Regulation of the Epithelial Mg2+ Channel TRPM6 by Estrogen and the Associated Repressor Protein of Estrogen Receptor Activity (REA)

The maintenance of the Mg2+ 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 Mg2+ 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 6th, 7th, and 8th β-sheets in its α-kinase domain. Importantly, REA and TRPM6 are coexpressed in renal Mg2+-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 17β-estradiol treatment disassociates the binding between these proteins. In agreement with this, 17β-estradiol treatment significantly stimulates the TRPM6-mediated current in HEK293 cells. These results suggest a rapid pathway for the effect of estrogen on Mg2+ 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 Mg2+ (re)absorption and provides new insight into the molecular mechanism of renal transepithelial Mg2+ transport. The maintenance of the Mg2+ 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 Mg2+ 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 6th, 7th, and 8th β-sheets in its α-kinase domain. Importantly, REA and TRPM6 are coexpressed in renal Mg2+-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 17β-estradiol treatment disassociates the binding between these proteins. In agreement with this, 17β-estradiol treatment significantly stimulates the TRPM6-mediated current in HEK293 cells. These results suggest a rapid pathway for the effect of estrogen on Mg2+ 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 Mg2+ (re)absorption and provides new insight into the molecular mechanism of renal transepithelial Mg2+ transport. Mg2+ is a central electrolyte important for many biological functions by its intervention in gene transcription, protein synthesis, nucleic acid stability, channel regulation, cell cycle, and numerous enzymatic systems (1Vetter T. Lohse M.J. Curr. Opin. Nephrol. Hypertens. 2002; 11: 403-410Crossref PubMed Scopus (89) Google Scholar, 2Grubbs R.D. Biometals. 2002; 15: 251-259Crossref PubMed Scopus (140) Google Scholar, 3Romani A. Scarpa A. Arch. Biochem. Biophys. 1992; 298: 1-12Crossref PubMed Scopus (320) Google Scholar, 4Konrad M. Schlingmann K.P. Gudermann T. Am. J. Physiol. Renal Physiol. 2004; 286: 599-605Crossref PubMed Scopus (188) Google Scholar). In most species, serum Mg2+ levels are kept within a narrow range between 0.8 and 1.1 mm, while the free intracellular Mg2+ concentration [Mg2+]i has been estimated around 0.5–1.0 mm (5Chubanov V. Gudermann T. Schlingmann K.P. Pflugers Arch. 2005; 451: 228-234Crossref PubMed Scopus (85) Google Scholar). Regulation of the Mg2+ balance principally resides within the kidney where Mg2+ excretion tightly matches the intestinal absorption of Mg2+ (5Chubanov V. Gudermann T. Schlingmann K.P. Pflugers Arch. 2005; 451: 228-234Crossref PubMed Scopus (85) Google Scholar). The majority of Mg2+ in the renal ultrafiltrate is reabsorbed passively in the proximal tubule and the thick ascending limb of the loop of Henle, while the final Mg2+ excretion is determined in the distal convoluted tubule (DCT) 3The abbreviations used are: DCT, distal convoluted tubule; TRPM6, Transient Receptor Potential Melastatin 6; REA, repressor of estrogen receptor activity; GST, glutathione S-transferase; HA, hemagglutinin; ER, estrogen receptor.3The abbreviations used are: DCT, distal convoluted tubule; TRPM6, Transient Receptor Potential Melastatin 6; REA, repressor of estrogen receptor activity; GST, glutathione S-transferase; HA, hemagglutinin; ER, estrogen receptor. via an active reabsorption process (5Chubanov V. Gudermann T. Schlingmann K.P. Pflugers Arch. 2005; 451: 228-234Crossref PubMed Scopus (85) Google Scholar). The Transient Receptor Potential Melastatin 6 (TRPM6) localizes along the apical membranes of DCT and intestinal cells where it plays a crucial role in active Mg2+ (re)absorption. Mutations in TRPM6 lead to hypomagnesemia with secondary hypocalcemia (HSH) indicating that this channel is important for the maintenance of the Mg2+ balance (6Walder R.Y. Landau D. Meyer P. Shalev H. Tsolia M. Borochowitz Z. Boettger M.B. Beck G.E. Englehardt R.K. Carmi R. Sheffield V.C. Nat. Genet. 2002; 31: 171-174Crossref PubMed Scopus (459) Google Scholar, 7Schlingmann K.P. Weber S. Peters M. Niemann Nejsum L. Vitzthum H. Klingel K. Kratz M. Haddad E. Ristoff E. Dinour D. Syrrou M. Nielsen S. Sassen M. Waldegger S. Seyberth H.W. Konrad M. Nat. Genet. 2002; 31: 166-170Crossref PubMed Scopus (630) Google Scholar, 8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Previous studies demonstrated that expression of TRPM6 is regulated by dietary Mg2+ and its channel activity is strongly inhibited by the intracellular Mg2+ concentration ([Mg2+]i) (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 9Groenestege W.M. Hoenderop J.G. van den Heuvel L. Knoers N. Bindels R.J. J. Am. Soc. Nephrol. 