Journal: Journal of Cellular and Molecular Medicine

Article Title: Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels

doi: 10.1111/j.1582-4934.2009.00890.x

Figure Lengend Snippet: Expression of TRPC1, TRPC3 and TRPC5 channels in mesenteric arterioles from SHR. Representative immunoblottings (A) and summary data (B) of TRPC1, TRPC3, TRPC4, TRPC5,TRPC6 channels and TRPC3 antibody with its respective antigenic peptide the protein expression in mesenteric arterioles from WKY (open bars) and SHR (filled bars). Data are mean ± S.E.M. of n = 4 independent experiments. * P < 0.05 or ** P < 0.01 by two-tailed Student’s t-test. (C) Light microscopy of mesenteric arterioles from SHR (upper panels) and immunohistochemistry (lower panels) of TRPC1, TRPC3 and TRPC5 protein expression in mesenteric arterioles from SHR. (D) Light microscopy of mesenteric arterioles from WKY (upper panels) and immunohistochemistry (lower panels) of TRPC1, TRPC3 and TRPC5 protein expression in mesenteric arterioles from WKY. Immunohistochemistry using specific antibodies labelled with green shows TRPC1, TRPC3 and TRPC5 protein expression in mesenteric arterioles. Control indicates immunohistochemistry after pre-incubation of the primary TRPC3 antibody with antigenic peptide for 12 hrs at 4°C. Bar denotes 200 μm. (43.0 ± 15%; P = n.s.; n = 6, ). In the presence of anti-TRPC5 antibodies the norepinephrine-induced vasomotion was not significantly affected (45.0 ± 11%; P = n.s. ). In the presence of anti-TRPC1 plus anti-TRPC3 antibodies the norepinephrine-induced vasomotion was significantly reduced to 29.0 ± 8% ( P < 0.05; n = 6, ). Furthermore, pre-incubation of mesenteric arterioles with anti-β-actin antibodies did not significantly affect norepinephrine-induced vasomotion when compared to control conditions (59.8 ± 15%, P = n.s.; n = 6, ). Incorporation of TRPC antibodies into mesenteric arteries by pinocytosis was facilitated by changing the bathing solution to a hypotonic solution according to recent literature .

Article Snippet: The membranes were incubated with primary rabbit antibodies against TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 (1:200; Alomone Labs), followed by incubation with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody.

Techniques: Expressing, Two Tailed Test, Light Microscopy, Immunohistochemistry, Incubation

Journal: Science Advances

Article Title: Cryo-EM structure of TRPC5 at 2.8-Å resolution reveals unique and conserved structural elements essential for channel function

doi: 10.1126/sciadv.aaw7935

Figure Lengend Snippet: ( A ) Side view of TRPC5’s pore region with chains A and C compared with TRPC4 (gray). The ion conduction pathway is shown as dots and mapped using HOLE with key amino acid residues labeled. ( B ) Pore radius along the central axis. The side chains of glycine form a narrow constriction at the selectivity filter. “INQ” motif forms the lower gate. ( C ) Side view of TRPC5 (blue) monomer subunit compared with TRPC4 (gray). Differences in the organizations of a linker (red arrowheads) of S5 and the small loop above the pore helix of the extracellular pore domain are enlarged. ( D ) Sequence of the mouse TRPC5 aligned to TRPC4 and other TRPC subfamily members (Clustal Omega) between S5 and S6 including the linker, pore helix, and pore loop. Regions corresponding to different extracellular pore domains are indicated by the red arrowheads. The two cysteines forming disulfide bonds, a conserved “LFW” motif, and the selectivity filter are highlighted. ( E to G ) Patch clamp recordings of TRPC5 mutants in response to channel activators Gd 3+ and EA.

Article Snippet: Primary antibodies against TRPC5 and TRPC4 were purchased from Sangon Biotech (Shanghai, China) and Proteintech (Wuhan, China), respectively.

