Journal: Pulmonary Circulation

Article Title: Stim-activated TRPC-ORAI channels in pulmonary hypertension induced by chronic intermittent hypoxia

doi: 10.1177/2045894020941484

Figure Lengend Snippet: Primers sequence’s characteristics uses in the analysis.

Article Snippet: After appropriate blocking (5% nonfat dried milk) the blots were probed with primary antibodies for TRPC1 (Santa Cruz, Dallas, TX, USA, sc-133076), TRPC6 (Santa Cruz, sc-515837), STIM1 (Santa Cruz, sc-166840), ORAI2 (Santa Cruz, sc-376757), and ORAI1 (Santa Cruz, sc-377281), diluted 1:1000 in 5% powdered nonfat dry milk overnight at 4°C.

Techniques: Sequencing

Journal: Pulmonary Circulation

Article Title: Stim-activated TRPC-ORAI channels in pulmonary hypertension induced by chronic intermittent hypoxia

doi: 10.1177/2045894020941484

Figure Lengend Snippet: Effect of CIH on pulmonary relative gene expression of STOC-forming subunits in rats. Pulmonary gene expression of (a) TRPC1, (b) TRPC4, (c) TRPC6, (d) ORAI1, and (e) ORAI 2 of control (empty bar, n = 6), 14d-CIH (lined bar, n = 6), 21d-CIH (gray bar, n = 7), and 28d-CIH (black bar, n = 7) animals. Values are expressed as 2−ΔΔCT in relation to the control. Values are mean ± SE. * p < 0.05 vs. control, † p < 0.05 vs. all. One-way ANOVA and Newman–Keuls post-test.

Article Snippet: After appropriate blocking (5% nonfat dried milk) the blots were probed with primary antibodies for TRPC1 (Santa Cruz, Dallas, TX, USA, sc-133076), TRPC6 (Santa Cruz, sc-515837), STIM1 (Santa Cruz, sc-166840), ORAI2 (Santa Cruz, sc-376757), and ORAI1 (Santa Cruz, sc-377281), diluted 1:1000 in 5% powdered nonfat dry milk overnight at 4°C.

Techniques: Gene Expression, Control

Journal: Pulmonary Circulation

Article Title: Stim-activated TRPC-ORAI channels in pulmonary hypertension induced by chronic intermittent hypoxia

doi: 10.1177/2045894020941484

Figure Lengend Snippet: Effect of the exposure to 28 days to CIH of the protein levels on STOC-forming subunits in the lungs. Quantification of the protein levels of (a) TRPC1, (b) TRPC6, (c) STIM1, (d) ORAI2, and (e) ORAI1 on control (empty bar, n = 6) and CIH-28d (black bar, n = 6) animals. Values are expressed as a ratio of β-actin. Values are mean ± SE. * p < 0.05 vs. control. Student’s unpaired t test.

Article Snippet: After appropriate blocking (5% nonfat dried milk) the blots were probed with primary antibodies for TRPC1 (Santa Cruz, Dallas, TX, USA, sc-133076), TRPC6 (Santa Cruz, sc-515837), STIM1 (Santa Cruz, sc-166840), ORAI2 (Santa Cruz, sc-376757), and ORAI1 (Santa Cruz, sc-377281), diluted 1:1000 in 5% powdered nonfat dry milk overnight at 4°C.

Techniques: Control

Journal: Open Biology

Article Title: TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

doi: 10.1098/rsob.120068

Figure Lengend Snippet: TRPC3 and TRPC6 are expressed in DRG neurons. In situ hybridization shows that TRPC3 ( a ) and TRPC6 ( b ) transcripts are found in almost all adult DRG neurons. The antisense TRPC3 probe does not stain neurons from TRPC3 knock-out mice and the antisense TRPC6 probe does not stain neurons from TRPC6 knock-out mice. See also electronic supplementary material, figure S1.

Article Snippet: Next, TRPC6 (NM_004621) was PCR-amplified from human cDNA clone SC310040 (OriGene) using the forward primer (5′ CGC GGA TCC TCG GGC GTT CCC GCC ATG AGC CAG) and the reverse primer (CCG GAA TTC ACG TTT TCT TGT TTA AAA GGT GG).

