Methionine sulfoxide reductase B1 (MsrB1) recovers TRPM6 channel activity during oxidative stress

Abstract

Mg(2+) is an essential ion for many cellular processes, including protein synthesis, nucleic acid stability, and numerous enzymatic reactions. Mg(2+) homeostasis in mammals depends on the equilibrium between intestinal absorption, renal excretion, and exchange with bone. The transient receptor potential melastatin type 6 (TRPM6) is an epithelial Mg(2+) channel, which is abundantly expressed in the luminal membrane of the renal and intestinal cells. It functions as the gatekeeper of transepithelial Mg(2+) transport. Remarkably, TRPM6 combines a Mg(2+)-permeable channel with an alpha-kinase domain. Here, by the Ras recruitment system, we identified methionine sulfoxide reductase B1 (MsrB1) as an interacting protein of the TRPM6 alpha-kinase domain. Importantly, MsrB1 and TRPM6 are both present in the renal Mg(2+)-transporting distal convoluted tubules. MsrB1 has no effect on TRPM6 channel activity in the normoxic conditions. However, hydrogen peroxide (H(2)O(2)) decreased TRPM6 channel activity. Co-expression of MsrB1 with TRPM6 attenuated the inhibitory effect of H(2)O(2) (TRPM6, 67 +/- 5% of control; TRPM6 + MsrB1, 81 +/- 5% of control). Cell surface biotinylation assays showed that H(2)O(2) treatment does not affect the expression of TRPM6 at the plasma membrane. Next, mutation of Met(1755) to Ala in TRPM6 reduced the inhibitory effect of H(2)O(2) on TRPM6 channel activity (TRPM6 M1755A: 84 +/- 10% of control), thereby mimicking the action of MsrB1. Thus, these data suggest that MsrB1 recovers TRPM6 channel activity by reducing the oxidation of Met(1755) and could, thereby, function as a modulator of TRPM6 during oxidative stress.

FIGURE 1.

Interaction and co-expression between TRPM6 and MsrB1. A, complementation of the cdc25-2 mutation through the interaction of TRPM6 α-kinase domain with MsrB1. The temperature-sensitive cdc25-2 yeast strain was co-transformed with TRPM6 α-kinase domain and MsrB1 or control plasmid (Mock) and incubated on galactose-containing plates either at 25 °C or at 37 °C. B, co-precipitation studies of GST and GST-α-kinase in MsrB1-expressing HEK293 cells (top panel). MsrB1 input (1%) expression was analyzed by immunoblotting (bottom panel). C, co-precipitation of GST-MsrB1 in TRPM6-expressing HEK293 cells (top panel). TRPM6 input (5%) and MsrB1 precipitation expression were analyzed by immunoblotting (middle panel and bottom panel). D, distribution of MsrB1 (top panel) mRNA expression analyzed by RT-PCR on various tissues. β-Actin was used as a positive control (bottom panel). E, immunohistochemical analysis of TRPM6 (upper panel) and MsrB1 (lower panel) in serial mouse kidney sections. * indicates overlapping immunopositive tubules for TRPM6 and MsrB1.

FIGURE 2.

H₂O₂ effect on TRPM6 channel activity and plasma membrane expression. A, time course of the current density (pA/pF) at +80 mV of TRPM6-transfected HEK293 cells, where 1 mm H₂O₂ was added to the bath solution after 200 s (arrow). B, current-voltage (I/V) relation of TRPM6-transfected cells perfused with standard extracellular solution (solid line) and 1 mm H₂O₂ (dashed line). C, the percentage of inhibition of the TRPM6 current at +80 mV after 1000 s in response to different H₂O₂ concentrations, normalized to 1 mm H₂O₂. D, TRPM6-expressing HEK293 cells were treated with 1 mm H₂O₂ and subsequently subjected to cell surface biotinylation assays. TRPM6 expression was analyzed by immunoblotting for the plasma membrane (PM) fraction (top panel) and for the total cell lysates (bottom panel). A representative immunoblot of three independent experiments is shown. As negative controls, mock-transfected cells (Mock) were used. E, densitometry quantification of TRPM6 surface expression with/without H₂O₂ treatment. OD, optical density.

FIGURE 3.

MsrB1 reduces the inhibitory effect of H₂O₂ on TRPM6. A, time course of the current density (pA/pF) at +80 mV of TRPM6 and TRPM6 + MsrB1-transfected HEK293 cells, where 1 mm H₂O₂ was added to the bath solution after 200 s (arrow). B, normalized values of the current at +80 mV after 800 s of TRPM6 and TRPM6 + MsrB1, treated with (open bars) or without (closed bars) H₂O₂. * indicates p < 0.05.

FIGURE 4.

TRPM6 M1755A is not significantly inhibited by H₂O₂. A, normalized values of the current at +80 mV after 800 s of TRPM6 and all TRPM6 Met/Ala mutants, treated with (open bars) or without (closed bars) 1 mm H₂O₂. * indicates p < 0.05. B, time course of the current density (pA/pF) at +80 mV of TRPM6 and TRPM6 M1755A-transfected HEK293 cells, where 1 mm H₂O₂ was added to the bath solution after 200 s (arrow).