter the surface-density of YFP-TRPC5 channels. YFP-tagged mTRPC5 or mTRPC5-T972A channels were expressed in HEK293T cells and studied by patch-clamp TIRF. The number of fluorescent particles was determined 200 s after whole-cell mode was established to allow dialysis of the cells with control solution (blue), 200 μM OAG (green) or 200 μM diC8-PIP 2 (purple). Cells were studied with or without optogenetic activation of 5’-ptaseOCRL (white stripped bars) or following incubation with 1 μM staurosporine (red). Bar graphs represent particle-density as mean ± s.e.m. number of fluorescent particles in the TIRF field in 3-6 random 10 x 10 μm squares per cell and from 4-6 cells per group. A ) TIRF image showing YFP-tagged wild type mTRPC5 channels at the cell surface. Four example particles corresponding to single TRPC5 channels are highlighted in cyan. B ) Bar graphs summarizing the density of fluorescent particles indicating no change from control values under any of the conditions studied. C ) TIRF image showing YFP-tagged mTRPC5-T972A channels at the cell surface. Four example particles corresponding to single TRPC5 channels are highlighted in cyan. D ) Bar graphs summarizing the density of fluorescent particles indicating no change from control values under any of the conditions studied.
    Figure Legend Snippet: Regulation by OAG or PIP 2 does not alter the surface-density of YFP-TRPC5 channels. YFP-tagged mTRPC5 or mTRPC5-T972A channels were expressed in HEK293T cells and studied by patch-clamp TIRF. The number of fluorescent particles was determined 200 s after whole-cell mode was established to allow dialysis of the cells with control solution (blue), 200 μM OAG (green) or 200 μM diC8-PIP 2 (purple). Cells were studied with or without optogenetic activation of 5’-ptaseOCRL (white stripped bars) or following incubation with 1 μM staurosporine (red). Bar graphs represent particle-density as mean ± s.e.m. number of fluorescent particles in the TIRF field in 3-6 random 10 x 10 μm squares per cell and from 4-6 cells per group. A ) TIRF image showing YFP-tagged wild type mTRPC5 channels at the cell surface. Four example particles corresponding to single TRPC5 channels are highlighted in cyan. B ) Bar graphs summarizing the density of fluorescent particles indicating no change from control values under any of the conditions studied. C ) TIRF image showing YFP-tagged mTRPC5-T972A channels at the cell surface. Four example particles corresponding to single TRPC5 channels are highlighted in cyan. D ) Bar graphs summarizing the density of fluorescent particles indicating no change from control values under any of the conditions studied.

    Techniques Used: Patch Clamp, Activation Assay, Incubation

    11) Product Images from "UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes"

    Article Title: UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201311094

    PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P
    Figure Legend Snippet: PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P

    Techniques Used: Transferring

    12) Product Images from "Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane"

    Article Title: Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200609576

    PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.
    Figure Legend Snippet: PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.

    Techniques Used: Transfection, Inhibition

    13) Product Images from "PIP2 regulation of TRPC5 channel activation and desensitization"