Techniques: In Situ Hybridization, Staining, Knock-Out

Journal: Open Biology

Article Title: TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

doi: 10.1098/rsob.120068

Figure Lengend Snippet: Selective deficits in innocuous mechanosensation in TRPC3/TRPC6 double knock-out (DKO) mice. ( a ) Fifty per cent threshold to mechanical stimulation using von Frey hairs in wild-type (WT) ( n = 8), TRPC3/TRPC6 DKO ( n = 9), TRPC3 knock-out (KO) ( n = 7) and TRPC6 KO ( n = 10) mice. ( b ) Responses of WT, TRPC3/TRPC6 DKO, TRPC3 KO and TRPC6 KO mice to a cotton bud applied to the plantar surface of the hind paw ( n = 6/group). ( c ) Response to noxious mechanical stimulation using a Randall–Selitto apparatus in WT ( n = 7), TRPC3/TRPC6 DKO ( n = 9), TRPC3 KO ( n = 6) and TRPC6 KO ( n = 7) mice. ( d ) Response to noxious thermal stimulation using Hargreaves’ apparatus in TRPC3/TRPC6 DKO ( n = 14), TRPC3 KO ( n = 11), TRPC6 KO ( n = 15) and WT ( n = 12) mice. Data are expressed as mean ± s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.001. See also electronic supplementary material, figure S2.

Article Snippet: Next, TRPC6 (NM_004621) was PCR-amplified from human cDNA clone SC310040 (OriGene) using the forward primer (5′ CGC GGA TCC TCG GGC GTT CCC GCC ATG AGC CAG) and the reverse primer (CCG GAA TTC ACG TTT TCT TGT TTA AAA GGT GG).

Techniques: Knock-Out, Randall–Selitto Test

Journal: Open Biology

Article Title: TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

doi: 10.1098/rsob.120068

Figure Lengend Snippet: Electrophysiological characterization of sensory neurons of single TRPC3 and TRPC6 knock-out mice. Mechanically evoked currents recorded from small-diameter DRG neurons with broad action potentials using the whole-cell patch clamp technique and classified based on their adaptation kinetic to a static mechanical stimulus applied to the soma. Currents are defined as rapidly adapting (RA), intermediately adapting (IA) and slowly adapting (SA). ( a ) The proportion of neurons expressing each current type from WT mice, TRPC3 knock-out mice and TRPC6 knock-out mice is shown. TRPC6 knock-out mice were statistically indistinguishable from WT (red, no response; black, RA; dark grey, IA; light grey, SA). ( b ) The magnitude of mechanically evoked peak currents in small-diameter DRG neurons voltage clamped at −70 mV. No significant difference between genotypes was observed (ANOVA, p > 0.05). Data are expressed as mean ± s.e.m.; ** p < 0.01 (black bars, WT; green bars, TRPC3 KO; blue bars, TRPC6 KO).

Article Snippet: Next, TRPC6 (NM_004621) was PCR-amplified from human cDNA clone SC310040 (OriGene) using the forward primer (5′ CGC GGA TCC TCG GGC GTT CCC GCC ATG AGC CAG) and the reverse primer (CCG GAA TTC ACG TTT TCT TGT TTA AAA GGT GG).

