The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics

Abstract

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+, and Ca2+, and thus shapes cellular excitability, plasticity, and metabolic activity. The molecular appearance and operation of TRPM7 channels in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative mass spectrometry (MS) analysis. We found that native TRPM7 channels are high-molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ADP-ribosylation factor-like protein 15 (ARL15). Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that complex formation effectively and specifically impacts TRPM7 activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.

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

“…Overexpression of ARL15 in renal carcinoma cells decreased cellular Mg2+ uptake, whereas KO of the protein increased it [33]. While our manuscript was under review, a preprint appeared on the bioRxiv server, reporting that macromolecular complexes of TRPM7, CNNMs, PRLs, and ARL15 can be detected in rodent brain [63]. The article, which was recently accepted for publication, demonstrated that heterologous overexpression of ARL15 with TRPM7 in HEK-293 cells suppressed TRPM7 channel activity [64].…”

“…While our manuscript was under review, a preprint appeared on the bioRxiv server, reporting that macromolecular complexes of TRPM7, CNNMs, PRLs, and ARL15 can be detected in rodent brain [63]. The article, which was recently accepted for publication, demonstrated that heterologous overexpression of ARL15 with TRPM7 in HEK-293 cells suppressed TRPM7 channel activity [64]. Using the Zn 2+ influx assay, we similarly found that overexpression of ARL15 inhibited TRPM7-mediated Zn 2+ influx (S12 Fig) . Conversely, we also find that knockdown of ARL15 in HEK-293 cells increased TRPM7-mediated Zn 2+ influx (S12 Fig) . Thus, PRLs and ARL15 appear to have opposing actions on TRPM7 function.…”

“…Interestingly, we find that CNNMs are capable of forming heteroligomers, with native CNNM3 and CNMM4 forming a complex together (S16 Fig) . The function of CNNM heteroligomers has not been investigated and whether they cooperatively regulate TRPM7 remains to be determined in future work. However, the discovery that TRPM7 has the potential to interact with multiple CNNM isoforms in vitro, as well as in native rodent brain [64], has uncovered a potential mechanism by which diverse control of the channel could be achieved in different tissues and cell types by varying the isoforms that makeup the CNNM-TRPM7 complex. We speculate that given Mg 2+ homeostasis profound impact on so many essential cellular activities, from cell metabolism to cell proliferation, and TRPM7's channel influence on disease events from cancer to stroke, CNNM-TRPM7 complexes of varying composition will likely be found contributing to diverse physiological and pathological processes.…”

Structural and functional comparison of magnesium transporters throughout evolution

“…A recent study has shown that CNNM proteins can interact with the Mg 2+ channel transient receptor potential receptor type 7 (TRPM7) in vivo and vitro. [ 117 ] ARL15 reduced TRPM7-mediated currents in X. laevis oocytes upon heterologous overexpression, suggesting a potential, complex regulatory mechanism of ARL15-CNNM regulation on TRPM7 channel activity.…”

The double lives of phosphatases of regenerating liver: A structural view of their catalytic and noncatalytic activities

“…There are many unanswered questions about the biological function of PRLs but also signs that the gaps in our understanding are closing. One promising development is the realization that CNNM proteins are associated with another family of proteins, the melastatin-related transient receptor potential ion channels, TRPM6 and TRPM7 ( 61 , 84 , 85 , 86 , 87 ). Like CNNMs, TRPM6/7 are important for magnesium homeostasis and have been implicated in cancer ( 55 , 88 , 89 , 90 ).…”