trpm6, lyophilized  (Alomone Labs)

Journal: Frontiers in Physiology

Article Title: Ghrelin enhances tubular magnesium absorption in the kidney

doi: 10.3389/fphys.2024.1363708

Figure Lengend Snippet: (A) The Ca 2+ channel TRPV5 is not stimulated by purified Ghrelin when co-expressed with GHSR in HEK293 cells (TRPV5+GHSR + control 583.2 ± 108.2 pA/pF vs. TRPV5+GHSR + Ghrelin vs. 524 ± 138 pA/pF; n. s. , n = six to eight each group). Typical current traces are shown below each specific group. Applied test pulses are shown at the bottom. (B) In contrast, the Mg 2+ channel TRPM6/7 displays increased current density when co-expressed with GHSR and treated with purified Ghrelin after 10, 30, and 60 min (TRPM6+GHSR + control 142.3 ± 21.9 pA/pF vs. TRPM6+GHSR + Ghrelin (10 min) 353.4 ± 80.2 pA/pF, p < 0.0001 , vs. TRPM6+GHSR + Ghrelin (30 min) 387.6 ± 52.3 pA/pF, p < 0.0001 , TRPM6+GHSR + Ghrelin (60 min) 368.6 ± 88.6 pA/pF, p < 0.0001 , n = six to seven each group). Typical current traces are shown below each specific group. Applied test pulses are shown at the bottom.

Article Snippet: Antibody against TRPM6 (ACC-046) (host rabbit, 1:1000) was purchased from Alomone (Jerusalem, Israel).

Techniques: Purification, Control

Journal: Frontiers in Physiology

Article Title: Ghrelin enhances tubular magnesium absorption in the kidney

doi: 10.3389/fphys.2024.1363708

Figure Lengend Snippet: (A) A dose-dependent increase in TRPM6/7 current density occurs with purified Ghrelin treatment at dosages from 1 to 100 nM 60 min after treatment (TRPM6+GHSR+0 nM Ghrelin 49.8 ± 25.2 pA/pF vs. TRPM6+GHSR+1 nM Ghrelin 106.4 ± 14.1 pA/pF, p < 0.01 , vs. TRPM6+GHSR+10 nM Ghrelin 161.9 ± 15.4 pA/pF, p < 0.001 , vs. TRPM6+GHSR+100 nM Ghrelin 269.4 ± 30.6 pA/pF, p < 0.0001 , n = five to six each group). No significant rise in TRPM6/7 current density is observed when the dose increased from 100 to 1000 nM (TRPM6+GHSR+100 nM Ghrelin 269.4 ± 30.6 pA/pF vs. TRPM6+GHSR+1000 nM Ghrelin 309.2 ± 64.3 pA/pF, n. s , n = five to six each group). (B) The stimulation of TRPM6/7 by Ghrelin is blocked by the GHSR-antagonist (D-Lys 3 )-GHRP6 (TRPM6+GHSR + control 81.5 ± 19.2 pA/pF vs. TRPM6+GHSR + Ghrelin 255.3 ± 66.4 pA/pF, p < 0.001 , vs. TRPM6+GHSR + Ghrelin+(D-Lys 3 )-GHRP6 113.2 ± 28 pA/pF, p < 0.01 , n = five to six each group). (C) TRPM6/7 current density is evoked by test pulses from −100 to +100 mV with 20 mV increments. The current–voltage (I/V) curves display a characteristic outwardly rectifying current typical for TRPM6. The I/V curve demonstrated more activity with Ghrelin stimulation as analyzed in (B) . (D) . TRPM6/7 current density is stimulated by the Ghrelin-mimetic (D-Trp 7 , Ala 8 ,D-Phe 10 )-α-MSH (6–11) amide (TRPM6+GHSR + control 84.3 ± 33.3 pA/pF vs. TRPM6+GHSR+(D-Trp 7 , Ala 8 ,D-Phe 10 )-α-MSH (6–11) amide 218.9 ± 31.9 pA/pF, p < 0.0001 ). This activation of TRPM6/7 current density is blocked by (D-Lys 3 )-GHRP6 (TRPM6+GHSR+(D-Trp 7 , Ala 8 ,D-Phe 10 )-α-MSH (6–11) amide 218.9 ± 31.9 pA/pF vs. TRPM6+GHSR+(D-Trp 7 , Ala 8 ,D-Phe 10 )-α-MSH (6–11) amide +(D-Lys 3 )-GHRP6 96.1 ± 48.5 pA/pF, p < 0.001 , n = five to six each group).