    Article Title: PIP2 regulation of TRPC5 channel activation and desensitization

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2021.100726

    Regulation by OAG or PIP 2 does not alter the surface density of YFP–TRPC5 channels. YFP-tagged mTRPC5 or mTRPC5–T972A channels were expressed in HEK293T cells and studied by patch clamp TIRF. The number of fluorescent particles was determined 200 s after whole-cell mode was established to allow dialysis of the cells with the control solution ( blue ), 200 μM OAG ( green ) or 200 μM diC8–PIP 2 ( purple ). Cells were studied with or without optogenetic activation of 5’-ptaseOCRL ( white stripped bars ) or after incubation with 1 μM staurosporine ( red ). Bar graphs represent particle density as the mean ± SD number of fluorescent particles in the TIRF field in 3 to 6 random 10 × 10 μm squares per cell and from 4 to 6 cells per group. A , TIRF image showing YFP-tagged WT mTRPC5 channels at the cell surface. Four example particles corresponding to single TRPC5 channels are highlighted in cyan . B , the bar graphs summarizing the density of fluorescent particles indicating no change from control values under any of the conditions studied. C , TIRF image showing YFP-tagged mTRPC5–T972A channels at the cell surface. Four example particles corresponding to single TRPC5 channels are highlighted in cyan . D , the bar graphs summarizing the density of fluorescent particles indicating no change from control values under any of the conditions studied. V alues reported as mean ± SD, p-values established using Students’ t test. diC 8 –PIP 2 , dioctanoyl-glycerol-PIP 2 ; TRPC5, transient receptor potential canonical type 5.
    Figure Legend Snippet: Regulation by OAG or PIP 2 does not alter the surface density of YFP–TRPC5 channels. YFP-tagged mTRPC5 or mTRPC5–T972A channels were expressed in HEK293T cells and studied by patch clamp TIRF. The number of fluorescent particles was determined 200 s after whole-cell mode was established to allow dialysis of the cells with the control solution ( blue ), 200 μM OAG ( green ) or 200 μM diC8–PIP 2 ( purple ). Cells were studied with or without optogenetic activation of 5’-ptaseOCRL ( white stripped bars ) or after incubation with 1 μM staurosporine ( red ). Bar graphs represent particle density as the mean ± SD number of fluorescent particles in the TIRF field in 3 to 6 random 10 × 10 μm squares per cell and from 4 to 6 cells per group. A , TIRF image showing YFP-tagged WT mTRPC5 channels at the cell surface. Four example particles corresponding to single TRPC5 channels are highlighted in cyan . B , the bar graphs summarizing the density of fluorescent particles indicating no change from control values under any of the conditions studied. C , TIRF image showing YFP-tagged mTRPC5–T972A channels at the cell surface. Four example particles corresponding to single TRPC5 channels are highlighted in cyan . D , the bar graphs summarizing the density of fluorescent particles indicating no change from control values under any of the conditions studied. V alues reported as mean ± SD, p-values established using Students’ t test. diC 8 –PIP 2 , dioctanoyl-glycerol-PIP 2 ; TRPC5, transient receptor potential canonical type 5.

    Techniques Used: Patch Clamp, Activation Assay, Incubation

    14) Product Images from "Hyperforin/HP-β-Cyclodextrin Enhances Mechanosensitive Ca2+ Signaling in HaCaT Keratinocytes and in Atopic Skin Ex Vivo Which Accelerates Wound Healing"

    Article Title: Hyperforin/HP-β-Cyclodextrin Enhances Mechanosensitive Ca2+ Signaling in HaCaT Keratinocytes and in Atopic Skin Ex Vivo Which Accelerates Wound Healing