Techniques: Labeling, Sequencing, Patch Clamp

Journal: Science Advances

Article Title: Cryo-EM structure of TRPC5 at 2.8-Å resolution reveals unique and conserved structural elements essential for channel function

doi: 10.1126/sciadv.aaw7935

Figure Lengend Snippet: ( A ) Superimposed side views of mouse TRPC5 subunit (blue) compared with other TRPC family members, including mouse TRPC4 [Protein Data Bank (PDB) ID code 5Z96, gray] , human TRPC3 (PDB ID 5ZBG, pink) , and human TRPC6 (PDB ID 5YX9, yellow) . ( B ) The conserved LFW motif in the pore helix from a π-π interaction that stabilizes the pore region. ( C ) Key pore loop disulfide bond between Cys 553 and Cys 548 in TRPC5 (black arrowhead) and the corresponding pore loop disulfide bond in TRPC4 that close to a linker (red arrowheads) of S5 and loop above the pore helix. This disulfide bond is not present in TRPC3 or TRPC6. ( D ) Differences in the organization of the S3 helix between the TRPC4/5 and TRPC3/6. The S3 helices of TRPC3 and TRPC6 are longer than those of TRPC5 and TRPC4. ( E ) Patch clamp recordings of wild-type (WT) TRPC5 and cysteine mutants in response to the reducing agent [dithiothreitol (DTT)] and channel activator (EA). ( F ) Localization of enhanced yellow fluorescent protein–tagged WT TRPC5 and cysteine mutants in HEK293 cells. ( G ) Quantification of cell surface expression of TRPC5 cysteine mutants ( n = 3). Mean values are shown as gray bars. By analysis of variance (ANOVA), there is no statistically significant difference. a.u., arbitrary units.

Article Snippet: Primary antibodies against TRPC5 and TRPC4 were purchased from Sangon Biotech (Shanghai, China) and Proteintech (Wuhan, China), respectively.

Techniques: Patch Clamp, Expressing

Journal: Science Advances

Article Title: Cryo-EM structure of TRPC5 at 2.8-Å resolution reveals unique and conserved structural elements essential for channel function

doi: 10.1126/sciadv.aaw7935

Figure Lengend Snippet: ( A to F and I ) The time course of whole-cell currents measured at +80 and −80 mV and representative I-V relationships in different conditions as labeled. TRPC5-C4motif, TRPC5 mutant carrying the TRPC4 motif ETKGLS; TRPC4-C5motif, TRPC4 mutant with the TRPC5 motif TRAIDEPNN; TRPC5-C4motif-C553A-C558A, TRPC5 mutant bearing the TRPC4 motif and double-cysteine mutations. ( G and H ) Time constants (τ) of channel activation and inactivation by 100 nM EA. τ values from each recording are plotted as dots. “∞” indicates that the current did not decay in the recording time frame. Mean τ values of each mutant are shown as gray bars. There is no significant difference by ANOVA in (G) ( n = 8 to 11).

Article Snippet: Primary antibodies against TRPC5 and TRPC4 were purchased from Sangon Biotech (Shanghai, China) and Proteintech (Wuhan, China), respectively.

Techniques: Labeling, Mutagenesis, Activation Assay

Journal: Science Advances

Article Title: Cryo-EM structure of TRPC5 at 2.8-Å resolution reveals unique and conserved structural elements essential for channel function

doi: 10.1126/sciadv.aaw7935

Figure Lengend Snippet: ( A ) A cation (purple sphere) on the cytosolic face is in the hydrophilic pocket of the S1-S4 domain, interacting with Glu 418 , Glu 421 , Asn 436 , and Asp 439 . Enlarged view of the cation binding site. ( B ) Comparable Ca 2+ (green sphere) binding site and an enlarged view of TRPM4 as “E/Q/N/D” (TRPC5, “E/E/N/D”). ( C ) Sequence of the mouse TRPC5 aligned to mTRPC4 and other representative TRP members (Clustal Omega). The key residues are highlighted as E/E/N/D, which are conserved in TRPC2/3/5/6/7 and TRPM7 but as E/Q/N/D in TRPC4 and TRPM4. ( D ) Patch clamp recordings of TRPC5 mutants in response to the channel activators Gd 3+ and EA. For the N436R mutant, 12 transfected cells showed no response to EA; weak activation was observed in three cells.

Article Snippet: Primary antibodies against TRPC5 and TRPC4 were purchased from Sangon Biotech (Shanghai, China) and Proteintech (Wuhan, China), respectively.

Techniques: Binding Assay, Sequencing, Patch Clamp, Mutagenesis, Transfection, Activation Assay