Article Snippet: Antibody against TRPM6 (ACC-046) (host rabbit, 1:1000) was purchased from Alomone (Jerusalem, Israel).

Techniques: Purification, Control, Activity Assay, Activation Assay

Journal: Frontiers in Physiology

Article Title: Ghrelin enhances tubular magnesium absorption in the kidney

doi: 10.3389/fphys.2024.1363708

Figure Lengend Snippet: (A) The PKA inhibitor H89 inhibits TRPM6/7 stimulation by Ghrelin. H89 has no effect on TRPM6/7 current density by itself (TRPM6+GHSR + control 53.2 ± 14.2 pA/pF vs. TRPM6+GHSR + control + H89 60.3 ± 15.8 pA/pF, n. s. ). Ghrelin activates TRPM6/7 current density and addition of H89 blocked the Ghrelin-mediated TRPM6 activity (TRPM6+GHSR + Ghrelin 193.5 ± 55.1 pA/pF vs. TRPM6+GHSR + Ghrelin + H89 40.6 ± 11.8 pA/pF, p < 0.001 ). (B) Only Gα s enhances TRPM6/7 current density (TRPM6+Gα s 184.7 ± 31.7 pA/pF vs. TRPM6+control 37.2 ± 17.3 pA/pF, p < 0.0001 ) but not Gα i2 , Gα q , or Gα 13 (TRPM6+Gα i2 42.2 ± 4.7 pA/pF vs. TRPM6+ Gα q 31 ± 10.3 pA/pF vs. TRPM6+ Gα 13 31.3 ± 6.7 pA/pF, n. s. , n = 5 each group). (C) . While Gα s activates TRPM6 current density the Gα s mutant Q227L cannot stimulate TRPM6 current density (TRPM6+control vs. TRPM6+Gα s vs. TRPM6+Gα sQ227L = 54.9 ± 13.9 pA/pF vs. 178.6 ± 25.9 pA/pF vs. 89.7 ± 10.9 pA/pF, p < 0.0001, n = 5 each group).

Article Snippet: Antibody against TRPM6 (ACC-046) (host rabbit, 1:1000) was purchased from Alomone (Jerusalem, Israel).

Techniques: Control, Activity Assay, Mutagenesis

Journal: Frontiers in Physiology

Article Title: Ghrelin enhances tubular magnesium absorption in the kidney

doi: 10.3389/fphys.2024.1363708

Figure Lengend Snippet: Model of how Ghrelin may stimulate TRPM6/7 based on our in vitro data. Given its low molecular weight Ghrelin may be filtered or synthesized in the kidney. Ghrelin binds to the GHSR receptor (which we hypothesize to be apically located) to induce Gα s signalling, activate adenylyl cyclase and raise cAMP levels. These result in PKA activation which then stimulates the Mg 2+ channel TRPM6/7 and allows for more Mg 2+ absorption. In Ghrelin deficiency the lack of TRPM6/7 stimulation may contribute to osteopenia. In addition, our in vivo experiments show that GHSR enhances transcription and translation of magnesiotropic genes after caloric restriction.

Article Snippet: Antibody against TRPM6 (ACC-046) (host rabbit, 1:1000) was purchased from Alomone (Jerusalem, Israel).

Techniques: In Vitro, Molecular Weight, Synthesized, Activation Assay, In Vivo

Journal: Frontiers in Physiology

Article Title: Ghrelin enhances tubular magnesium absorption in the kidney

doi: 10.3389/fphys.2024.1363708

Figure Lengend Snippet: Ghrelin (GHRL) and GHSR mRNA is detected in microdissected TAL and DCT. In microdissected TAL and DCT from WT mice mRNAs of marker genes for the TAL (e.g., NKCC2) and DCT (e.g., NCC and TRPM6) were identified. Expression of mRNA is normalized to GAPDH. GHRL mRNA was two-fold higher in TAL vs. DCT. GHSR mRNA expression was similar in TAL and DCT. As anticipated NKCC2 was most abundant in TAL, whereas NCC and TRPM6 were strongly expressed in the DCT. n = 10 tubules for each group.

Article Snippet: Antibody against TRPM6 (ACC-046) (host rabbit, 1:1000) was purchased from Alomone (Jerusalem, Israel).

Techniques: Marker, Expressing

Journal: Frontiers in Physiology

Article Title: Ghrelin enhances tubular magnesium absorption in the kidney

doi: 10.3389/fphys.2024.1363708

Figure Lengend Snippet: Transcription of magnesiotropic genes in GHSR-null and in WT mice after caloric restriction. Expression of mRNA levels of magnesiotropic genes such as Claudin-16 (Cldn16), Claudin-19 (Cldn19), Hnf1b, Fxyd2b, and Parvalbumin were significantly lower in GHSR-null mice compared to WT mice. No significant differences were found for Trpm6 or epidermal growth factor mRNA levels (Egf) after caloric restriction.

