Regulation of the epithelial Mg2+ channel TRPM6 by estrogen and the associated repressor protein of estrogen receptor activity (REA)

Abstract

The maintenance of the Mg(2+) balance of the body is essential for neuromuscular excitability, protein synthesis, nucleic acid stability, and numerous enzymatic systems. The Transient Receptor Potential Melastatin 6 (TRPM6) functions as the gatekeeper of transepithelial Mg(2+) transport. However, the molecular regulation of TRPM6 channel activity remains elusive. Here, we identified the repressor of estrogen receptor activity (REA) as an interacting protein of TRPM6 that binds to the 6(th), 7(th), and 8(th) beta-sheets in its alpha-kinase domain. Importantly, REA and TRPM6 are coexpressed in renal Mg(2+)-transporting distal convoluted tubules (DCT). We demonstrated that REA significantly inhibits TRPM6, but not its closest homologue TRPM7, channel activity. This inhibition occurs in a phosphorylation-dependent manner, since REA has no effect on the TRPM6 phosphotransferase-deficient mutant (K1804R), while it still binds to this mutant. Moreover, activation of protein kinase C by phorbol 12-myristate 13-acetate-PMA potentiated the inhibitory effect of REA on TRPM6 channel activity. Finally, we showed that the interaction between REA and TRPM6 is a dynamic process, as short-term 17beta-estradiol treatment disassociates the binding between these proteins. In agreement with this, 17beta-estradiol treatment significantly stimulates the TRPM6-mediated current in HEK293 cells. These results suggest a rapid pathway for the effect of estrogen on Mg(2+) homeostasis in addition to its transcriptional effect. Together, these data indicate that REA operates as a negative feedback modulator of TRPM6 in the regulation of active Mg(2+) (re)absorption and provides new insight into the molecular mechanism of renal transepithelial Mg(2+) transport.

FIGURE 1.

REA interacts with the α-kinase domain in TRPM6. A, GST pull-down assay between [³⁵S]methionine-labeled REA protein and GST or GST fused to the α-kinase domain. B, co-precipitation studies of GST and GST-α-kinase in REA-expressing HEK293 cells (top panel). REA input (1%) expression was analyzed by immunoblotting (bottom panel). C, mapping of the REA binding site within the α-kinase domain. Predicted three-dimensional structure model of the α-kinase domain with different truncations of the α-kinase. D, Coomassie Blue staining of the SDS-PAGE gel. E, co-precipitation studies of GST and GST-α-kinase in REA-expressing HEK293 cells in the presence of different amounts of RACK1 (dilution 1:10 (0.1), 1:2 (0.5), and non-diluted (1.0)) (top panel). REA and RACK1 input (1%) expression was analyzed by immunoblotting (bottom panels).

FIGURE 2.

Tissue distribution and localization of REA. A, distribution of REA (top panel) mRNA expression analyzed by RT-PCR on various tissues. β-actin was used as a positive control (bottom panel). B, immunohistochemical analysis of REA (left panel) and TRPM6 (right panel) in mouse kidney serial sections. * indicates overlapping immunopositive tubules for TRPM6 and REA.

FIGURE 3.

Functional effect of REA on TRPM6 activity. A, time course of the current density (pA/pF) at +80 mV of TRPM6 and mock (▪), and TRPM6 and REA (•) transfected HEK293 cells. B, averaged values of the current density at +80 mV after 200 s of TRPM6 and mock (n = 17), and TRPM6 and REA (n = 17). * indicates p < 0.05 compared with TRPM6 and mock. C, time course of the current density (pA/pF) at +80 mV of TRPM7 and mock (□), and TRPM7 and REA (○) transfected HEK293 cells. D, averaged values of the current density at +80 mV after 200 s of TRPM7 and mock (n = 18), and TRPM7 and REA (n = 17).

FIGURE 4.

REA inhibits TRPM6 channel activity in a rapid and phosphorylation-dependent manner. A, Coomassie Blue staining of purified GST and GST-REA. B, current-voltage (I/V) relation of TRPM6-transfected cells infused with GST (solid trace) or GST-REA (dashed trace). C, averaged values of the current density at +100 mV after 600 s of GST infused (n = 16) and GST-REA infused (n = 18) cells. * indicates p < 0.05. D, current-voltage (I/V) relations of TRPM6 K1804R transfected cells infused with GST (solid trace) or GST-REA (dashed trace). E, averaged values of the current density at +100 mV after 600 s of GST infused (n = 26) and GST-REA infused (n = 21) cells. F, co-precipitation studies of GST, GST-α-kinase, and GST-α-kinase K1804R in REA-expressing HEK293 cells (top panel). REA input (1%) expression was analyzed by immunoblotting (bottom panel). G, in vitro protein kinase assay of TRPM6 and TRPM6 and REA (left panel), analyzed for total expression by immunoblotting (right panels). H, effect of PKC activation by PMA (100 nm, 5 min pretreatment) on the current density at +80 after 200 s of TRPM6 and mock (n = 17–44), and TRPM6 and REA (n = 17–36). * indicates p < 0.05 compared with TRPM6 and mock without pretreatment, and # indicates p < 0.05 compared with TRPM6 and REA without pretreatment.

FIGURE 5.

Effect of 17β-estradiol on the binding between REA on TRPM6 and on TRPM6 channel activity. A, GST-fused TRPM6 α-kinase domain, GST (control) and REA were co-expressed in HEK293 cells. After treatment with 50 nm 17β-estradiol (17β-E2) or vehicle, REA was co-precipitated with GST-fused α-kinase domain and visualized using the anti-REA antibody (top panel). Immunoblotting (bottom panel) and densitometry quantification (right panel) of binding between TRPM6 α-kinase domain and REA. * indicates p < 0.05 compared with vehicle treatment. B, time course of the current density (pA/pF) at +80 mV of TRPM6 transfected HEK293 cells in control condition (▪) and treated with 50 nm 17β-estradiol (E2) for 10 min at 37 °C (▴). C, averaged values of the current density at +80 mV after 200 s of TRPM6 (n = 37) and TRPM6 pretreated with 50 nm of 17β-estradiol (E2) (n = 37). * indicates p < 0.05 compared with control condition. D, TRPM6 expressing HEK293 cells were treated with 50 nm 17β-estradiol (E2) or vehicle and subsequently subjected to cell surface biotinylation assay. TRPM6 expression was analyzed by immunoblot for the plasma membrane fraction (top panel) and for the total cell lysates (bottom panel). Surface expression of TRPM6 was quantified by densitometry (right panel). As negative controls, mock cells were used.