Methionine Sulfoxide Reductase B1 (MsrB1) Recovers TRPM6 Channel Activity during Oxidative Stress*

Mg2+ is an essential ion for many cellular processes, including protein synthesis, nucleic acid stability, and numerous enzymatic reactions. Mg2+ 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 Mg2+ channel, which is abundantly expressed in the luminal membrane of the renal and intestinal cells. It functions as the gatekeeper of transepithelial Mg2+ transport. Remarkably, TRPM6 combines a Mg2+-permeable channel with an α-kinase domain. Here, by the Ras recruitment system, we identified methionine sulfoxide reductase B1 (MsrB1) as an interacting protein of the TRPM6 α-kinase domain. Importantly, MsrB1 and TRPM6 are both present in the renal Mg2+-transporting distal convoluted tubules. MsrB1 has no effect on TRPM6 channel activity in the normoxic conditions. However, hydrogen peroxide (H2O2) decreased TRPM6 channel activity. Co-expression of MsrB1 with TRPM6 attenuated the inhibitory effect of H2O2 (TRPM6, 67 ± 5% of control; TRPM6 + MsrB1, 81 ± 5% of control). Cell surface biotinylation assays showed that H2O2 treatment does not affect the expression of TRPM6 at the plasma membrane. Next, mutation of Met1755 to Ala in TRPM6 reduced the inhibitory effect of H2O2 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 Met1755 and could, thereby, function as a modulator of TRPM6 during oxidative stress.

To maintain a physiological extra-and intracellular Mg 2ϩ concentration is of great importance to keep the accurate function of more than 300 enzymatic systems and the subsequent various biological and physiological processes (1)(2)(3)(4). The kidney is the principal organ responsible for the regulation of the body Mg 2ϩ balance. Around 80% of the total plasma Mg 2ϩ is ultrafiltered through the glomeruli and subsequently reabsorbed passively in the proximal tubule and the thick ascending limb of Henle's loop (5). The final urinary Mg 2ϩ concentration is defined by active Mg 2ϩ reabsorption in the distal convoluted tubule (DCT) 3 (6).
The transient receptor potential melastatin type 6 (TRPM6) is a cation channel playing a crucial role in Mg 2ϩ homeostasis. Mutations in TRPM6 cause hypomagnesemia with secondary hypocalcemia (7,8). Interestingly, mice deficient of TRPM6 (TRPM6 Ϫ/Ϫ mice) were essentially embryonically lethal, and the incidental TRPM6 Ϫ/Ϫ mice that survived had neural tube defects (9). TRPM6 and its closest homologue TRPM7 uniquely combine an ion channel pore-forming region with a serine/ threonine protein kinase domain. It is located at the carboxyl terminus and has similarities with members of the ␣-kinase family (10,11). Previous studies demonstrated that receptor for activated C-kinase 1 (RACK1) and repressor of estrogen receptor activity (REA) interact with this domain and inhibit channel activity in an (auto)phosphorylation-dependent manner (12,13). Moreover, modulation of TRPM6 channel activity by intracellular ATP requires the ATP-binding motif in the ␣-kinase domain (14). Although the phosphorylation activity of the TRPM6/7 ␣-kinase domains has been well determined, the role of these domains in regulating channel activity remains elusive (12,(15)(16)(17)(18).
Over the last years, several studies have implicated TRPM channels in ischemia (19,20). Sun et al. (21) showed that decreased TRPM7 channel expression significantly reduced neuronal cell death after global ischemia. Furthermore, TRPM4 channel activation in vascular smooth muscle has been shown to contribute to cell death of vascular cells during ischemic injury, and TRPM2 has been well studied in relation to oxidative stress (22)(23)(24)(25). Accumulating evidence suggests that reactive oxygen species are not only harmful side products of cellular metabolism but also central players in cell signaling and regulation (26 -29). Interestingly, renal DCT cells contain the largest number of mitochondria. However, the effect of oxidative stress on the epithelial Mg 2ϩ channel TRPM6, expressed at the apical membrane of the DCT, has not been studied.
The aim of the present study was to investigate the role of the ␣-kinase domain in TRPM6 channel activity by the identification of associated proteins. To this end, the Ras recruitment system (RRS), a novel yeast two-hybrid screening system, which is designed to screen for partners of plasma membrane proteins, was applied (30). Here, we identified methionine sulfoxide reductase B1 (MsrB1) as a TRPM6-associated protein, bind-ing to the TRPM6 ␣-kinase domain. Using biochemical, immunohistochemical, and electrophysiological analyses, we demonstrated a novel operation mode for MsrB1 in the regulation of TRPM6 channel activity in oxidative stress through modulating methionine oxidation in TRPM6.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfection-Human embryonic kidney (HEK) 293 cells were grown and transfected as described previously (31), and electrophysiological recordings were performed 48 h after transfection.
