trpm7  (Alomone Labs)


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    Structured Review

    Alomone Labs trpm7
    Possible association of zinc with neuronal death after pilocarpine-induced SE. This schematic drawing represents several chain reactions that may occur after carvacrol and 2-APB treatment in pilocarpine-induced SE. ( A ) These are the possible cellular pathways through which neuronal death occurs after pilocarpine-induced SE. ( B ) Blocking <t>TRPM7</t> by carvacrol and 2-APB can inhibit several chain reactions that are thought to occur following pilocarpine-induced SE.
    Trpm7, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trpm7/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trpm7 - by Bioz Stars, 2022-05
    93/100 stars

    Images

    1) Product Images from "The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity"

    Article Title: The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21217897

    Possible association of zinc with neuronal death after pilocarpine-induced SE. This schematic drawing represents several chain reactions that may occur after carvacrol and 2-APB treatment in pilocarpine-induced SE. ( A ) These are the possible cellular pathways through which neuronal death occurs after pilocarpine-induced SE. ( B ) Blocking TRPM7 by carvacrol and 2-APB can inhibit several chain reactions that are thought to occur following pilocarpine-induced SE.
    Figure Legend Snippet: Possible association of zinc with neuronal death after pilocarpine-induced SE. This schematic drawing represents several chain reactions that may occur after carvacrol and 2-APB treatment in pilocarpine-induced SE. ( A ) These are the possible cellular pathways through which neuronal death occurs after pilocarpine-induced SE. ( B ) Blocking TRPM7 by carvacrol and 2-APB can inhibit several chain reactions that are thought to occur following pilocarpine-induced SE.

    Techniques Used: Blocking Assay

    2-APB treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 5–6 from each seizure group). * p
    Figure Legend Snippet: 2-APB treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 5–6 from each seizure group). * p

    Techniques Used: Over Expression, Immunofluorescence

    Carvacrol treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 7 from each seizure group). * p
    Figure Legend Snippet: Carvacrol treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 7 from each seizure group). * p

    Techniques Used: Over Expression, Immunofluorescence

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    Alomone Labs rabbit anti trpm7
    Possible association of zinc with neuronal death after pilocarpine-induced SE. This schematic drawing represents several chain reactions that may occur after carvacrol and 2-APB treatment in pilocarpine-induced SE. ( A ) These are the possible cellular pathways through which neuronal death occurs after pilocarpine-induced SE. ( B ) Blocking <t>TRPM7</t> by carvacrol and 2-APB can inhibit several chain reactions that are thought to occur following pilocarpine-induced SE.
    Rabbit Anti Trpm7, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti trpm7/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti trpm7 - by Bioz Stars, 2022-05
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    Image Search Results


    Possible association of zinc with neuronal death after pilocarpine-induced SE. This schematic drawing represents several chain reactions that may occur after carvacrol and 2-APB treatment in pilocarpine-induced SE. ( A ) These are the possible cellular pathways through which neuronal death occurs after pilocarpine-induced SE. ( B ) Blocking TRPM7 by carvacrol and 2-APB can inhibit several chain reactions that are thought to occur following pilocarpine-induced SE.

    Journal: International Journal of Molecular Sciences

    Article Title: The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity

    doi: 10.3390/ijms21217897

    Figure Lengend Snippet: Possible association of zinc with neuronal death after pilocarpine-induced SE. This schematic drawing represents several chain reactions that may occur after carvacrol and 2-APB treatment in pilocarpine-induced SE. ( A ) These are the possible cellular pathways through which neuronal death occurs after pilocarpine-induced SE. ( B ) Blocking TRPM7 by carvacrol and 2-APB can inhibit several chain reactions that are thought to occur following pilocarpine-induced SE.

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-TRPM7 (diluted 1:400; Alomone labs, Jerusalem, Israel), mouse andti-4HNE (diluted 1:500; Alpha Diagnostic Intl.

    Techniques: Blocking Assay

    2-APB treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 5–6 from each seizure group). * p

    Journal: International Journal of Molecular Sciences

    Article Title: The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity

    doi: 10.3390/ijms21217897

    Figure Lengend Snippet: 2-APB treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 5–6 from each seizure group). * p

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-TRPM7 (diluted 1:400; Alomone labs, Jerusalem, Israel), mouse andti-4HNE (diluted 1:500; Alpha Diagnostic Intl.

    Techniques: Over Expression, Immunofluorescence

    Carvacrol treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 7 from each seizure group). * p

    Journal: International Journal of Molecular Sciences

    Article Title: The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity

    doi: 10.3390/ijms21217897

    Figure Lengend Snippet: Carvacrol treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 7 from each seizure group). * p

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-TRPM7 (diluted 1:400; Alomone labs, Jerusalem, Israel), mouse andti-4HNE (diluted 1:500; Alpha Diagnostic Intl.

    Techniques: Over Expression, Immunofluorescence

    CNNMs interact with TRPM7 and regulate its activity. (A) HA-TRPM7 was coexpressed with FLAG-tagged CNNM1-4 in HEK-293T cells, and the channel was immunoprecipitated with HA-agarose. CNNM1 and CNNM3 strongly interacted with TRPM7. CNNM2 and CNNM4 also interacted with TRPM7, but lowered TRPM7 expression nonspecifically. CNNMs were not coimmunoprecipitated from HEK-293T cells N.T. with HA-TRPM7. Supplementation of the growth medium with 20 mM MgCl 2 boosted TRPM7 expression, revealing the channel’s capacity to interact with CNNM2 and CNNM4. (B) FLAG-tagged TRPM7 robustly coimmunoprecipitated native CNNM3 and CNNM4, whereas FLAG-TRPM2 did not. (C) A Zinc influx assay using the FluoZin-3 Zn 2 + indicator was used to monitor TRPM7 function in intact 293-TRPM7 cells (293-M7 cells), which express FLAG-tagged TRPM7 upon tetracycline treatment. Coexpression of CNNMs with TRPM7 increased intracellular free Zn 2 + more than expression of TRPM7 alone. The T-REx-293 cell line (293), which expresses the Tet Repressor protein, was used as the negative control. Shown are images taken at a time point between 5 and 10 minutes after application of 30 μM ZnCl 2 . White scale bar = 100 μM. (D) Quantification of the results from (C). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (E) Western blot showing expression of TRPM7 in the sample from (C). (F) Separate time course measurements were also acquired to further demonstrate that cells coexpressing CNNMs with TRPM7 have a higher rate of Zn 2+ influx compared to 293-TRPM7 cells expressing TRPM7 or CNNMs alone. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data . HBSS, Hanks’ balanced salt solution; N.T., not transfected.