Article Snippet: Antibody against TRPM6 (ACC-046) (host rabbit, 1:1000) was purchased from Alomone (Jerusalem, Israel).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Detection of TRPM6 and TRPM7 Proteins in Normal and Diseased Cardiac Atrial Tissue and Isolated Cardiomyocytes

doi: 10.3390/ijms232314860

Figure Lengend Snippet: Specificity of antibody-based detection of TRPM6 and TPRM7 in pig right atrial (RA) cardiomyocytes: ( A , B ) Immunofluorescence signals in cells incubated with anti-TRPM6 (red, when using Alexa Fluor 546) or anti-TRPM7 (green, when using Alexa Fluor 488) antibodies used alone (upper panels). The immunofluorescence of TRPM6 was increased post-acquisition by a factor of 2 for better visualization of the red staining. Bottom panels show merged images in the same cells stained with either anti-TRPM6 or anti-TRPM7 as well as Phalloidin-CF 405 for filamentous actin (F-actin) cytoskeleton (in surrogate grey) and Hoechst 33342 for nuclei (in surrogate white); ( C , D ) immunofluorescence signals in cells incubated with anti-TRPM6 or anti-TRPM7 antibodies in the presence of antibody-blocking peptides (upper panels). Bottom panels show merged images in the same cells stained with either anti-TRPM6 or anti-TRPM7, together with the blocking peptides, as well as Phalloidin-CF 405 for F-actin cytoskeleton (in surrogate grey) and Hoechst 33342 for nuclei (in surrogate white). There was no staining with anti-TRPM6 (red) or anti-TRPM7 (green) in the presence of antibody-blocking peptides; ( E , F ) immunofluorescence signals in cells incubated with anti-TRPM6 antibodies in the presence of the TRPM7-blocking peptide or with anti-TRPM7 antibodies in the presence of the TRPM6-blocking peptide (upper panels). Bottom panels show merged images in the same cells stained with the anti-TRPM antibodies together with the off-target blocking peptides, as well as Phalloidin-CF 405 for F-actin cytoskeleton (in surrogate grey) and Hoechst 33342 for nuclei (in surrogate white). Staining was similar to that in conditions where there is no blocking peptide. Scale bars indicate 20 µm.

Article Snippet: The blocking peptide for anti-TRPM7 antibody (#BLP-CC047) and the blocking peptide for anti-TRPM6 (#BPL-CC046) was created by Alomone Labs for the antibody TRPM6 or TRPM7.

Techniques: Immunofluorescence, Incubation, Staining, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Detection of TRPM6 and TRPM7 Proteins in Normal and Diseased Cardiac Atrial Tissue and Isolated Cardiomyocytes

doi: 10.3390/ijms232314860

Figure Lengend Snippet: Detection of TRPM6 and TRPM7 in the same cell and cellular localization of TRPM7: ( A , B ) Images of human RA cell illustrating the localization of TRPM6 (red) and TRPM7 (green) proteins in the same cardiomyocyte obtained using primary antibodies conjugated with Alexa Fluor 546 and Alexa Fluor 488, respectively, at different scanning layers [( A ), near the cell surface and ( B ), deeper in the cell towards the middle of its thickness]. Phalloidin-CF 405 for F-actin cytoskeleton and Hoechst 33342 for nuclei appear in surrogate grey; ( C , D ) immunofluorescence images of TRPM7 (stained in green when using Alexa Fluor 488) and F-actin cytoskeleton (stained in red when using Phalloidin-Alexa Fluor 546) were used for calculating fluorescence intensity in human ( C ) and pig ( D ) RA cardiomyocytes presented in ( E , F ). Hoechst 33342 for nuclei appears in blue. Scale bars indicate 20 µm; ( E , F ) constructed graphs of the fluorescence intensity using stacks of 12 slices for the localization of TRPM7 (green) and F-actin (red) along lines of interest on the cardiomyocytes merged images. The sampling distance in depth between scanned slices was 1 µm. The calculated correlation coefficient between F-actin and TRPM7 peaks (at a place as indicated) was 0.32 for human ( E ) and 0.11 for pig ( F ) RA cardiomyocytes.

Article Snippet: The blocking peptide for anti-TRPM7 antibody (#BLP-CC047) and the blocking peptide for anti-TRPM6 (#BPL-CC046) was created by Alomone Labs for the antibody TRPM6 or TRPM7.

