Defective Transient Receptor Potential Channel TRPM7 Impairs Cellular Glycolysis Via GLUT3 Inhibition

Objective:Most cells require energy for growth mainly from glucose catabolism,especially glycolysis,which not only provides energy for cellular life activities directly,but also regulates cellular functions by producing metabolites such as lactate,participating in the construction of acidic microenvironment and protein modification and thus regulating pathophysiological processes and disease occurrence,such as tumorigenic and vasoproliferative diseases.Although many regulatory molecules such as metabolic enzymes and transcription factors have been identified in cells,the role of ion channels,as important transmembrane molecules responsible for signal transduction,in the regulation of cellular glycolytic metabolism has not been revealed.Therefore,identifying the key ion channels that regulate cellular glycolytic metabolism and elucidating the mechanisms could help expand the understanding of the regulatory network of cellular metabolism and provide potential targets for the treatment of diseases such as tumors and abnormal vascular proliferation.Methods:Firstly,we used TRP channel family members as the main candidate ion channel proteins and performed RT-q PCR in human breast cancer cells（MCF7）and human bladder cancer cells（T24）with high basal glycolytic metabolism to determine the expression abundance,with the highest abundance of TRPM7 as the main candidate molecule for study.Subsequently,we established a TRPM7 knockout T24 cell line based on CRISPR/Cas9 technology and explored the metabolic changes in TRPM7knockdown cells by using glucose uptake assays,quantitative lactate ELISA,Seahorse live cell metabolic function assays and isotope glucose metabolic flow tracing to clarify the TRPM7 and cellular glucose metabolism association and to examine cell growth.Subsequently,transcriptomic and bioinformatics analyses were used to identify the potential mechanisms by which TRPM7 affects cellular glycolysis-Ca²⁺/Calcineurin/CRTC2/CREB/GLUT3-and subsequently revert to the key signaling pathway node molecules CRTC2/CREB and the direct target molecule GLUT3 by lentivirus in TRPM7 knockout cells and tested whether the reversion of key molecules improved the metabolism and growth of TRPM7 knockdown cells.Finally,these mechanisms were validated in nude mouse transplantation tumor and retinal angiogenesis models to determine whether TRPM7 is involved in tumor growth and retinal angiogenesis through this mechanism and to clarify its potential value as a therapeutic target for disease.Results:TRPM7 is the most abundantly expressed TRP channel family member in human bladder cancer cells（T24）and human breast cancer cells（MCF7）.In cells after TRPM7 knockdown by CRISPR/Cas9,reduced cellular glucose uptake and glycolytic metabolism and decreased lactate production were found,accompanied by diminished cell proliferation and migration capacity.Mechanistically,TRPM7 knockdown reduced extracellular Ca2+inward flow,decreased activation of the calcineurin,inhibited CRTC2 entry into the nucleus,impaired CREB1 phosphorylation,and led to impaired transcription of the glucose transporter GLUT3,triggering glucose transport across the membrane,glycolytic metabolism,and cellular dysfunction.In addition,defective TRPM7 in vascular endothelial cells can also conservatively trigger impaired GLUT3transcription and reduce endothelial glucose transport across membranes,glycolytic metabolism and endothelial function,leading to impaired angiogenesis.Conclusion:TRPM7 deficiency leads to impaired GLUT3transcription through inhibition of Ca²⁺/calcineurin/CRTC2/CREB1signaling pathway,inhibiting cellular glycolytic metabolism and impairing cell proliferation.