DNA Constructs-The ␣-kinase domain of mouse (1759 -2028) TRPM6 was cloned into the pMet425-Myc-Ras (kind gift from Dr. A. Aronheim, Haifa, Israel) by PCR using mouse kidney cDNA as template. The ␣-kinase domain of human (1759 -2022) TRPM6 was cloned into the pEBG vector using human TRPM6 in pCINeo/IRES-GFP (32) as template. Full-length mouse MsrB1 cDNA was cloned into pCB7 by PCR using mouse kidney cDNA and FLAG-tagged at the N-terminal tail. Wild-type human TRPM6 in the pCINeo/IRES-GFP vector was HA-tagged at the N-terminal tail as described previously (32). TRPM6 M1755A, TRPM6 M1775A, TRPM6 M1904A, and TRPM6 M1947A mutants were created using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. All constructs were verified by sequence analysis.
RRS Screening-RRS screening was performed as described previously (30). Briefly, cdc25-2 strains (kind gift from Dr. A. Aronheim) were co-transformed with the pMet425-Myc-Ras-TRPM6 ␣-kinase domain and mouse kidney cDNA library. Transformants were grown on selectable minimal glucose plates for 5 days at 25°C followed by subsequent replica plating onto minimal galactose plates, incubating for 5-7 days at 37°C. Library plasmids of positive colonies growing on galactose plates at 37°C were isolated and further analyzed by DNA sequencing. The positive colonies were co-transformed with the pMet425-Myc-Ras-TRPM6 ␣-kinase domain into cdc25-2 cells to confirm specificity of interaction.
RT-PCR-Total RNA isolation from mouse tissue and reverse transcription were performed as described previously (33). MsrB1 and ␤-actin were amplified by PCR and subsequently analyzed by agarose gel electrophoresis.
Electrophysiology-Patch clamp experiments were performed in the tight seal whole-cell configuration at room temperature using an EPC-10 patch clamp amplifier computer controlled by the Pulse software (HEKA Elektronik, Lambrecht, Germany). Electrode resistances were 2-5 megaohms, and capacitance and access resistance were monitored continuously. A ramp protocol, consisting of linear voltage ramp from Ϫ100 to ϩ100 mV (within 450 ms), was applied every 2 s from a holding potential of 0 mV. Current densities were obtained by normalizing the current amplitude to the cell membrane capacitance. The time course of current development was determined by measuring the current at ϩ80 and Ϫ80 mV. I/V relations were established from the ramp protocols. The analysis and display of patch clamp data were performed using Igor Pro software (WaveMetrics, Lake Oswego, OR). The standard pipette solution contained 150 mM NaCl, 10 mM EDTA, and 10 mM HEPES-NaOH, pH 7.2. The extracellular solution contained 150 mM NaCl, 10 mM HEPES-NaOH, pH 7.4, supplemented with 1 mM CaCl 2 . To avoid breakdown, hydrogen peroxide (H 2 O 2 ) was stored at 4°C prior to use and added to the perfusate immediately (Ͻ1 min) prior to making recordings.
Cell Surface Labeling with Biotin-HEK293 cells, in poly-Llysine (Sigma)-coated 10-cm dishes, were transiently transfected with 15 g of HA-TRPM6. 72 h after transfection, cells were treated with 1 mM H 2 O 2 for 10 min at 37°C. Cell surface labeling with NHS-LC-LC-biotin (Pierce, Etten-Leur, The Netherlands) was performed as described previously (34). 1 h after homogenizing, biotinylated proteins were precipitated using NeutrAvidin-agarose beads (Pierce). TRPM6 expression was analyzed by immunoblot for the precipitates (plasma membrane fraction) and for the total cell lysates using the mouse anti-HA antibody.
Immunoblotting-Protein samples were denatured by incubation for 30 min at 37°C in Laemmli buffer and then subjected to SDS-PAGE. Immunoblots were incubated with either mouse anti-HA or rabbit anti-MsrB1 antibody. Subsequently, blots were incubated with sheep horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Sigma) and then visualized using the enhanced chemiluminescence system.