Techniques: Immunofluorescence, Staining, Fluorescence, Construct, Sampling

Journal: International Journal of Molecular Sciences

Article Title: Detection of TRPM6 and TRPM7 Proteins in Normal and Diseased Cardiac Atrial Tissue and Isolated Cardiomyocytes

doi: 10.3390/ijms232314860

Figure Lengend Snippet: Cellular localization of TRPM6: ( A , B ) The immunofluorescence images of TRPM6 (stained in green when using Alexa Fluor 488) and F-actin cytoskeleton (stained in red when using Phalloidin-Alexa Fluor 546) were used for calculating fluorescence intensity in human ( A ) and porcine ( B ) RA cardiomyocytes presented in ( C , D ). Hoechst 33342 for nuclei appears in blue. Scale bars indicate 20 µm; ( C , D ) constructed graphs of the fluorescence intensity for the localization of TRPM6 (green) and F-actin (red) along lines of interest on the merged images of cardiomyocytes as indicated by the zoomed-in area. The calculated correlation coefficient between F-actin and TRPM6 peaks (at a place as indicated) was 0.25 for human ( C ) and 0.33 for pig ( D ) RA cardiomyocytes.

Article Snippet: The blocking peptide for anti-TRPM7 antibody (#BLP-CC047) and the blocking peptide for anti-TRPM6 (#BPL-CC046) was created by Alomone Labs for the antibody TRPM6 or TRPM7.

Techniques: Immunofluorescence, Staining, Fluorescence, Construct

Journal: International Journal of Molecular Sciences

Article Title: Detection of TRPM6 and TRPM7 Proteins in Normal and Diseased Cardiac Atrial Tissue and Isolated Cardiomyocytes

doi: 10.3390/ijms232314860

Figure Lengend Snippet: Effect of TRP channel modulating drugs carvacrol (CAR) or 2-aminoethoxydiphenyl borate (2-APB) on detected TRPM6 and TRPM7 in pig and human RA cardiomyocytes: ( A – F ) Immunofluorescence of TRPM6 and TPRM7 in pig cardiomyocytes under control conditions and in the presence of CAR or 2-APB. Scale bars indicate 20 µm; ( G , H ) quantification of the immunodetected TRPM7 and TRPM6 proteins in pig RA cardiomyocytes in the absence (unfilled symbols) or in the presence (filled symbols) of either CAR ( G ) or 2-APB ( H ); ( I , J ) quantification of the immunodetected TRPM7 ( I ) and TRPM6 ( J ) proteins in human RA cardiomyocytes in the absence (unfilled symbols) or presence (filled symbols) of 2-APB at either 500 µM or 2 mM. Fluorescence intensity expressed in arbitrary units (a.u.). * p < 0.001 for drug vs. no drug. Notice opposite changes on TRPM7 with 2-APB at 500 µM vs. at 2 mM.

Article Snippet: The blocking peptide for anti-TRPM7 antibody (#BLP-CC047) and the blocking peptide for anti-TRPM6 (#BPL-CC046) was created by Alomone Labs for the antibody TRPM6 or TRPM7.

Techniques: Immunofluorescence, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Detection of TRPM6 and TRPM7 Proteins in Normal and Diseased Cardiac Atrial Tissue and Isolated Cardiomyocytes

doi: 10.3390/ijms232314860

Figure Lengend Snippet: Tissue-level and subcellular localization of TRPM6 and TRPM7 by immunohistochemistry in the pig RA: ( A – D ) Confocal microscopy images obtained from perpendicular ( A , B ) and longitudinal ( C , D ) tissue slices. Images shown at higher magnification in the lower panels correspond to zoomed-in areas delimited by squares at a lower scale in the upper panels. TRPM6 and TRPM7 proteins were detected with AF-488-conjugated antibodies (green), the cell surface was labeled with AF-555 for wheat germ agglutinin (WGA, in red), whereas the nucleus staining (with Hoechst 33342) appears in blue. Notice that TRPM6 and TRPM7 are detected both at the cell membrane (filled arrows) and intracellularly (unfilled arrows). Scale bars indicate 20 µm.

Article Snippet: The blocking peptide for anti-TRPM7 antibody (#BLP-CC047) and the blocking peptide for anti-TRPM6 (#BPL-CC046) was created by Alomone Labs for the antibody TRPM6 or TRPM7.