    Journal: BioMed Research International

    doi: 10.1155/2017/8701801

    The effects of various inhibitors on the stretch-induced Ca 2+ responses and wound closure. (a) The time course of changes in the fluorescence intensity of Fluo-8 due to a transient 20% stretch in hyperforin/HP- β -CD-treated HaCaT cells (control). Each color trace indicated the data at different distances of 0, 60, 120, 180, and 240 from the wound edge (inset image). The intensity was normalized to the peak value obtained with ionomycin treatment at the end of each experiment. (b) The effects of Gd 3+ on the stretch-induced Ca 2+ response as a typical example of the blocking effects of the inhibitors. Gd 3+ (10 μ M) was applied at 10 min before the application of a 20% stretch. (c) The effects of various inhibitors on 20% transient stretch-induced Ca 2+ responses in hyperforin/HP- β -CD-treated HaCaT cells. The intensity traces at each distance from the wound edge were averaged and normalized to the peak intensity obtained with ionomycin treatment. The data show the average of the peak values obtained in 3–6 separate experiments. Various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), U73122 (10 μ M), diC8-PIP 2 (5 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M), were applied at 10 min before the stretch stimulation. A Ca 2+ -free condition was achieved by changing the medium to Ca 2+ -free medium that contained 0.5 μ M EGTA. All of the quantitative data are shown as the mean (±SEM). (d) The effects of various inhibitors on the stretch facilitated wound closure in hyperforin/HP- β -CD-treated HaCaT cells. Confluent cell cultures were scratched and allowed to migrate for 6 h under a sustained 20% stretch in a medium that contained various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M) or in nominally Ca 2+ -free medium. shTRPC6 was applied to the cells for 3 h; the cells were then grown to confluence. Representative DIC images (upper panel) and the means of 3–8 wound closure experiments at 6 h after scratching (lower panel) are shown. All of the quantitative data are shown as the mean (±SEM).
    Figure Legend Snippet: The effects of various inhibitors on the stretch-induced Ca 2+ responses and wound closure. (a) The time course of changes in the fluorescence intensity of Fluo-8 due to a transient 20% stretch in hyperforin/HP- β -CD-treated HaCaT cells (control). Each color trace indicated the data at different distances of 0, 60, 120, 180, and 240 from the wound edge (inset image). The intensity was normalized to the peak value obtained with ionomycin treatment at the end of each experiment. (b) The effects of Gd 3+ on the stretch-induced Ca 2+ response as a typical example of the blocking effects of the inhibitors. Gd 3+ (10 μ M) was applied at 10 min before the application of a 20% stretch. (c) The effects of various inhibitors on 20% transient stretch-induced Ca 2+ responses in hyperforin/HP- β -CD-treated HaCaT cells. The intensity traces at each distance from the wound edge were averaged and normalized to the peak intensity obtained with ionomycin treatment. The data show the average of the peak values obtained in 3–6 separate experiments. Various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), U73122 (10 μ M), diC8-PIP 2 (5 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M), were applied at 10 min before the stretch stimulation. A Ca 2+ -free condition was achieved by changing the medium to Ca 2+ -free medium that contained 0.5 μ M EGTA. All of the quantitative data are shown as the mean (±SEM). (d) The effects of various inhibitors on the stretch facilitated wound closure in hyperforin/HP- β -CD-treated HaCaT cells. Confluent cell cultures were scratched and allowed to migrate for 6 h under a sustained 20% stretch in a medium that contained various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M) or in nominally Ca 2+ -free medium. shTRPC6 was applied to the cells for 3 h; the cells were then grown to confluence. Representative DIC images (upper panel) and the means of 3–8 wound closure experiments at 6 h after scratching (lower panel) are shown. All of the quantitative data are shown as the mean (±SEM).

    Techniques Used: Fluorescence, Blocking Assay

    15) Product Images from "UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes"

    Article Title: UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201311094

    PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P
    Figure Legend Snippet: PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P

    Techniques Used: Transferring

    16) Product Images from "UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes"

    Article Title: UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201311094

    PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P
    Figure Legend Snippet: PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P

    Techniques Used: Transferring

    17) Product Images from "UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes"

    Article Title: UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201311094

    PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P
    Figure Legend Snippet: PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P

    Techniques Used: Transferring

    18) Product Images from "Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane"

    Article Title: Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200609576

    PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.
    Figure Legend Snippet: PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.

    Techniques Used: Transfection, Inhibition

    19) Product Images from "UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes"

    Article Title: UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201311094

    PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P
    Figure Legend Snippet: PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P

    Techniques Used: Transferring

    20) Product Images from "A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function *"

    Article Title: A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.645671

    Modulation of Ca v 1.2S and Ca v 1.2S 4E currents by wortmannin/m-3M3FBS or diC8-PIP 2 treatment. A , Ca v 1.2S, perfused with bath solution (control; gray circles ; data are shown for seven cells; due to different recording duration, means ± S.E. reflect n = 7 until 410 s, n = 6 until 450 s), 100 μ m intracellular diC8-PIP2 ( gray triangles ; means ± S.E., n = 4), or extracellular 20 μ m wortmannin and 50 μ m m-3M3FBS ( black squares ; means ± S.E. for n = 8 until 280 s, n = 3 until 450 s). B , Ca v 1.2S 4E , control ( red circles ; n = 9 cells until 440 s, n = 8 cells until 450 s), 100 μ m intracellular diC8 ( red triangles ; n = 4 until 420 s, n = 3 until 450 s), or extracellular 20 μ m wortmannin and 50 μ m m-3M3FBS ( white squares ; n = 8 until 320 s, n = 6 until 450 s). Statistical analysis was performed using two-way analysis of variance of all data sets followed by Bonferroni post hoc test. Wortmannin/m-3M3FBS treatment significantly enhanced current decrease over time for both constructs ( a , p
    Figure Legend Snippet: Modulation of Ca v 1.2S and Ca v 1.2S 4E currents by wortmannin/m-3M3FBS or diC8-PIP 2 treatment. A , Ca v 1.2S, perfused with bath solution (control; gray circles ; data are shown for seven cells; due to different recording duration, means ± S.E. reflect n = 7 until 410 s, n = 6 until 450 s), 100 μ m intracellular diC8-PIP2 ( gray triangles ; means ± S.E., n = 4), or extracellular 20 μ m wortmannin and 50 μ m m-3M3FBS ( black squares ; means ± S.E. for n = 8 until 280 s, n = 3 until 450 s). B , Ca v 1.2S 4E , control ( red circles ; n = 9 cells until 440 s, n = 8 cells until 450 s), 100 μ m intracellular diC8 ( red triangles ; n = 4 until 420 s, n = 3 until 450 s), or extracellular 20 μ m wortmannin and 50 μ m m-3M3FBS ( white squares ; n = 8 until 320 s, n = 6 until 450 s). Statistical analysis was performed using two-way analysis of variance of all data sets followed by Bonferroni post hoc test. Wortmannin/m-3M3FBS treatment significantly enhanced current decrease over time for both constructs ( a , p

