Mg·ATP insensitivity of TRPM6/TRPM7 heteromeric ion channels

The Transient Receptor Potential Melastatin member 7 (TRPM7) and member 6 (TRPM6) are divalent cation channel kinases essential for magnesium (Mg) homeostasis in vertebrates. It remains unclear how TRPM6 affects divalent cation transport and whether this involves functional homomeric TRPM6 plasma membrane channels or heteromeric channel assemblies with TRPM7. We show that homomeric TRPM6 is highly sensitive to intracellular free Mg and therefore unlikely to be active at physiological levels of [Mg]i. Coexpression of TRPM7 and TRPM6 produces heteromeric TRPM7/M6 channels with altered pharmacology and sensitivity to intracellular Mg·ATP compared to homomeric TRPM7. Strikingly, the activity of heteromeric TRPM7/M6 channels is independent of intracellular Mg·ATP concentrations, essentially uncoupling channel activity from cellular energy status. Disruption of TRPM6 kinase phosphorylation activity re-introduces Mg·ATP sensitivity to the heteromeric channel similar to that of TRPM7. Thus, TRPM6 modulates the functionality of TRPM7, and the TRPM6 kinase plays a critical role in tuning the phenotype of the TRPM7/M6 channel complex. TRPM7 and TRPM6 belong to the melastatinrelated subfamily of TRP channels and represent unique bifunctional proteins with both ion channel and protein kinase function (1–4). The two proteins are highly homologous to each other with >50% sequence identity. TRPM7and TRPM6-mediated membrane currents exhibit a http://www.jbc.org/cgi/doi/10.1074/jbc.M113.512285 The latest version is at JBC Papers in Press. Published on January 2, 2014 as Manuscript M113.512285 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Mg·ATP insensitivity of TRPM6/TRPM7 heteromeric ion channels 2 highly nonlinear current-voltage (I/V) relationship with pronounced outward rectification (3, 5). The small inward currents at physiologically relevant negative membrane voltages are carried by divalent cations, including Mg and Ca, and the large outward currents at positive voltages are due to efflux of monovalent cations (5, 6). Considerable efforts have been directed towards understanding the physiological functions of TRPM7 (7). TRPM7 represents the only known ion channel that is essential for cell viability (3, 8, 9). Its channel function is inhibited by both intracellular free Mg and Mg·ATP (3, 10) and both factors serve as the major physiological mechanisms controlling TRPM7-mediated divalent cation transport. Information is more limited for TRPM6 and some of it remains controversial. Conflicting results have been reported concerning TRPM6 expression in heterologous expression systems. Two independent groups were able to measure homomeric TRPM6 currents in HEK-293 cells (5, 11–13), whereas the other two show that TRPM6 is retained intracellularly when expressed alone (14–17). Hence, it has been proposed that TRPM6 may function in association with TRPM7 by forming a heteromeric TRPM7/M6 channel complex (14–17). Channel kinase heteromerization results in a slightly altered permeation and pharmacology profile, pH sensitivity and single channel conductance (11, 12). However, important questions about regulation and functionality of heteromeric channel kinases remain unresolved. EXPERIMENTAL PROCEDURES Cell culture and transfection Wild-type HEK-293 cells were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS) (Invitrogen, USA). The cells were transiently transfected with N-terminally hemagglutinin (HA)-tagged human TRPM6 constructs using Lipofectamine 2000 (Invitrogen, USA). Human TRPM6 phosphotransferasedeficient mutant (TRPM6-K1804R) and human TRPM6 ∆kinase were made from the original wild-type pCINeo-IRES-GFP-TRPM6 construct (TRPM6-WT, GenBank accession number 017662) and kindly provided by Dr. J. Hoenderop and Dr. R. Bindels (5, 13). Whole-cell patch clamp experiments were carried out 30 hours posttransfection. Expression of various human TRPM6 constructs was identified by green fluorescence. For expression in tetracycline (tet)-inducible HEK293 cells, human TRPM6 WT and TRPM6 mutants were cloned into the pcDNA5/TO-FLAG vector and human TRPM7 WT into the pcDNA4/TO-HA vector as previously described (8, 17). Tet-inducible HEK-293 cells encoding human TRPM7 + human TRPM6 WT (TRPM7/M6), human TRPM7 + human TRPM6 K1804R (TRPM7/M6 K1804R) and human TRPM7 + human TRPM6 ∆kinase (TRPM7/M6 ∆kinase) were maintained in DMEM media containing 10% FBS, blasticidin (5 μg/ml), zeocin (0.4 mg/ml) and hygromycin (0.5 mg/ml) (17). Tetracycline-inducible human TRPM7 HEK-293 cells were cultured in DMEM media containing 10% FBS, blasticidin (5 μg/ml) and zeocin (0.4 mg/ml) (8). Tetracycline-inducible human TRPM6 HEK-293 cells were maintained in DMEM media containing 10% FBS, blasticidin (5 μg/ml) and hygromycin (0.5 mg/ml) (Invitrogen, USA) (17). The proteins were induced by adding 1 μg/ml tetracycline to the culture media. Current measurements were performed 14 24 hours following tetracycline induction.