TRPM7 Knockout Cell Line (HEK293)

Magnesium is essential for cellular life, but how it is homeostatically controlled still remains poorly understood. Here, we report that members of CNNM family, which have been controversially implicated in both cellular Mg2+ influx and efflux, selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells. Coexpression of CNNMs with the channel markedly increased uptake of divalent cations, which is prevented by an inactivating mutation to the channel's pore. Knockout (KO) of TRPM7 in cells or application of the TRPM7 channel inhibitor NS8593 also interfered with CNNM-stimulated divalent cation uptake. Conversely, KO of CNNM3 and CNNM4 in HEK-293 cells significantly reduced TRPM7-mediated divalent cation entry, without affecting TRPM7 protein expression or its cell surface levels. Furthermore, we found that cellular overexpression of phosphatases of regenerating liver (PRLs), known CNNMs binding partners, stimulated TRPM7-dependent divalent cation entry and that CNNMs were required for this activity. Whole-cell electrophysiological recordings demonstrated that deletion of CNNM3 and CNNM4 from HEK-293 cells interfered with heterologously expressed and native TRPM7 channel function. We conclude that CNNMs employ the TRPM7 channel to mediate divalent cation influx and that CNNMs also possess separate TRPM7-independent Mg2+ efflux activities that contribute to CNNMs' control of cellular Mg2+ homeostasis.

BACKGROUND:The local anesthetic lidocaine suppresses some cancer cell lines but the mechanism is unclear. The melastatin-like transient receptor potential 7 (TRPM7) ion channel is aberrantly expressed in some cancers and may play a role in the disease. Hence, we suggested that lidocaine affects the viability and migration of breast cancer cells by regulating TRPM7. METHODS:We measured the effects of lidocaine on TRPM7 function in HEK293 with exogenous TRPM7 expression (HEK-M7) using whole-cell patch-clamp and fura-2AM-based quench assay. We measured the effect of lidocaine on TRPM7 function, cell viability, and migration in TRPM7 expressing human breast cancer cell lines using fura-2AM-based quench, MTT, and wound-healing assays respectively. We compared cell viability and migration of wild type HEK293 cells (WT-HEK) with HEK-M7 and wild type MDA-MB-231 (WT-231) with TRPM7 knockout MDA-MB-231 (KO-231). RESULTS:Lidocaine (1-3 mM) inhibited the viability and migration of all of these breast cancer cell lines. Functional evidence for TRPM7 was confirmed in the MDA-MB-231, AU565, T47D, and MDA-MB-468 cell lines where lidocaine at 0.3-3 mM suppressed the TRPM7 function. Lidocaine preferentially suppressed viability and migration of HEK-M7 over WT-HEK and WT-231 over KO-231. CONCLUSIONS:Lidocaine differentially reduced the viability and migration of human breast cancer cell lines tested. TRPM7 is one of the potential targets for the effects of lidocaine on viability and migration in MDA-MB-231, AU565, T47D, and MDA-MB-468.