Techniques: Immunohistochemistry, Confocal Microscopy, Labeling, Staining

Journal: International Journal of Molecular Sciences

Article Title: Detection of TRPM6 and TRPM7 Proteins in Normal and Diseased Cardiac Atrial Tissue and Isolated Cardiomyocytes

doi: 10.3390/ijms232314860

Figure Lengend Snippet: Tissue-level and subcellular localization of TRPM6 and TRPM7 by immunohistochemistry in the RA of an undiseased human heart from car victim. ( A – D ) Confocal microscopy images obtained from perpendicular ( A , B ) and longitudinal ( C , D ) tissue slices. Images shown at higher magnification in the lower panels correspond to zoomed-in areas delimited by squares at a lower scale in the upper panels. TRPM6 and TRPM7 proteins were detected with AF-488-conjugated antibodies (green), the cell surface was labeled with AF-555 for wheat germ agglutinin (WGA, in red), whereas the nucleus staining (with Hoechst 33342) appears in blue. Notice that TRPM6 and TRPM7 are detected both at the cell membrane (filled arrows) and intracellularly (unfilled arrows). Scale bars indicate 20 µm.

Article Snippet: The blocking peptide for anti-TRPM7 antibody (#BLP-CC047) and the blocking peptide for anti-TRPM6 (#BPL-CC046) was created by Alomone Labs for the antibody TRPM6 or TRPM7.

Techniques: Immunohistochemistry, Confocal Microscopy, Labeling, Staining

Journal: International Journal of Molecular Sciences

Article Title: Detection of TRPM6 and TRPM7 Proteins in Normal and Diseased Cardiac Atrial Tissue and Isolated Cardiomyocytes

doi: 10.3390/ijms232314860

Figure Lengend Snippet: TRPM6 and TRPM7 detection by immunohistochemistry in diseased human RA tissue: ( A – D ) Representative images obtained from longitudinal slices of RA tissues from two different patients: an ischemic heart disease (IHD) patient ( A , B ) and an AF patient ( C , D ). The area labeled with the square bars corresponds to the images presented on a larger scale in the panels below them. AF-488 for the TRPM6 and TRPM7 protein appears in green. AF-555 for the cell membrane surface labeling with WGA appear in red, and Hoechst 33342 for the nucleus staining appear in blue. Arrows highlight the presence of TRP proteins. Notice, qualitatively, the amorphous distribution of TRPs and less colocalization with the cell membrane in both the IHD ( A , B ) and AF ( C , D ) hearts as compared with the control (car victim’s heart in ).

Article Snippet: The blocking peptide for anti-TRPM7 antibody (#BLP-CC047) and the blocking peptide for anti-TRPM6 (#BPL-CC046) was created by Alomone Labs for the antibody TRPM6 or TRPM7.

Techniques: Immunohistochemistry, Labeling, Staining

Journal: International Journal of Molecular Sciences

Article Title: Detection of TRPM6 and TRPM7 Proteins in Normal and Diseased Cardiac Atrial Tissue and Isolated Cardiomyocytes

doi: 10.3390/ijms232314860

Figure Lengend Snippet: Effect of cardiac disease conditions on TRPM6 and TRPM7: ( A – C ) TRPM fluorescence intensity levels in RA cells ( A ) or tissues ( B , C ). Fluorescence intensity levels of TRPM6 (unfilled) and TRPM7 (filled) channels in isolated cardiomyocytes of the human heart, grouped according to the heart rhythm (SR vs. AF, ( A )) or according to the absence or presence of ischemic heart disease (non-IHD vs. IHD, ( B )). Fluorescence intensity levels of TRPM6 (unfilled) and TRPM7 (filled) channels in histological slices grouped according to the heart rhythm (SR vs. AF, ( C )); ( D ) TRPM7 and TRPM6 protein levels in human heart tissue homogenates grouped according to the heart rhythm and the presence of ischemic disease. Mean data of immunofluorescence provided in arbitrary units (a.u) for TRPM6 and TRPM7: * and ** p < 0.05 AF vs. SR, respectively, in isolated cells; # and ## p < 0.01 IHD vs. non-IHD, respectively, in tissue sections; $ and $$ p < 0.01 AF vs. SR, respectively, in tissue sections, and mean data of TRPM6 and TRPM7 protein concentrations in pg/mL, † and †† p < 0.05 AF vs. SR, ‡ and ‡‡ p < 0.05 IHD vs. SR (non-IHD), respectively.

Article Snippet: The blocking peptide for anti-TRPM7 antibody (#BLP-CC047) and the blocking peptide for anti-TRPM6 (#BPL-CC046) was created by Alomone Labs for the antibody TRPM6 or TRPM7.

Techniques: Fluorescence, Isolation, Immunofluorescence