We propose a unique mode of TRPM6 regulation in which the Mg(2+) influx is controlled by RACK1 through its interaction with the alpha-kinase and the phosphorylation state of the threonine(1851) residue.

-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 17␤-estradiol treatment disassociates the binding between these proteins. In agreement with this, 17␤-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.

Mg2؉ 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. 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 (1-4). 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 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-

Urinary proteins that leak through the abnormal glomerulus in nephrotic syndrome may affect tubular transport by interacting with membrane transporters on the luminal side of tubular epithelial cells. Patients with nephrotic syndrome can develop nephrocalcinosis, which animal models suggest may develop from impaired transcellular Ca 2+ reabsorption via TRPV5 in the distal convoluted tubule (DCT). In nephrotic-range proteinuria, filtered plasminogen reaches the luminal side of DCT, where it is cleaved into active plasmin by urokinase. In this study, we found that plasmin purified from the urine of patients with nephrotic-range proteinuria inhibits Ca 2+ uptake in TRPV5-expressing human embryonic kidney 293 cells through the activation of protease-activated receptor-1 (PAR-1). Preincubation with a plasmin inhibitor, a PAR-1 antagonist, or a protein kinase C (PKC) inhibitor abolished the effect of plasmin on TRPV5. In addition, ablation of the PKC phosphorylation site S144 rendered TRPV5 resistant to the action of plasmin. Patchclamp experiments showed that a decreased TRPV5 pore size and a reduced open probability accompany the plasmin-mediated reduction in Ca 2+ uptake. Furthermore, high-resolution nuclear magnetic resonance spectroscopy demonstrated specific interactions between calmodulin and residues 133-154 of the N-terminus of TRPV5 for both wild-type and phosphorylated (S144pS) peptides. In summary, PAR-1 activation by plasmin induces PKC-mediated phosphorylation of TRPV5, thereby altering calmodulin-TRPV5 binding, resulting in decreased channel activity. These results indicate that urinary plasmin could contribute to the downstream effects of proteinuria on the tubulointerstitium by negatively modulating TRPV5. 23: 182423: -183423: , 201223: . doi: 10.1681 In the kidney, the fine regulation of Ca 2+ balance occurs through the activity of the epithelial Ca 2+ channel TRPV5. 1 TRPV5 is mostly expressed in the distal convoluted tubule (DCT) and connecting tubule of the nephron, where it constitutes the apical entry mechanism for transcellular Ca 2+ reabsorption. TRPV5 is a constitutively active ion channel that bears unique electrophysiologic characteristics, including calmodulin (CaM) and Ca 2+ -dependent inactivation and high selectivity for Ca 2+ . 2,3 The activity of TRPV5 is tightly controlled at multiple levels by an array of different factors, including parathyroid hormone and the serine protease tissue kallikrein. Both parathyroid hormone and tissue kallikrein initiate the phosphorylation of TRPV5 through the cAMP/protein kinase A (PKA) and phospholipase C (PLC)/ diacylglycerol (DAG)/protein kinase C (PKC) signaling cascades, respectively. 4,5 J Am Soc Nephrol

Trpv5 plays an important role in calcium (Ca2+) homeostasis, among others by mediating renal calcium reabsorption. Accordingly, Trpv5 deficiency strongly stresses Ca2+ homeostasis in order to maintain stable serum Ca2+. We addressed the impact of lifelong challenge of calcium homeostasis on the bone phenotype of these mice.Aging significantly increased serum 1,25(OH)2D3 and PTH levels in both genotypes but they were more elevated in Trpv5−/− mice, whereas serum Ca2+ was not affected by age or genotype. Age-related changes in trabecular and cortical bone mass were accelerated in Trpv5−/− mice, including reduced trabecular and cortical bone thickness as well as reduced bone mineralization. No effect of Trpv5 deficiency on bone strength was observed. In 78-week-old mice no differences were observed between the genotypes regarding urinary deoxypyridinoline, osteoclast number, differentiation and activity as well as osteoclast precursor numbers, as assessed by flow cytometry.In conclusion, life-long challenge of Ca2+ homeostasis present in Trpv5−/− mice causes accelerated bone aging and a low cortical and trabecular bone mass phenotype. The phenotype of the Trpv5−/− mice suggests that maintenance of adequate circulatory Ca2+ levels in patients with disturbances in Ca2+ homeostasis should be a priority in order to prevent bone loss at older age.