    Journal: PLoS Biology

    Article Title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

    doi: 10.1371/journal.pbio.3001496

    Figure Lengend Snippet: CNNMs interact with TRPM7 and regulate its activity. (A) HA-TRPM7 was coexpressed with FLAG-tagged CNNM1-4 in HEK-293T cells, and the channel was immunoprecipitated with HA-agarose. CNNM1 and CNNM3 strongly interacted with TRPM7. CNNM2 and CNNM4 also interacted with TRPM7, but lowered TRPM7 expression nonspecifically. CNNMs were not coimmunoprecipitated from HEK-293T cells N.T. with HA-TRPM7. Supplementation of the growth medium with 20 mM MgCl 2 boosted TRPM7 expression, revealing the channel’s capacity to interact with CNNM2 and CNNM4. (B) FLAG-tagged TRPM7 robustly coimmunoprecipitated native CNNM3 and CNNM4, whereas FLAG-TRPM2 did not. (C) A Zinc influx assay using the FluoZin-3 Zn 2 + indicator was used to monitor TRPM7 function in intact 293-TRPM7 cells (293-M7 cells), which express FLAG-tagged TRPM7 upon tetracycline treatment. Coexpression of CNNMs with TRPM7 increased intracellular free Zn 2 + more than expression of TRPM7 alone. The T-REx-293 cell line (293), which expresses the Tet Repressor protein, was used as the negative control. Shown are images taken at a time point between 5 and 10 minutes after application of 30 μM ZnCl 2 . White scale bar = 100 μM. (D) Quantification of the results from (C). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (E) Western blot showing expression of TRPM7 in the sample from (C). (F) Separate time course measurements were also acquired to further demonstrate that cells coexpressing CNNMs with TRPM7 have a higher rate of Zn 2+ influx compared to 293-TRPM7 cells expressing TRPM7 or CNNMs alone. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data . HBSS, Hanks’ balanced salt solution; N.T., not transfected.

    Article Snippet: Fig 1E: The information of the anti-TRPM7 antibody should be provided in the methods section.

    Techniques: Activity Assay, Immunoprecipitation, Expressing, Negative Control, Western Blot, Fluorescence, Transfection

    Functional assessment of CNNMs on TRPM7 channel activity. TRPM7 currents were recorded in 293-TRPM7 cells ( n = 30), 293-M7-ΔCNNM3/4 cells ( n = 30), and 293-M7-ΔCNNM3/4 cells ( n = 40) with stable episomal expression of CNNM4 to keep CNNM4 protein levels low so that it would not interfere with TRPM7 protein expression. (A) Western blot demonstrating CNNM4 expression in the cell groups used in (B–D). (B) Representative TRPM7 whole-cell currents from the 3 cell lines recorded with an internal pipette solution containing 0 Mg 2+ /Mg-ATP to achieve full TRPM7 channel activity. (C) Average current density of the different groups from (A). (D) Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in cells from (B,C). White scale bar = 100 μM. Shown are images taken at a time point 5 to 10 minutes after application of 30 μM ZnCl 2 . (E) Quantification of results from (D). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (F) Native TRPM7 currents were recorded in the T-REx-293 Cell Line (293), which was the parental cell line of 293-ΔCNNM3/4 cells. Shown are representative TRPM7 whole-cell currents from the 2 cell lines recorded with an internal pipette solution containing 0 Mg 2+ /Mg-ATP to achieve full TRPM7 channel activity. (G) Average current density of the different groups from (F) ( n = 16 per group). Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data .

    Journal: PLoS Biology

    Article Title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

    doi: 10.1371/journal.pbio.3001496

    Figure Lengend Snippet: Functional assessment of CNNMs on TRPM7 channel activity. TRPM7 currents were recorded in 293-TRPM7 cells ( n = 30), 293-M7-ΔCNNM3/4 cells ( n = 30), and 293-M7-ΔCNNM3/4 cells ( n = 40) with stable episomal expression of CNNM4 to keep CNNM4 protein levels low so that it would not interfere with TRPM7 protein expression. (A) Western blot demonstrating CNNM4 expression in the cell groups used in (B–D). (B) Representative TRPM7 whole-cell currents from the 3 cell lines recorded with an internal pipette solution containing 0 Mg 2+ /Mg-ATP to achieve full TRPM7 channel activity. (C) Average current density of the different groups from (A). (D) Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in cells from (B,C). White scale bar = 100 μM. Shown are images taken at a time point 5 to 10 minutes after application of 30 μM ZnCl 2 . (E) Quantification of results from (D). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (F) Native TRPM7 currents were recorded in the T-REx-293 Cell Line (293), which was the parental cell line of 293-ΔCNNM3/4 cells. Shown are representative TRPM7 whole-cell currents from the 2 cell lines recorded with an internal pipette solution containing 0 Mg 2+ /Mg-ATP to achieve full TRPM7 channel activity. (G) Average current density of the different groups from (F) ( n = 16 per group). Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data .

    Article Snippet: Fig 1E: The information of the anti-TRPM7 antibody should be provided in the methods section.

    Techniques: Functional Assay, Activity Assay, Expressing, Western Blot, Transferring

    CNNMs have TRPM7-dependent and independent activities. (A) A 25 Mg uptake assay was employed to assay TRPM7 channel function and its dependence on CNNMs. Moreover, 293-TRPM7 cells have significantly higher 25 Mg uptake than the negative control T-REx-293 cell line (293), which expresses the Tet Repressor protein. Compared to 293-TRPM7 cells, 293-M7-ΔCNNM3/4 exhibited significantly lower 25 Mg uptake, which was increased by reexpression of CNNM4. * = p

    Journal: PLoS Biology

    Article Title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

    doi: 10.1371/journal.pbio.3001496

    Figure Lengend Snippet: CNNMs have TRPM7-dependent and independent activities. (A) A 25 Mg uptake assay was employed to assay TRPM7 channel function and its dependence on CNNMs. Moreover, 293-TRPM7 cells have significantly higher 25 Mg uptake than the negative control T-REx-293 cell line (293), which expresses the Tet Repressor protein. Compared to 293-TRPM7 cells, 293-M7-ΔCNNM3/4 exhibited significantly lower 25 Mg uptake, which was increased by reexpression of CNNM4. * = p

    Article Snippet: Fig 1E: The information of the anti-TRPM7 antibody should be provided in the methods section.