Immunohistochemistry-Immunohistochemistry was performed as described previously (35). Briefly, mouse kidney sections were incubated for 16 h at 4°C with rabbit anti-MsrB1 and guinea pig anti-TRPM6. To visualize TRPM6, tyramide signal amplification kit (PerkinElmer Life Sciences, Zaventem, Belgium) was used after incubation with biotin-coated goat antimouse secondary antibody. Images were taken with a Bio-Rad MRC 100 confocal laser scanning microscope.
Statistical Analysis-Values are expressed as mean Ϯ S.E. Statistical significance between groups was determined by analysis of variance followed by Bonferroni's multiple comparison test. Differences between the means of two groups were analyzed by an unpaired Student's t test. p Ͻ 0.05 was considered statistically significant.

MsrB1
Interacts with TRPM6 ␣-Kinase Domain-To identify proteins interacting with the ␣-kinase domain of TRPM6, we applied the RRS. When compared with the conventional yeast two-hybrid screening system, RRS is more appropriate to detect interaction partners of plasma membrane proteins (30). In this approach, MsrB1, a methionine sulfoxide reductase (36), was identified as an interacting protein of the TRPM6 ␣-kinase domain. Subsequently, MsrB1 cDNA was co-transformed with the ␣-kinase domain of TRPM6 into cdc25-2 yeast strain to confirm the interaction. As shown in Fig. 1A, whereas the cdc25-2 strains cotransformed with MsrB1 and the TRPM6 ␣-kinase domain grow at 37°C, yeast co-transformed with the control vector and TRPM6 ␣-kinase domain only survived at 24°C. The association between TRPM6 and MsrB1 was further substantiated by co-precipitation studies of glutathione S-transferase (GST) and GST-TRPM6-kinase in MsrB1-expressing HEK293 cells. MsrB1 coprecipitated with the GST-␣-kinase but not with GST alone (Fig. 1B,  upper panel). MsrB1 was equally expressed in the tested conditions (Fig. 1B, lower panel). Furthermore, co-precipitation studies of fulllength TRPM6 with MsrB1 in HEK293 cells showed that fulllength TRPM6 associates with GST-MsrB1 but not with GST alone (Fig. 1C, upper panel). TRPM6 and MsrB1 were expressed in all conditions tested (Fig. 1C, middle  and bottom panel).
MsrB1 Co-expresses with TRPM6 in Kidney-To address the tissue distribution of MsrB1, reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed on a panel of mouse tissues. The expected DNA fragment of MsrB1 was detected in all tissues as indicated in Fig. 1D. The integrity of the cDNA was confirmed by the detection of ␤-actin. To further study the co-expression of MsrB1 and TRPM6 in kidney, immunohistochemistry was performed on serial mouse kidney sections. This analysis indicated 70% immunopositive staining for MsrB1 in the TRPM6-expressing DCT segment, which has been implicated in active Mg 2ϩ reabsorption (32) (Fig. 1E).
H 2 O 2 Inhibits TRPM6 Channel Activity-Considering that MsrB1 is a stress protein that mainly exerts its function during oxidative stress (36), we hypothesized that MsrB1 regulates TRPM6 channel activity during oxidative stress. Therefore, we examined the effect of H 2 O 2 on TRPM6 channel activity. To this end, HEK293 cells expressing TRPM6 were treated with 1 mM H 2 O 2 during whole-cell patch clamp recordings. As shown in Fig. 2, A and B, H 2 O 2 caused a significant inhibition of the TRPM6-mediated current (67 Ϯ 5% of control, n ϭ 13, p Ͻ 0.05) when compared with the non-treated cells (n ϭ 11). Furthermore, we demonstrate that H 2 O 2 inhibits TRPM6 channel activity in a dose-dependent effect with an IC 50 of 148 M (Fig. 2C).
H 2 O 2 Treatment Does Not Affect TRPM6 Cell Surface Expression-Next, the influence of H 2 O 2 on the amount of TRPM6 channels at the plasma membrane was investigated by cell surface biotinylation experiments. As shown in Fig. 2D (upper   panel), treatment with H 2 O 2 did not affect the plasma membrane abundance of TRPM6. Of note, the protein levels of TRPM6 were equal in all tested conditions as verified by the total cell lysates (Fig. 2D, (Fig. 3, A and B).

DISCUSSION
In the present study, we identified MsrB1 as a new TRPM6associated protein and showed that MsrB1 recovers TRPM6 4 H. Venselaar, personal communication. channel activity via reducing the oxidation state of Met 1755 during oxidative stress. First, MsrB1 directly binds to the TRPM6 ␣-kinase domain and co-precipitates full-length TRPM6. Second, MsrB1 is co-expressed with TRPM6 in the renal DCT. Third, H 2 O 2 inhibits TRPM6 channel activity without affecting the plasma membrane expression. Fourth, MsrB1 prevents the inhibitory effect of H 2 O 2 on TRPM6 channel activity. Finally, H 2 O 2 has no significant effect on the TRPM6 M1755A mutant.