Techniques: Knock-Out, Patch Clamp, Expressing

Journal: Open Biology

Article Title: TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

doi: 10.1098/rsob.120068

Figure Lengend Snippet: Electrophysiological characterization of sensory neurons of TRPC3/TRPC6 DKO mice. ( a ) Exemplar whole-cell voltage clamp trace from a large-diameter (narrow action potential) mouse DRG neuron in response to a 7.8 μm membrane deformation (holding potential −70 mV). All large-diameter neurons responded with a rapidly adapting current (black line, WT; red line, TRPC3/TRPC6 DKO). ( b ) The stimulus response curve of mechanically evoked peak currents in large-diameter WT ( n = 12, black squares) and TRPC3/TRPC6 DKO ( n = 12, red circles) mouse DRG neurons with action potential widths less than 1 ms voltage clamped at −70 mV. ( c ) The magnitude of currents evoked by a 7.8 μm stimulus in small-diameter DRG neurons with action potential width more than 1 ms (holding potential −70 mV) (black bars, WT; red bars, TRPC3/TRPC6 DKO). ( d ) Small-diameter DRG neurons had mechanically activated currents which could be classified based on their adaptation kinetics to a static mechanical stimulus: rapidly adapting (RA), intermediately adapting (IA), slowly adapting (SA). The proportion of small-diameter mouse DRG neurons expressing each current type from WT and TRPC3/TRPC6 DKO mice is shown. A significant reduction in the number of neurons displaying RA currents and an increase in number of non-responsive neurons was observed in TRPC3/TRPC6 DKO mice ( χ 2 -test, p < 0.05) (red, no response; black, RA; dark grey, IA; light grey, SA).

Article Snippet: Next, TRPC6 (NM_004621) was PCR-amplified from human cDNA clone SC310040 (OriGene) using the forward primer (5′ CGC GGA TCC TCG GGC GTT CCC GCC ATG AGC CAG) and the reverse primer (CCG GAA TTC ACG TTT TCT TGT TTA AAA GGT GG).

Techniques: Expressing

Journal: Open Biology

Article Title: TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

doi: 10.1098/rsob.120068

Figure Lengend Snippet: Hair cells in mouse cochlea express TRPC3 and TRPC6 and are normal in TRPC3/TRPC6 DKO mice. ( a ) Exemplar surface confocal sections of organ of Corti from WT and TRPC3/TRPC6 DKO mice (11 weeks old). Rhodamine-phalloidin (red) + nuclear DAPI (blue) staining of the cochlea showing outer hair cells and pillar cells of the basal turn (20× magnification). Inset shows 63× magnification. ( b ) In situ hybridization shows that TRPC3 and TRPC6 are found in the hair cells of P21 mice. Arrows indicate the location of inner hair cells (IHC). Sense probes for TRPC3 and TRPC6 produced little or no staining (see also electronic supplementary material, figure S3).

Article Snippet: Next, TRPC6 (NM_004621) was PCR-amplified from human cDNA clone SC310040 (OriGene) using the forward primer (5′ CGC GGA TCC TCG GGC GTT CCC GCC ATG AGC CAG) and the reverse primer (CCG GAA TTC ACG TTT TCT TGT TTA AAA GGT GG).

Techniques: Staining, In Situ Hybridization, Produced

Journal: Open Biology

Article Title: TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

doi: 10.1098/rsob.120068

Figure Lengend Snippet: Selective hearing deficit in TRPC3/TRPC6 DKO mice. ( a ) Preyer reflex score of WT, TRPC3/TRPC6 DKO, TRPC3 KO and TRPC6 KO mice ( n = 6 in all groups) to a click box device that emits a 90 dSB toneburst centred around 20 kHz frequency. ( b ) Reaching response, a measure of vestibular function, of WT, TRPC3/TRPC6 DKO, TRPC3 KO and TRPC6 KO mice ( n = 6 in all groups). ( c ) Swim test ability, number of head submersions in 1 min ( n = 6 in all groups). ( d ) Auditory brain-stem recording (ABR) response thresholds to tone bursts of WT ( n = 8, black squares) and TRPC3/TRPC6 DKO mice ( n = 9, red circles). Data are expressed as mean ± s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.001. See also electronic supplementary material, movies S1–S5 for exemplar videos of the click box and swim test.

Article Snippet: Next, TRPC6 (NM_004621) was PCR-amplified from human cDNA clone SC310040 (OriGene) using the forward primer (5′ CGC GGA TCC TCG GGC GTT CCC GCC ATG AGC CAG) and the reverse primer (CCG GAA TTC ACG TTT TCT TGT TTA AAA GGT GG).