    Techniques: Negative Control

    CNNMs are required for TRPM7-mediated Zn 2+ influx. (A) A Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in intact cells under the same conditions described in Fig 1 . KO of CNNM3 , CNNM4 , and both CNNM3 and CNNM4 from 293-TRPM7 cells (293-M7-ΔCNNM3, 293-M7-ΔCNNM4, and 293-M7-ΔCNNM3/4) reduced TRPM7 channel function, which could be rescued to varying degrees by reexpression of CNNM3 and/or CNNM4. The T-REx-293 cell line (293), which expresses the Tet Repressor protein, was used as the negative control. All the cells in the assay were treated with tetracycline to induce TRPM7 expression. White scale bar = 100 μM. (B) Quantification of the results from (A). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. Images taken 5 to 10 minutes after application of 30 μM ZnCl 2 . (C) Separate time course measurements were also acquired to demonstrate that 293-TRPM7 cells lacking CNNMs have a reduced rate of Zn 2+ influx compared to 293-TRPM7 expressing TRPM7. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. (D) CRISPR/Cas-9 was used to KO CNNM3 , CNNM4 , and both CNNM3 4 from 293-TRPM7 cells, which express TRPM7. Western blotting of 2 independent clones showed specific KO of CNNM isoforms and similar expression of TRPM7 among the lines. Note that the antibody used to detected CNNM4 detects an unrelated protein as indicated. (E) Cell surface biotinylation of 293-TRPM7 WT and 293-M7-ΔCNNM3/4 cells demonstrated that surface level of TRPM7 in the 2 cell lines is similar. Shown are SA purified (SA purified) proteins, the input lysate for each cell type, and the proteins not bound to SA (SA unbound). (F) Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in intact cells. Shown are images taken at a time point between 5 and 10 minutes after application of 30 μM ZnCl 2 . Overexpression of PRL-2 stimulates TRPM7 channels function, which requires to varying degrees CNNM3 and CNNM4. White scale bar = 100 μM. (G) Western blot demonstrating expression of TRPM7 (Anti-FLAG) and PRL-2 under the conditions described in (F). (H) Quantification of the results from (F). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (I) Separate time course measurements were also acquired to demonstrate that PRL overexpression stimulates an increased rate of Zn 2+ influx compared to 293-TRPM7 expressing TRPM7 and that CNNM3 and CNNM4 are required for this activity. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data . HBSS, Hanks’ balanced salt solution; KO, knockout; PRL, phosphatase of regenerating liver; PRL-2, phosphatase of regenerating liver 2; SA, streptavidin agarose.

    Journal: PLoS Biology

    Article Title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

    doi: 10.1371/journal.pbio.3001496

    Figure Lengend Snippet: CNNMs are required for TRPM7-mediated Zn 2+ influx. (A) A Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in intact cells under the same conditions described in Fig 1 . KO of CNNM3 , CNNM4 , and both CNNM3 and CNNM4 from 293-TRPM7 cells (293-M7-ΔCNNM3, 293-M7-ΔCNNM4, and 293-M7-ΔCNNM3/4) reduced TRPM7 channel function, which could be rescued to varying degrees by reexpression of CNNM3 and/or CNNM4. The T-REx-293 cell line (293), which expresses the Tet Repressor protein, was used as the negative control. All the cells in the assay were treated with tetracycline to induce TRPM7 expression. White scale bar = 100 μM. (B) Quantification of the results from (A). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. Images taken 5 to 10 minutes after application of 30 μM ZnCl 2 . (C) Separate time course measurements were also acquired to demonstrate that 293-TRPM7 cells lacking CNNMs have a reduced rate of Zn 2+ influx compared to 293-TRPM7 expressing TRPM7. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. (D) CRISPR/Cas-9 was used to KO CNNM3 , CNNM4 , and both CNNM3 4 from 293-TRPM7 cells, which express TRPM7. Western blotting of 2 independent clones showed specific KO of CNNM isoforms and similar expression of TRPM7 among the lines. Note that the antibody used to detected CNNM4 detects an unrelated protein as indicated. (E) Cell surface biotinylation of 293-TRPM7 WT and 293-M7-ΔCNNM3/4 cells demonstrated that surface level of TRPM7 in the 2 cell lines is similar. Shown are SA purified (SA purified) proteins, the input lysate for each cell type, and the proteins not bound to SA (SA unbound). (F) Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in intact cells. Shown are images taken at a time point between 5 and 10 minutes after application of 30 μM ZnCl 2 . Overexpression of PRL-2 stimulates TRPM7 channels function, which requires to varying degrees CNNM3 and CNNM4. White scale bar = 100 μM. (G) Western blot demonstrating expression of TRPM7 (Anti-FLAG) and PRL-2 under the conditions described in (F). (H) Quantification of the results from (F). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (I) Separate time course measurements were also acquired to demonstrate that PRL overexpression stimulates an increased rate of Zn 2+ influx compared to 293-TRPM7 expressing TRPM7 and that CNNM3 and CNNM4 are required for this activity. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data . HBSS, Hanks’ balanced salt solution; KO, knockout; PRL, phosphatase of regenerating liver; PRL-2, phosphatase of regenerating liver 2; SA, streptavidin agarose.

    Article Snippet: Fig 1E: The information of the anti-TRPM7 antibody should be provided in the methods section.

    Techniques: Negative Control, Expressing, Fluorescence, CRISPR, Western Blot, Clone Assay, Purification, Over Expression, Activity Assay, Knock-Out

    CHBP inhibited TRPM7-like currents in TCMK-1 cells. The voltage ramp protocol invoking TRPM7 currents and the representative TRPM7 currents recorded in TCMK-1 cells ( a ). Time-dependent running up of TRPM7-like currents after break-in when dialyzed with Mg 2+ free internal solution by whole cell patch clamp ( b ). Blockade of TRPM7-like current (at +100 mV) by CHBP (100 nmol/l) ( c ). The periods of exposure to the normal extracellular solutions and CHBP were indicated by horizontal bars. Outward TRPM7-like currents were inhibited by CHBP, but reversibly changed by CHBP washout. (n = 6, ( d )).

    Journal: Scientific Reports

    Article Title: TRPM7 in CHBP-induced renoprotection upon ischemia reperfusion-related injury

    doi: 10.1038/s41598-018-22852-2

    Figure Lengend Snippet: CHBP inhibited TRPM7-like currents in TCMK-1 cells. The voltage ramp protocol invoking TRPM7 currents and the representative TRPM7 currents recorded in TCMK-1 cells ( a ). Time-dependent running up of TRPM7-like currents after break-in when dialyzed with Mg 2+ free internal solution by whole cell patch clamp ( b ). Blockade of TRPM7-like current (at +100 mV) by CHBP (100 nmol/l) ( c ). The periods of exposure to the normal extracellular solutions and CHBP were indicated by horizontal bars. Outward TRPM7-like currents were inhibited by CHBP, but reversibly changed by CHBP washout. (n = 6, ( d )).

    Article Snippet: Blots were blocked with 5% milk and incubated overnight with anti-TRPM7 antibodies (ACC-047, Alomone Labs, Jerusalem, Israel; Ab729, Abcam, Cambridge, UK), monoclonal anti-β-actin (Abcam, 1:10,000) antibodies, and anti-HMGB1 antibody (ab11354, Abcam, 1:1000), anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Beverly, MA), anti-bax antibody (1:1000, Cell Signaling Technology) and anti-bcl-2 antibody (1:1000, Cell Signaling Technology).

    Techniques: Patch Clamp

    The positive correlation between TRPM7 protein, supernatant LDH and cellular HMGB1 in HR TCMK-1 cells. Cytotoxicity was assessed by LDH release in TCMK-1 cells exposed to 12-h H and followed by 6-h, 12-h, and 24-h R ( a ). The positive correlation between TRPM7 expression and LDH in TCMK-1 cells ( b ). The expression of HMGB1 was also detected by western blotting in HR TCMK-1 cells ( c ). The positive correlation between TRPM7 and HMGB1 protein was revealed in TCMK-1 cells (n = 6, ( e )).

