Investigation of the TRPM7 COMPLEX in mouse embryonic stem cells
Creators: name: Nethramangalath, Thushara
Publisher: name: No Publisher Supplied
Year: 2024

description: Transient Receptor Potential Melastatin 7 (TRPM7) is an ion channel and kinase with broad functions in cell physiology, including cellular magnesium homeostasis, cell proliferation, and cytoskeletal remodeling. TRPM7 is permeable to the essential divalent cations Mg2+, Ca2+ and Zn2, which are fundamental to the channel’s function. TRPM7’s kinase has been shown to phosphorylate multiple substrates and when cleaved from the channel, regulate gene expression events in the nucleus. TRPM7 is also the first ion channel shown to be indispensable for embryonic development. Loss of the channel-kinase in mouse and Xenopus laevis, results in embryonic lethality. Despite extensive research on the channel over the past decades, how TRPM7 exerts its many cellular functions, particularly during embryonic development, is poorly understood. Recently TRPM7 was found to assemble in rodent brain with newly identified interacting partners: CNNMs (cyclin and CBS domain divalent metal cation transport mediators), ARL15 (ADP ribosylation factor-like protein 15), and PRLs (Phosphatases of Regenerating Liver). We hypothesize that TRPM7 assembles with these proteins in embryonic stem cells to regulate TRPM7’s diverse functions. To test our hypothesis, we used mouse embryonic stem cells (mESCs) as our model system to investigate TRPM7’s role in early developmental processes. Using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and RNA sequencing (RNAseq), we demonstrate that Cnnm2 and Cnnm3 are the predominant CNNM members in mESCs. Mass spectrometry analysis further reveals that CNNM2 and CNNM3 are the dominant CNNM proteins that interact with the channel in mESCs. TRPM7 knockout (KO) mESCs exhibit impaired proliferation, despite magnesium supplementation in the growth media. Our experiments also demonstrate that TRPM7 KO mESCs display altered gene expression of pluripotency markers, including Nanog and Sox2. Moreover, RNAseq analysis of TRPM7 KO mESCs reveals significant transcriptional changes, with over 500 differentially expressed genes (DEGs). We generated CNNM3 homozygous and heterozygous knockout mESCs and show that they retain viability and express key pluripotency markers. Nevertheless, deletion of CNNM3 from mESC significantly reduces TRPM7 channel activity. Differentiation assays using mESCs demonstrate that TRPM7 KO cells exhibit widespread changes in gene expression and potentially influences lineage specification in mESCs, with TRPM7 KO cells showing upregulation of mesodermal and endodermal markers. Neuroectodermal (NE) differentiation of TRPM7 KO mESCs show altered morphology of TRPM7 KO NE cells and significant changes in gene expression compared to WT NE cells. Mass spectrometry analysis of NE cells identify CNNM4, CNNM3, CNNM2 and ARL15 as the main TRPM7 interacting proteins in NE cells. Together, these findings advance the current understanding of TRPM7’s interactome in early embryonic cells, revealing new insights into TRPM7 and CNNM’s roles in mESC pluripotency, proliferation, and lineage specification.