Techniques:

Journal: Open Biology

Article Title: TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

doi: 10.1098/rsob.120068

Figure Lengend Snippet: Mechano-electrical transduction in WT and TRPC3/TRPC6 DKO OHCs. ( a – c ) MET currents in response to 45 Hz sinusoidal force stimuli from a fluid jet in WT and TRPC3/TRPC6 OHCs. The holding potential was −84 mV and the membrane potential was stepped in 20 mV increments from −164 mV to +96 mV. Driver voltage (DV, amplitude 40 V) waveform to the fluid jet is shown above the current traces. Positive DV moves the hair bundles in the excitatory direction towards the kinocilium. Recordings in a – c are averages from two stimulus presentations each. ( a ) WT OHC, mid-basal coil P2 + 1. C m 5.6 pF; R s 0.80 MΩ. ( b ) TRPC3/TRPC6 DKO OHC, basal end of apical coil P2 + 2. C m 7.4 pF; R s 0.63 MΩ. ( c ) TRPC3/TRPC6 DKO OHC, mid-basal coil P2 + 2. C m 6.9 pF; R s 0.74 MΩ. ( d ) Current-voltage curves averaged from 4 WT OHCs from the basal coil (black squares), 5 apical-coil TRPC3/TRPC6 DKO OHCs (blue circles) and 8 basal-coil TRPC3/TRPC6 DKO OHCs (red circles).

Article Snippet: Next, TRPC6 (NM_004621) was PCR-amplified from human cDNA clone SC310040 (OriGene) using the forward primer (5′ CGC GGA TCC TCG GGC GTT CCC GCC ATG AGC CAG) and the reverse primer (CCG GAA TTC ACG TTT TCT TGT TTA AAA GGT GG).

Techniques: Transduction

Journal: Open Biology

Article Title: TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

doi: 10.1098/rsob.120068

Figure Lengend Snippet: Heterologous expression of TRPC3 with or without TRPC6 confers mechanosensitivity on some cell lines. Cells were transfected with constructs encoding TRPC3-IRES-GFP + TRPC6-IRES-DsRed2, TRPC3-IRES-GFP alone, TRPC6-IRES-DsRed2 alone or IRES-GFP and/or IRES-DsRed2. Cells were selected based on their fluorescence and voltage-clamped at −70 mV in whole-cell configuration. The magnitude of mechanically evoked currents in ( a ) HEK293a cells, ( b ) CHO K1 (( a , b ): black dots, empty vector ( n = 9); red circles, TRPC3/TRPC6 ( n = 9)) or ( c ) ND-C cells, a sensory neuron cell line is presented (black dots, empty vector ( n = 33); red circles, TRPC3/TRPC6 ( n = 11); green triangles, TRPC3 ( n = 12); blue diamonds, TRPC6 ( n = 13)). ( d ) Exemplar traces from ( c ) induced by a 9.9 μm membrane displacement (top plot: movement of the probe). ND-C cell were transfected with TRPC3 and TRPC6 and voltage-clamped at −70 mV in whole-cell configuration. Mechanically activated currents were elicited by an approximately 14 µm displacement of a glass probe at 10 μm distance from the cell (black lines, empty vector; red lines, TRPC3/TRPC6; green lines, TRPC3; blue lines, TRPC6). ( e ) Normalized current amplitudes from three cells before, during and 30 s after application of SKF96365. ( f ) Normalized current amplitudes ( n = 7) before and after application of FM1-43. ( g ) Representative current traces before (black), during (red) and 30 s after (blue) application of 100 μM SKF96365 or 15 μM FM1-43. Top plot shows movement of displacement of glass probe. Data represent mean ± s.e.m. ** p < 0.01; *** p < 0.001 (see also electronic supplementary material, figure S4).

Article Snippet: Next, TRPC6 (NM_004621) was PCR-amplified from human cDNA clone SC310040 (OriGene) using the forward primer (5′ CGC GGA TCC TCG GGC GTT CCC GCC ATG AGC CAG) and the reverse primer (CCG GAA TTC ACG TTT TCT TGT TTA AAA GGT GG).