    Journal: Scientific Reports

    Article Title: TRPM7 in CHBP-induced renoprotection upon ischemia reperfusion-related injury

    doi: 10.1038/s41598-018-22852-2

    Figure Lengend Snippet: The positive correlation between TRPM7 protein, supernatant LDH and cellular HMGB1 in HR TCMK-1 cells. Cytotoxicity was assessed by LDH release in TCMK-1 cells exposed to 12-h H and followed by 6-h, 12-h, and 24-h R ( a ). The positive correlation between TRPM7 expression and LDH in TCMK-1 cells ( b ). The expression of HMGB1 was also detected by western blotting in HR TCMK-1 cells ( c ). The positive correlation between TRPM7 and HMGB1 protein was revealed in TCMK-1 cells (n = 6, ( e )).

    Article Snippet: Blots were blocked with 5% milk and incubated overnight with anti-TRPM7 antibodies (ACC-047, Alomone Labs, Jerusalem, Israel; Ab729, Abcam, Cambridge, UK), monoclonal anti-β-actin (Abcam, 1:10,000) antibodies, and anti-HMGB1 antibody (ab11354, Abcam, 1:1000), anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Beverly, MA), anti-bax antibody (1:1000, Cell Signaling Technology) and anti-bcl-2 antibody (1:1000, Cell Signaling Technology).

    Techniques: Expressing, Western Blot

    Positive correlations between TRPM7 protein, inflammation and apoptosis-related markers subjected to TRPM7 manipulation. TRPM7 expression was positively related to HMGB1 ( a ) and Bax/Bcl-2 ( b ) in HR TCMK-1 cells, as well as 17 kDa cleaved caspase-3 in both HR TCMK-1 cells ( c ) and mouse IR injury kidneys ( d ).

    Journal: Scientific Reports

    Article Title: TRPM7 in CHBP-induced renoprotection upon ischemia reperfusion-related injury

    doi: 10.1038/s41598-018-22852-2

    Figure Lengend Snippet: Positive correlations between TRPM7 protein, inflammation and apoptosis-related markers subjected to TRPM7 manipulation. TRPM7 expression was positively related to HMGB1 ( a ) and Bax/Bcl-2 ( b ) in HR TCMK-1 cells, as well as 17 kDa cleaved caspase-3 in both HR TCMK-1 cells ( c ) and mouse IR injury kidneys ( d ).

    Article Snippet: Blots were blocked with 5% milk and incubated overnight with anti-TRPM7 antibodies (ACC-047, Alomone Labs, Jerusalem, Israel; Ab729, Abcam, Cambridge, UK), monoclonal anti-β-actin (Abcam, 1:10,000) antibodies, and anti-HMGB1 antibody (ab11354, Abcam, 1:1000), anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Beverly, MA), anti-bax antibody (1:1000, Cell Signaling Technology) and anti-bcl-2 antibody (1:1000, Cell Signaling Technology).

    Techniques: Expressing

    TRPM7 siRNA inhibited inflammation and apoptosis in mouse IR kidneys. TRPM7 siRNA decreased the expression of TRPM7 mRNA ( a ) and the level of serum creatinine ( b ). TRPM7, HMGB1, 32, 17 and 12 kDa caspase-3, Bax and Bcl-2 were detected by western blotting ( c ). In IR kidneys, TRPM7 siRNA significantly down-regulated TRPM7, HMGB1, 17 and 12 kDa caspase-3 and Bax/Bcl-2 ratio, but not 32 kDa caspase-3 ( d ).

    Journal: Scientific Reports

    Article Title: TRPM7 in CHBP-induced renoprotection upon ischemia reperfusion-related injury

    doi: 10.1038/s41598-018-22852-2

    Figure Lengend Snippet: TRPM7 siRNA inhibited inflammation and apoptosis in mouse IR kidneys. TRPM7 siRNA decreased the expression of TRPM7 mRNA ( a ) and the level of serum creatinine ( b ). TRPM7, HMGB1, 32, 17 and 12 kDa caspase-3, Bax and Bcl-2 were detected by western blotting ( c ). In IR kidneys, TRPM7 siRNA significantly down-regulated TRPM7, HMGB1, 17 and 12 kDa caspase-3 and Bax/Bcl-2 ratio, but not 32 kDa caspase-3 ( d ).

    Article Snippet: Blots were blocked with 5% milk and incubated overnight with anti-TRPM7 antibodies (ACC-047, Alomone Labs, Jerusalem, Israel; Ab729, Abcam, Cambridge, UK), monoclonal anti-β-actin (Abcam, 1:10,000) antibodies, and anti-HMGB1 antibody (ab11354, Abcam, 1:1000), anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Beverly, MA), anti-bax antibody (1:1000, Cell Signaling Technology) and anti-bcl-2 antibody (1:1000, Cell Signaling Technology).

    Techniques: Expressing, Western Blot

    Specific TRMP7 agonist bradykinin aggravated inflammation and apoptosis in TCMK-1 cells. TRPM7, HMGB1, 32, 17 and 12 kDa caspase-3, Bax and Bcl-2 were detected by western blotting ( a ). In HR TCMK-1 cells, CHBP decreased the expression of TRPM7, HMGB1, 32 and17 kDa caspase-3 and Bax/Bcl-2 ratio, while bradykinin and bradykinin + CHBP significantly increased the expression of TRPM7, HMGB1, 17 kDa caspase-3 and Bax/Bcl-2 ratio ( b ). There was no significant difference in 12 kDa caspase-3 between all groups, and in 32 kDa caspase-3 between groups with or without Bradykinin. Apoptotic cells were labelled by Annexin V/PI staining and quantitatively assessed by flow cytometer ( c ). The histograms showed that the increase of early apoptotic cells in the HR group was inhibited by CHBP, but further increased by bradykinin. There was no statistical significance between HR and HR + CHBP + Bradykinin (n = 3, ( d )).

    Journal: Scientific Reports

    Article Title: TRPM7 in CHBP-induced renoprotection upon ischemia reperfusion-related injury

    doi: 10.1038/s41598-018-22852-2

    Figure Lengend Snippet: Specific TRMP7 agonist bradykinin aggravated inflammation and apoptosis in TCMK-1 cells. TRPM7, HMGB1, 32, 17 and 12 kDa caspase-3, Bax and Bcl-2 were detected by western blotting ( a ). In HR TCMK-1 cells, CHBP decreased the expression of TRPM7, HMGB1, 32 and17 kDa caspase-3 and Bax/Bcl-2 ratio, while bradykinin and bradykinin + CHBP significantly increased the expression of TRPM7, HMGB1, 17 kDa caspase-3 and Bax/Bcl-2 ratio ( b ). There was no significant difference in 12 kDa caspase-3 between all groups, and in 32 kDa caspase-3 between groups with or without Bradykinin. Apoptotic cells were labelled by Annexin V/PI staining and quantitatively assessed by flow cytometer ( c ). The histograms showed that the increase of early apoptotic cells in the HR group was inhibited by CHBP, but further increased by bradykinin. There was no statistical significance between HR and HR + CHBP + Bradykinin (n = 3, ( d )).