TRPM6 belongs to the TRPM subfamily of the TRP channels and is so far the only known channel directly mediating active transepithelial Mg 2ϩ transport (4,7,8,32). However, the molecular regulation of this channel remains elusive. Here, we used a novel yeast two-hybrid procedure, RRS, to screen proteins interacting with the TRPM6 ␣-kinase domain. Our data showed that MsrB1 binds to the ␣-kinase domain of TRPM6, resulting in the recruitment of Ras to the membrane and subsequent complementation of the temperature-sensitive cdc25-2 mutation, so we identified MsrB1 as a new interacting protein of the TRPM6 ␣-kinase domain. This interaction has been confirmed by a subsequent GST co-precipitation assay in HEK293 cells with full-length TRPM6. Importantly, MsrB1 and TRPM6 are co-expressed in the renal DCT, which further substantiates the physiological relevance of the interaction between both proteins.
MsrB1 is an oxidoreductase that catalyzes the thiol-dependent reduction of methionine sulfoxide (36). MsrB1 belongs to the Msr family composed of MsrA and MsrB. Mammals contain one MsrA and three MsrBs that are highly abundant in kidney, liver, heart, and nervous tissue (37)(38)(39)(40)(41). These enzymes protect cells from oxidative stress via the repair of oxidative damage to proteins and thereby restore biological activity. They can be involved in reactive oxygen species-mediated signal transduction through modulation of the function of target proteins (36,(42)(43)(44). Accumulating data showed that reversible methionine oxidation and reduction play a dynamic role in a variety of cellular signaling pathways (45,46). For example, the methionine residues of Helix-3, Ca 2ϩ /calmodulin-dependent protein kinase II (CamKII), shaker voltage-dependent K ϩ channel, and Slo1 K ϩ channels can be oxidized and hereby regulate their function (46 -49). It has been shown that oxidation of a methionine residue in the shaker voltage-dependent K ϩ channel disrupts its inactivation. This effect can be reversed by coexpression with MsrA1 (47).
In the present study, we showed that MsrB1 interacts with the TRPM6 ␣-kinase domain but does not affect the channel activity in normoxic conditions. However, H 2 O 2 is a product during oxidative stress and has been studied in relation to potassium channel function and the TRPM2 channel (reviewed in Refs. 50 and 51). Here, we demonstrated that H 2 O 2 significantly decreases the TRPM6-mediated current in HEK293 cells in a dose-dependent manner. As H 2 O 2 did not change the surface expression of TRPM6, it possibly regulates TRPM6 channel activity directly through modulation of the channel conductance. Importantly, the decreased TRPM6 channel activity can be partly recovered by co-expression with MsrB1. This par-  tial recovery could be explained by the fact that Msrs function optimally at 37°C (52), whereas our experiments were performed at room temperature. Another possible explanation is the time lapse of MsrB1 functioning, which is likely slower than the oxidation by H 2 O 2 .
Free and protein-bound methionine residues are among the most susceptible to oxidation by reactive oxygen species (53). It is proposed that surface-exposed methionine residues in a protein constitute an antioxidant defense mechanism because various oxidants can easily react with these residues to form methionine sulfoxide. Reduction back to methionine by methionine sulfoxide reductases could catalytically drive this antioxidant system as has been suggested by several studies (54 -56). Therefore, the methionine residues located on the periphery of the TRPM6 ␣-kinase domain three-dimensional structure 4 (57). TRPM6 is predominantly expressed in DCT and is inhibited by H 2 O 2 . Therefore, it is conceivable that the metabolic state of DCT cells, which influences the abundance of reactive oxygen species generated from mitochondria, modulates the Mg 2ϩ reabsorption via TRPM6.
Taken together, our study demonstrated that H 2 O 2 inhibits TRPM6 channel activity and pointed out Met 1775 as an important target for channel oxidation. Moreover, its interacting protein MsrB1 dynamically regulates this oxidative effect on TRPM6. These data provide insight into the molecular basis of TRPM6 channel regulation and transepithelial Mg 2ϩ (re)absorption. Our findings present the first example of modulating TRP channel activity by methionine oxidation and contribute to a further understanding of the regulation of TRP channels by novel post-translational modifications.