Techniques: Expressing, Transfection, Construct, Fluorescence, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: The store-operated Ca 2+ channel Orai1α is required for agonist-evoked NF-κB activation by a mechanism dependent on PKCβ2

doi: 10.1016/j.jbc.2023.102882

Figure Lengend Snippet: TRPC1 and Orai3 are involved in carbachol (CCh)-evoked NF-κB activation in Orai1-KO cells. A, Orai1-KO HEK-293 cells (O1KO) transfected with either shTRPC1 (O1KO (shT1)), shTRPC6 (O1KO (shT6)), shOrai3 (O1KO (shO3)), or scramble plasmid (O1KO) were transfected with pNL3.2.NFkB-RE[NlucP/NF-kB-RE/Hygro]. Forty-eight hours later, cells were suspended in HBS containing 1 mM Ca 2+ and then stimulated for 5 h with 100 μM CCh or the vehicle (control) and lysed. Luciferase activity of the lysates was measured as described in the section. The luminescence in relative light units (RLUs) of unstimulated O1KO, O1KO(shT1), O1KO(shT6), and O1KO(shO3) HEK-293 cells were 598,259 ± 47,521, 601,025 ± 51,248, 599,987 ± 32,581, and 601,277 ± 39,985, respectively). B, O1KO, O1KO (shT1), and O1KO (shT6) HEK-293 cells were lysed and then subjected to 10% SDS-PAGE and Western blotting with anti-TRPC1 ( left panels ) or anti-TRPC6 ( right panels ) antibody. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of TRPC1 and TRPC6 bands was normalized to β-actin and quantified ( bar graph ). C, WT HEK-293 cells (WT), WT cells transfected with shOrai3 (shO3), O1KO cells, O1KO cells transfected with shOrai3 (O1KO (shO3)), and Orai1/Orai2/Orai3 triple-KO cells (TKO) transfected with pNL3.2.NFkB-RE[NlucP/NF-kB-RE/Hygro]. Forty-eight hours later, cells were suspended in HBS containing 1 mM Ca 2+ and stimulated for 5 h with 100 μM CCh or the vehicle (control) and lysed. Luciferase activity was measured as described in the section. The luminescence in RLUs of unstimulated WT, O1KO, O1KO(shO3), and TKO HEK-293 cells were 610,982 ± 48,875, 607,588 ± 47,510, 592,115 ± 37,514, and 597,323 ± 37,899, respectively. D, WT HEK-293 cells (WT) and WT cells transfected with shOrai3 (shO3) were lysed and subjected to 10% SDS-PAGE and Western blotting with anti-Orai3 antibody. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of Orai3 bands was normalized to β-actin and quantified ( bar graph ). E, WT, O1KO, TKO, and O1KO (shO3) HEK-293 cells were lysed and then subjected to 10% SDS-PAGE and Western blotting with anti-Orai3. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of Orai3 bands was normalized to β-actin and quantified ( bar graph ). For A, from left to right , n = 18, 14, 18, 13, 18, 12, 18, and 13; for C, from left to right , n = 30, 24, 12, 12, 18, 13, 18, 14, 18, and 12; n values correspond to separate experiments. Scatter plots are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn's test). ∗ p < 0.05 and ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as compared with CCh-treated O1KO HEK-293 cells ( A ) or CCh-treated WT HEK-293 cells ( C ). $ p < 0.05, $$ p < 0.01, and $$$$ p < 0 .0001 as compared with their respective control (untreated) cells. HBS, Hepes-buffered saline; HEK-293, human embryonic kidney 293 cell line.

Article Snippet: Rabbit polyclonal anti-TRPC6 antibody (catalog number: TA328771, epitope: amino acid residues 573–586 of TRPC6) was from Origene Technologies, Inc. Rabbit monoclonal anti-PKCβ2 antibody (clone Y125, epitope located in the region near the C terminus of PKCβ2; catalog number: ab32026) and rabbit monoclonal anti-Orai3 antibody (clone EPR22575-17, catalog number: ab254260) were purchased from Abcam.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, SDS Page, Western Blot, Saline