    Article Snippet: Blots were blocked with 5% milk and incubated overnight with anti-TRPM7 antibodies (ACC-047, Alomone Labs, Jerusalem, Israel; Ab729, Abcam, Cambridge, UK), monoclonal anti-β-actin (Abcam, 1:10,000) antibodies, and anti-HMGB1 antibody (ab11354, Abcam, 1:1000), anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Beverly, MA), anti-bax antibody (1:1000, Cell Signaling Technology) and anti-bcl-2 antibody (1:1000, Cell Signaling Technology).

    Techniques: Western Blot, Expressing, Staining, Flow Cytometry, Cytometry

    The positive correlation between TRPM7 protein and parameters of renal IR injury. TRPM7 expression was positively related to serum creatinine ( a ), blood urea nitrogen ( b ), inflammation ( d ) and apoptosis ( e ) in mouse IR injury kidneys, only marginally associated with tubulointerstitial damage score ( c ).

    Journal: Scientific Reports

    Article Title: TRPM7 in CHBP-induced renoprotection upon ischemia reperfusion-related injury

    doi: 10.1038/s41598-018-22852-2

    Figure Lengend Snippet: The positive correlation between TRPM7 protein and parameters of renal IR injury. TRPM7 expression was positively related to serum creatinine ( a ), blood urea nitrogen ( b ), inflammation ( d ) and apoptosis ( e ) in mouse IR injury kidneys, only marginally associated with tubulointerstitial damage score ( c ).

    Article Snippet: Blots were blocked with 5% milk and incubated overnight with anti-TRPM7 antibodies (ACC-047, Alomone Labs, Jerusalem, Israel; Ab729, Abcam, Cambridge, UK), monoclonal anti-β-actin (Abcam, 1:10,000) antibodies, and anti-HMGB1 antibody (ab11354, Abcam, 1:1000), anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Beverly, MA), anti-bax antibody (1:1000, Cell Signaling Technology) and anti-bcl-2 antibody (1:1000, Cell Signaling Technology).

    Techniques: Expressing

    TRPM7 siRNA downregulated its mRNA expression and TRPM7-like current in TCMK-1 cells. Compared to the cells treated by the NC siRNA, the expression of TRPM7 mRNA was reduced by TRPM7 siRNA sequence 3239 and 1793 in TCMK-1 cells ( P

    Journal: Scientific Reports

    Article Title: TRPM7 in CHBP-induced renoprotection upon ischemia reperfusion-related injury

    doi: 10.1038/s41598-018-22852-2

    Figure Lengend Snippet: TRPM7 siRNA downregulated its mRNA expression and TRPM7-like current in TCMK-1 cells. Compared to the cells treated by the NC siRNA, the expression of TRPM7 mRNA was reduced by TRPM7 siRNA sequence 3239 and 1793 in TCMK-1 cells ( P

    Article Snippet: Blots were blocked with 5% milk and incubated overnight with anti-TRPM7 antibodies (ACC-047, Alomone Labs, Jerusalem, Israel; Ab729, Abcam, Cambridge, UK), monoclonal anti-β-actin (Abcam, 1:10,000) antibodies, and anti-HMGB1 antibody (ab11354, Abcam, 1:1000), anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Beverly, MA), anti-bax antibody (1:1000, Cell Signaling Technology) and anti-bcl-2 antibody (1:1000, Cell Signaling Technology).

    Techniques: Expressing, Sequencing

    The inhibited role of CHBP on increased TRPM7 at mRNA and protein levels in HR cells and IR kidneys. TRPM7 mRNA level was increased by 24-h reperfusion in both TCMK-1 ( a ) and HK-2 cells ( b ) subjected to 12-h HR, While TRPM7 protein level was also increased in TCMK-1 cells subjected to 24-h reoxygenation in 12-h H TCMK-1 cells ( c ), and in the mouse kidneys followed 12 h, 24 h, and 7 days reperfusion ( d ). In addition, the expression of TRPM7 mRNA (A) and protein ( c ) was reduced in the 12-h, 24-h HR TCMK-1 cells by 30 nM CHBP at the onset of hypoxia. The TRPM7 protein was significantly reduced by 8 nmol/Kg CHBP intraperitoneally injected in the 5-d, 7-d IR mouse kidneys ( d ). The volume density was quantitatively analyzed using 42 kDa β-actin as the loading control (n = 6).

    Journal: Scientific Reports

    Article Title: TRPM7 in CHBP-induced renoprotection upon ischemia reperfusion-related injury

    doi: 10.1038/s41598-018-22852-2

    Figure Lengend Snippet: The inhibited role of CHBP on increased TRPM7 at mRNA and protein levels in HR cells and IR kidneys. TRPM7 mRNA level was increased by 24-h reperfusion in both TCMK-1 ( a ) and HK-2 cells ( b ) subjected to 12-h HR, While TRPM7 protein level was also increased in TCMK-1 cells subjected to 24-h reoxygenation in 12-h H TCMK-1 cells ( c ), and in the mouse kidneys followed 12 h, 24 h, and 7 days reperfusion ( d ). In addition, the expression of TRPM7 mRNA (A) and protein ( c ) was reduced in the 12-h, 24-h HR TCMK-1 cells by 30 nM CHBP at the onset of hypoxia. The TRPM7 protein was significantly reduced by 8 nmol/Kg CHBP intraperitoneally injected in the 5-d, 7-d IR mouse kidneys ( d ). The volume density was quantitatively analyzed using 42 kDa β-actin as the loading control (n = 6).

    Article Snippet: Blots were blocked with 5% milk and incubated overnight with anti-TRPM7 antibodies (ACC-047, Alomone Labs, Jerusalem, Israel; Ab729, Abcam, Cambridge, UK), monoclonal anti-β-actin (Abcam, 1:10,000) antibodies, and anti-HMGB1 antibody (ab11354, Abcam, 1:1000), anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Beverly, MA), anti-bax antibody (1:1000, Cell Signaling Technology) and anti-bcl-2 antibody (1:1000, Cell Signaling Technology).

    Techniques: Expressing, Injection

    TRMP7 siRNA and inhibitor reduced inflammation and apoptosis inTCMK-1 cells. The protein expression of TRPM7, HMGB1, 32, 17 and 12 kDa caspase-3, Bax and Bcl-2 in the HR TCMK-1 cells was detected by western blotting ( a ). TRPM7 siRNA significantly reversed increased TRPM7, HMGB1, 32 and 17 kDa caspase-3 and Bax/Bcl-2 ratio, but not 12 kDa caspase-3 upon HR ( b ). Apoptotic cells were labelled by Annexin V/PI and quantitatively assessed by flow cytometer ( c , e ). The histograms showed that the increase of early apoptotic cells was inhibited by TRPM7 siRNA and its inhibitor 2-APB (n = 3, ( d , f )).