    Techniques Used: Construct

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    Echelon Biosciences dic8 pip2
    PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue <t>diC8-PIP</t> 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with <t>diC8-PIP2</t> had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P
    Dic8 Pip2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P

    Journal: The Journal of General Physiology

    Article Title: UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes

    doi: 10.1085/jgp.201311094

    Figure Lengend Snippet: PIP 2 regulates UVR-activated TRPA1 currents. (A) The UVR (240 mJ/cm 2 )-induced whole-cell current of a representative HEM dialyzed with the PIP 2 analogue diC8-PIP 2 and measured at +80 mV immediately after break-in was significantly reduced after 5 min of dialysis to allow diC8-PIP 2 to diffuse into the cell. (B) HEMs dialyzed with diC8-PIP2 had reduced UVR photocurrent densities at all voltages when stimulated after 5 min of dialysis, compared with immediately after break-in. (C) HEMs exposed to a submaximal UVR dose (160 mJ/cm 2 ) elicited a small but significant increase in whole-cell current at +80 mV (first trace). Including poly-lysine (polyK, 50 mg/ml) in the patch pipette did not alter the baseline current, but significantly potentiated the UVR (160 mJ/cm 2 )-induced current after 5 min of dialysis (second and third trace from the same representative cell). The augmented UVR-induced current measured in the presence of polyK was abolished by treatment with the TRPA1 antagonist HC-030031 (HC; 100 µM; fourth trace). (D) Dialysis with diC8-PIP 2 reduced mean peak UVR photocurrents by ∼93% compared with control. n = 7 cells, P

    Article Snippet: DiC8-PIP2 was from Echelon Biosciences.

    Techniques: Transferring

    PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.

    Journal: The Journal of General Physiology

    Article Title: Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

    doi: 10.1085/jgp.200609576

    Figure Lengend Snippet: PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine ( > 300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels ( Zhang et al., 2005b ). Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.

    Article Snippet: Other potential explanations include nonspecific effects of polylysine and greater activity of DiC8-PIP2 compared with native PIP2.

    Techniques: Transfection, Inhibition

    SNX27 binds PtdIns P  lipids through its FERM domain.  The binding of SNX27 or the SNX27 FERM domain to water-soluble PtdIns P  species was measured by ITC. The full-length SNX27 protein is able to bind to all PtdIns P s tested, whereas the FERM domain binds

    Journal: Journal of Cell Science

    Article Title: Phosphoinositide binding by the SNX27 FERM domain regulates its localization at the immune synapse of activated T-cells

    doi: 10.1242/jcs.158204

    Figure Lengend Snippet: SNX27 binds PtdIns P lipids through its FERM domain. The binding of SNX27 or the SNX27 FERM domain to water-soluble PtdIns P species was measured by ITC. The full-length SNX27 protein is able to bind to all PtdIns P s tested, whereas the FERM domain binds

    Article Snippet: Soluble diC8 PtdIns P s were purchased from Echelon Biosciences.

    Techniques: Binding Assay