    Journal: Scientific Reports

    Article Title: TRPM7 in CHBP-induced renoprotection upon ischemia reperfusion-related injury

    doi: 10.1038/s41598-018-22852-2

    Figure Lengend Snippet: TRMP7 siRNA and inhibitor reduced inflammation and apoptosis inTCMK-1 cells. The protein expression of TRPM7, HMGB1, 32, 17 and 12 kDa caspase-3, Bax and Bcl-2 in the HR TCMK-1 cells was detected by western blotting ( a ). TRPM7 siRNA significantly reversed increased TRPM7, HMGB1, 32 and 17 kDa caspase-3 and Bax/Bcl-2 ratio, but not 12 kDa caspase-3 upon HR ( b ). Apoptotic cells were labelled by Annexin V/PI and quantitatively assessed by flow cytometer ( c , e ). The histograms showed that the increase of early apoptotic cells was inhibited by TRPM7 siRNA and its inhibitor 2-APB (n = 3, ( d , f )).

    Article Snippet: Blots were blocked with 5% milk and incubated overnight with anti-TRPM7 antibodies (ACC-047, Alomone Labs, Jerusalem, Israel; Ab729, Abcam, Cambridge, UK), monoclonal anti-β-actin (Abcam, 1:10,000) antibodies, and anti-HMGB1 antibody (ab11354, Abcam, 1:1000), anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Beverly, MA), anti-bax antibody (1:1000, Cell Signaling Technology) and anti-bcl-2 antibody (1:1000, Cell Signaling Technology).

    Techniques: Expressing, Western Blot, Flow Cytometry, Cytometry

    Screening of cysteines in mTRPM7 that were responsible for the oxidative stress-induced inhibition. (A) Schematic diagram of 34 cysteines that are conserved in mouse and human TRPM7. S1107 and D1510 were also depicted (red dots). (B) Expression of TRPM7-wt and its mutants. Immunoblotting of TRPM7 and calnexin (loading control) was performed with HEK293 whole-cell lysates. Numbers on the left represent molecular mass of standards (in kDa). (C) The effect of H 2 O 2 (500 µM) on mutant TRPM7 current at 0.2 mM [Mg 2+ ] i . Each bar represents the mean ± SEM (vertical bar) of 6–13 observations. * p

    Journal: bioRxiv

    Article Title: Zinc-binding motif acts as an oxidative stress sensor to regulate TRPM7 channel activity

    doi: 10.1101/2020.09.28.316125

    Figure Lengend Snippet: Screening of cysteines in mTRPM7 that were responsible for the oxidative stress-induced inhibition. (A) Schematic diagram of 34 cysteines that are conserved in mouse and human TRPM7. S1107 and D1510 were also depicted (red dots). (B) Expression of TRPM7-wt and its mutants. Immunoblotting of TRPM7 and calnexin (loading control) was performed with HEK293 whole-cell lysates. Numbers on the left represent molecular mass of standards (in kDa). (C) The effect of H 2 O 2 (500 µM) on mutant TRPM7 current at 0.2 mM [Mg 2+ ] i . Each bar represents the mean ± SEM (vertical bar) of 6–13 observations. * p

    Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047; epitope 1146-1165 of human TRPM7, Alomone Labs, Jerusalem, Israel) was used.

    Techniques: Inhibition, Expressing, Mutagenesis

    Schematic illustration of TRPM7 regulation by oxidative stress. (Left) Under basal conditions, Mg 2+ -inhibition of TRPM7 current is attenuated by the kinase domain possibly via the interaction with the channel domain. (Right) Oxidative stress causes the oxidation of the cysteines (C1809 and C1813) in the zinc-binding motif, which may disrupt the interaction between the channel domain and the kinase domain and enhance the inhibition of TRPM7 by intracellular Mg 2+ .

    Journal: bioRxiv

    Article Title: Zinc-binding motif acts as an oxidative stress sensor to regulate TRPM7 channel activity

    doi: 10.1101/2020.09.28.316125

    Figure Lengend Snippet: Schematic illustration of TRPM7 regulation by oxidative stress. (Left) Under basal conditions, Mg 2+ -inhibition of TRPM7 current is attenuated by the kinase domain possibly via the interaction with the channel domain. (Right) Oxidative stress causes the oxidation of the cysteines (C1809 and C1813) in the zinc-binding motif, which may disrupt the interaction between the channel domain and the kinase domain and enhance the inhibition of TRPM7 by intracellular Mg 2+ .

    Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047; epitope 1146-1165 of human TRPM7, Alomone Labs, Jerusalem, Israel) was used.

    Techniques: Inhibition, Binding Assay

    Functional reconstitution of the TRPM7 current by co-expression of M7kd in M7cd-expressing cells. (A) Transient expression of M7kd-wt in M7cd-expressing cells resulted in an increase in the M7cd current density that was inhibited by H 2 O 2 (500 μM) in the presence of 0.2 mM [Mg 2+ ] i (closed circles), but not in the absence of intracellular Mg 2+ (open circles). Each symbol represents the mean ± SEM (vertical bar) of 8–9 recordings. (B, C) Representative I–V relationships of the M7cd current that was recorded in M7cd-expressing cells transfected with M7kd-wt in the presence (B) or absence of 0.2 mM [Mg 2+ ] i (C), before (black line) or 4 min after (red line) the application of H 2 O 2 (500 μM). (D) [Mg 2+ ] i -dependent inhibition of M7cd with co-expression of M7kd-wt in the control (black squares) and H 2 O 2 -treated (red squares) conditions. The data fit well with a biphasic concentration–response curve, assuming the fraction of high affinity inhibition of 0.314 estimated for full-length TRPM7-wt (in Figure 1D ). Under control conditions, the current was inhibited by intracellular free Mg 2+ with an IC 50(1) of 7.6 μM and an IC 50(2) of 986 μM. After H 2 O 2 treatment, the current was inhibited by intracellular free Mg 2+ with an IC 50 of 3.0 μM. Each symbol represents the mean ± SEM (vertical bar) of 5–28 observations. (E) The time course of the M7cd current when M7kd-wt was transfected by either lipofection (closed circles) or baculovirus infection (open squares) in the presence of 0.2 mM [Mg 2+ ] i . Each symbol represents the mean ± SEM (vertical bar) of 8–20 observations. (F) The effect of H 2 O 2 (500 μM) on the M7cd current in the presence of 0.2 mM [Mg 2+ ] i in cells that were transfected with M7kd-wt by baculovirus infection. Each symbol represents the mean ± SEM (vertical bar) of eight recordings. (G) The effect of NMM (100 μM) on M7cd current in the presence of 0.2 mM [Mg 2+ ] i in cells that were transfected with M7kd-wt by baculovirus infection. Each symbol represents the mean ± SEM (vertical bar) of six recordings. (H) The effect of co-expression with kinase-inactive mutant M7kd-1645R on M7cd current. Each symbol represents the mean ± SEM (vertical bar) of four recordings. (I) The effect of TRPM6 kinase domain (M6kd) co-expression on M7cd current. Each symbol represents the mean ± SEM (vertical bar) of eight recordings.

    Journal: bioRxiv

    Article Title: Zinc-binding motif acts as an oxidative stress sensor to regulate TRPM7 channel activity

    doi: 10.1101/2020.09.28.316125

    Figure Lengend Snippet: Functional reconstitution of the TRPM7 current by co-expression of M7kd in M7cd-expressing cells. (A) Transient expression of M7kd-wt in M7cd-expressing cells resulted in an increase in the M7cd current density that was inhibited by H 2 O 2 (500 μM) in the presence of 0.2 mM [Mg 2+ ] i (closed circles), but not in the absence of intracellular Mg 2+ (open circles). Each symbol represents the mean ± SEM (vertical bar) of 8–9 recordings. (B, C) Representative I–V relationships of the M7cd current that was recorded in M7cd-expressing cells transfected with M7kd-wt in the presence (B) or absence of 0.2 mM [Mg 2+ ] i (C), before (black line) or 4 min after (red line) the application of H 2 O 2 (500 μM). (D) [Mg 2+ ] i -dependent inhibition of M7cd with co-expression of M7kd-wt in the control (black squares) and H 2 O 2 -treated (red squares) conditions. The data fit well with a biphasic concentration–response curve, assuming the fraction of high affinity inhibition of 0.314 estimated for full-length TRPM7-wt (in Figure 1D ). Under control conditions, the current was inhibited by intracellular free Mg 2+ with an IC 50(1) of 7.6 μM and an IC 50(2) of 986 μM. After H 2 O 2 treatment, the current was inhibited by intracellular free Mg 2+ with an IC 50 of 3.0 μM. Each symbol represents the mean ± SEM (vertical bar) of 5–28 observations. (E) The time course of the M7cd current when M7kd-wt was transfected by either lipofection (closed circles) or baculovirus infection (open squares) in the presence of 0.2 mM [Mg 2+ ] i . Each symbol represents the mean ± SEM (vertical bar) of 8–20 observations. (F) The effect of H 2 O 2 (500 μM) on the M7cd current in the presence of 0.2 mM [Mg 2+ ] i in cells that were transfected with M7kd-wt by baculovirus infection. Each symbol represents the mean ± SEM (vertical bar) of eight recordings. (G) The effect of NMM (100 μM) on M7cd current in the presence of 0.2 mM [Mg 2+ ] i in cells that were transfected with M7kd-wt by baculovirus infection. Each symbol represents the mean ± SEM (vertical bar) of six recordings. (H) The effect of co-expression with kinase-inactive mutant M7kd-1645R on M7cd current. Each symbol represents the mean ± SEM (vertical bar) of four recordings. (I) The effect of TRPM6 kinase domain (M6kd) co-expression on M7cd current. Each symbol represents the mean ± SEM (vertical bar) of eight recordings.

    Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047; epitope 1146-1165 of human TRPM7, Alomone Labs, Jerusalem, Israel) was used.

    Techniques: Functional Assay, Expressing, Transfection, Inhibition, Concentration Assay, Infection, Mutagenesis

    Localization of TRPM7 or its mutants in HEK293 cells. Representative confocal images showing the localization of TRPM7 (green) in doxycycline-untreated (Dox (−)) and mTRPM7-wt (WT), -C721A, -C738A, -C721S, -C738S, -C1809S, and -C1813S-expressing HEK293 cells. Phase contrast images are also shown. Scale bar, 20 μm

    Journal: bioRxiv

    Article Title: Zinc-binding motif acts as an oxidative stress sensor to regulate TRPM7 channel activity

    doi: 10.1101/2020.09.28.316125

    Figure Lengend Snippet: Localization of TRPM7 or its mutants in HEK293 cells. Representative confocal images showing the localization of TRPM7 (green) in doxycycline-untreated (Dox (−)) and mTRPM7-wt (WT), -C721A, -C738A, -C721S, -C738S, -C1809S, and -C1813S-expressing HEK293 cells. Phase contrast images are also shown. Scale bar, 20 μm

    Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047; epitope 1146-1165 of human TRPM7, Alomone Labs, Jerusalem, Israel) was used.

    Techniques: Expressing

    Functional expression of the TRPM7 channel domain (M7cd) in HEK293 cells. (A) Time course of M7cd currents in the absence (open circles) or presence of 2.8 μM (blue squares), 20.9 μM (green triangles), and 0.2 mM [Mg 2+ ] i (black circles) in HEK293 cells that were treated with doxycycline. Each symbol represents the mean ± SEM (vertical bar) of 9–16 recordings. (B) Representative I–V relationship of M7cd current recorded in the presence of various [Mg 2+ ] i at 2 min after break-in. (C) [Mg 2+ ] i -dependent inhibition of M7cd current at 2 min. The current was inhibited by intracellular free Mg 2+ with an IC 50 of 3.0 μM. Each symbol represents the mean ± SEM (vertical bar) of 14–36 recordings. The dashed line is the [Mg 2+ ] i -dependent curve of the full-length TRPM7-wt current (from Figure 1D ). (D) M7cd-S1107E current density was similar in the absence (open inversed triangles) and presence of 0.2 mM [Mg 2+ ] i (closed inversed triangles). Each symbol represents the mean ± SEM (vertical bar) of 7–10 recordings. (E) The effect of H 2 O 2 (500 μM) on M7cd current in the absence of intracellular Mg 2+ (open circles). Each symbol represents the mean ± SEM (vertical bar) of 19 recordings. (F) The time course of the M7cd current in the presence of 0.8 μM [Mg 2+ ] i with (red circles) or without (black circles) H 2 O 2 application at 2 min. Each symbol represents the mean ± SEM (vertical bar) of 17–35 recordings. The current decreased over time even in the control conditions (black circles), and repeated-measures ANOVA revealed that H 2 O 2 has no significant effect on the current decrease (degrees-of-freedom were corrected using the Greenhouse– Geisser estimate of epsilon, p = 0.247).

    Journal: bioRxiv

    Article Title: Zinc-binding motif acts as an oxidative stress sensor to regulate TRPM7 channel activity

    doi: 10.1101/2020.09.28.316125

    Figure Lengend Snippet: Functional expression of the TRPM7 channel domain (M7cd) in HEK293 cells. (A) Time course of M7cd currents in the absence (open circles) or presence of 2.8 μM (blue squares), 20.9 μM (green triangles), and 0.2 mM [Mg 2+ ] i (black circles) in HEK293 cells that were treated with doxycycline. Each symbol represents the mean ± SEM (vertical bar) of 9–16 recordings. (B) Representative I–V relationship of M7cd current recorded in the presence of various [Mg 2+ ] i at 2 min after break-in. (C) [Mg 2+ ] i -dependent inhibition of M7cd current at 2 min. The current was inhibited by intracellular free Mg 2+ with an IC 50 of 3.0 μM. Each symbol represents the mean ± SEM (vertical bar) of 14–36 recordings. The dashed line is the [Mg 2+ ] i -dependent curve of the full-length TRPM7-wt current (from Figure 1D ). (D) M7cd-S1107E current density was similar in the absence (open inversed triangles) and presence of 0.2 mM [Mg 2+ ] i (closed inversed triangles). Each symbol represents the mean ± SEM (vertical bar) of 7–10 recordings. (E) The effect of H 2 O 2 (500 μM) on M7cd current in the absence of intracellular Mg 2+ (open circles). Each symbol represents the mean ± SEM (vertical bar) of 19 recordings. (F) The time course of the M7cd current in the presence of 0.8 μM [Mg 2+ ] i with (red circles) or without (black circles) H 2 O 2 application at 2 min. Each symbol represents the mean ± SEM (vertical bar) of 17–35 recordings. The current decreased over time even in the control conditions (black circles), and repeated-measures ANOVA revealed that H 2 O 2 has no significant effect on the current decrease (degrees-of-freedom were corrected using the Greenhouse– Geisser estimate of epsilon, p = 0.247).

    Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047; epitope 1146-1165 of human TRPM7, Alomone Labs, Jerusalem, Israel) was used.

    Techniques: Functional Assay, Expressing, Inhibition

    Mouse TRPM7 is inhibited by oxidative stress that is induced by H 2 O 2 in an [Mg 2+ ] i -dependent manner. (A) Effect of H 2 O 2 (500 μM) on mTRPM7 current at +80 mV in the absence (open circles) or the presence of 0.2 mM [Mg 2+ ] i (closed circles). Under the whole-cell clamp mode, ramp command pulses from −100 to +100 V (1-s duration) were applied every 10 s. Each symbol represents the mean ± SEM (vertical bar) of 8–14 recordings. (B) Representative I–V relationship of mTRPM7-wt current recorded in the presence of 0.2 mM [Mg 2+ ] i , before (black line) or 4 min after (red line) the application of H 2 O 2 (500 μM). (C) Representative I–V relationship of mTRPM7-wt current recorded in the absence of intracellular Mg 2+ , before (black line) or 4 min after (red line) the application of H 2 O 2 (500 μM). (D) [Mg 2+ ] i -dependent inhibition of mTRPM7 current under control (black circles) and H 2 O 2 -treated (red circles) conditions. The data are presented as a relative current to the mean current that was recorded at 2 min after break-in (i.e. just before application of H 2 O 2 ) in the absence of intracellular Mg 2+ . Under control conditions (just before H 2 O 2 application), the current was inhibited by intracellular free Mg 2+ with an IC 50(1) of 5.6 μM and an IC 50(2) of 558 μM (black line). After H 2 O 2 treatment for 4 min, the current was inhibited by intracellular free Mg 2+ with IC 50 at 3.4 μM (red line). Each symbol represents the mean ± SEM (vertical bar) of 4–22 observations. (E) A Mg 2+ -insensitive mutant, mTRPM7-S1107E, was not inhibited by H 2 O 2 (500 μM) in the absence (open squares) or the presence of 0.2 mM [Mg 2+ ] i (closed squares). Each symbol represents the mean ± SEM (vertical bar) of 13–16 recordings. (F) Mean mTRPM7-S1107E current density did not differ in the absence (open bars) or presence (closed bars) of H 2 O 2 . Each bar represents the mean ± SEM of 8–15 observations. (G) A caspase cleavage-resistant mutant mTRPM7-D1510A was inhibited by H 2 O 2 (500 μM) in the presence of 0.2 mM [Mg 2+ ] i (closed triangles). Each symbol represents the mean ± SEM (vertical bar) of six recordings. (H) The inhibitory effect of N-methylmaleimide (NMM) (100 μM) on the mTRPM7 current in the presence of 0.2 mM [Mg 2+ ] i (closed circles). Each symbol represents the mean ± SEM (vertical bar) of five recordings. (I) NMM (100 μM) inhibited mTRPM7 current in the presence of 0.2 mM [Mg 2+ ] i (n = 9), but not in the absence of intracellular Mg 2+ (n = 4). Each bar represents the mean ± SEM (vertical bar). * p

    Journal: bioRxiv

    Article Title: Zinc-binding motif acts as an oxidative stress sensor to regulate TRPM7 channel activity

    doi: 10.1101/2020.09.28.316125

    Figure Lengend Snippet: Mouse TRPM7 is inhibited by oxidative stress that is induced by H 2 O 2 in an [Mg 2+ ] i -dependent manner. (A) Effect of H 2 O 2 (500 μM) on mTRPM7 current at +80 mV in the absence (open circles) or the presence of 0.2 mM [Mg 2+ ] i (closed circles). Under the whole-cell clamp mode, ramp command pulses from −100 to +100 V (1-s duration) were applied every 10 s. Each symbol represents the mean ± SEM (vertical bar) of 8–14 recordings. (B) Representative I–V relationship of mTRPM7-wt current recorded in the presence of 0.2 mM [Mg 2+ ] i , before (black line) or 4 min after (red line) the application of H 2 O 2 (500 μM). (C) Representative I–V relationship of mTRPM7-wt current recorded in the absence of intracellular Mg 2+ , before (black line) or 4 min after (red line) the application of H 2 O 2 (500 μM). (D) [Mg 2+ ] i -dependent inhibition of mTRPM7 current under control (black circles) and H 2 O 2 -treated (red circles) conditions. The data are presented as a relative current to the mean current that was recorded at 2 min after break-in (i.e. just before application of H 2 O 2 ) in the absence of intracellular Mg 2+ . Under control conditions (just before H 2 O 2 application), the current was inhibited by intracellular free Mg 2+ with an IC 50(1) of 5.6 μM and an IC 50(2) of 558 μM (black line). After H 2 O 2 treatment for 4 min, the current was inhibited by intracellular free Mg 2+ with IC 50 at 3.4 μM (red line). Each symbol represents the mean ± SEM (vertical bar) of 4–22 observations. (E) A Mg 2+ -insensitive mutant, mTRPM7-S1107E, was not inhibited by H 2 O 2 (500 μM) in the absence (open squares) or the presence of 0.2 mM [Mg 2+ ] i (closed squares). Each symbol represents the mean ± SEM (vertical bar) of 13–16 recordings. (F) Mean mTRPM7-S1107E current density did not differ in the absence (open bars) or presence (closed bars) of H 2 O 2 . Each bar represents the mean ± SEM of 8–15 observations. (G) A caspase cleavage-resistant mutant mTRPM7-D1510A was inhibited by H 2 O 2 (500 μM) in the presence of 0.2 mM [Mg 2+ ] i (closed triangles). Each symbol represents the mean ± SEM (vertical bar) of six recordings. (H) The inhibitory effect of N-methylmaleimide (NMM) (100 μM) on the mTRPM7 current in the presence of 0.2 mM [Mg 2+ ] i (closed circles). Each symbol represents the mean ± SEM (vertical bar) of five recordings. (I) NMM (100 μM) inhibited mTRPM7 current in the presence of 0.2 mM [Mg 2+ ] i (n = 9), but not in the absence of intracellular Mg 2+ (n = 4). Each bar represents the mean ± SEM (vertical bar). * p

    Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047; epitope 1146-1165 of human TRPM7, Alomone Labs, Jerusalem, Israel) was used.

    Techniques: Inhibition, Mutagenesis