2006; 17: 1035-1043Crossref PubMed Scopus (184) Google Scholar). Generally, Mg2+-free solutions are used to measure the characteristic outwardly rectifying TRPM6 currents (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 10Li M. Du J. Jiang J. Ratzan W.J. Su L.T. Runnels L.W. Yue L. J. Biol. Chem. 2007; 282: 25817-25830Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 11Chubanov V. Schlingmann K.P. Waring J. Heinzinger J. Kaske S. Waldegger S. Mederos y Schnitzler M. Gudermann T. J. Biol. Chem. 2007; 282: 7656-7667Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Remarkably, TRPM6 contains a C-terminal α-kinase domain, of which the regulatory role on channel activity remains elusive (12Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (611) Google Scholar, 13Drennan D. Ryazanov A.G. Prog. Biophys. Mol. Biol. 2004; 85: 1-32Crossref PubMed Scopus (102) Google Scholar, 14Montell C. Curr. Biol. 2003; 13: R799-801Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). It has been shown that RACK1 can interact with this domain and inhibit channel activity in a phosphorylation-dependent manner (15Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). A recent study demonstrated that the ATP binding motif in the α-kinase domain is required for modulation of TRPM6 channel activity by intracellular ATP (16Thebault S. Cao G. Venselaar H. Xi Q. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2008; 283: 19999-20007Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Previous studies suggested that Mg2+ uptake in DCT is stimulated by hormones, such as parathyroid hormone, calcitonin, arginine vasopressin, insulin and prostaglandins (17Dai L.J. Ritchie G. Kerstan D. Kang H.S. Cole D.E. Quamme G.A. Physiol. Rev. 2001; 81: 51-84Crossref PubMed Scopus (258) Google Scholar). A recent study reported that epidermal growth factor (EGF) acts as an autocrine/paracrine magnesiotropic hormone via stimulating its receptor on the basolateral membrane of DCT cells, thereby specifically increasing TRPM6 current (18Groenestege W.M. Thebault S. van der Wijst J. van den Berg D. Janssen R. Tejpar S. van den Heuvel L.P. van Cutsem E. Hoenderop J.G. Knoers N.V. Bindels R.J. J. Clin. Investig. 2007; 117: 2260-2267Crossref PubMed Scopus (287) Google Scholar). Further, Groenestege et al. (9Groenestege W.M. Hoenderop J.G. van den Heuvel L. Knoers N. Bindels R.J. J. Am. Soc. Nephrol. 2006; 17: 1035-1043Crossref PubMed Scopus (184) Google Scholar) demonstrated that the renal TRPM6 mRNA level in ovariectomized rats was significantly reduced, whereas 17β-estradiol treatment normalized TRPM6 mRNA levels. Next to this classical transcriptional pathway, accumulating evidence suggests also rapid estrogen effects that within via a K. 2006; PubMed Scopus Google Scholar, T. 2007; PubMed Scopus Google Scholar, T. P. N. 2006; PubMed Scopus Google Scholar, Mol. Biol. 2005; PubMed Scopus Google Scholar). has the to rapid activation of such as the receptor Science. 2005; PubMed Scopus Google Scholar). In it has been demonstrated that the classical estrogen receptor can to the membrane in to estrogen by interaction with and S. L. G. M. P. S. N. L. E. G. P. J. 2004; 117: PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar). The of the study was to the regulation of the α-kinase domain on TRPM6 channel activity by of specifically interacting with the α-kinase domain. a and we demonstrated a for rapid estrogen regulation on TRPM6 channel activity via the inhibitory effect of the repressor of estrogen receptor activity and cells and as Groenestege Thebault S. van den Berg D. Nilius B. Hoenderop J.G. Bindels R.J. 2007; PubMed Scopus Google and and kinase domain of and TRPM6 was into by kidney TRPM6 in (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar) as The α-kinase domain of TRPM6 was into the T. R. K. A. T. T. S. K. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). REA was into and by kidney TRPM6 in the was the as (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). and TRPM6 phosphotransferase-deficient mutant and mutant the to the by REA was in as J.G. van der Kemp A.W. A. van Bindels R.J. Biochem. Biophys. PubMed Scopus Google Scholar). of by GST, and α-kinase interacting with and and by as M. Mol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). by a to a as M. Mol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). and identified the to a of the data data with to and of and α-kinase protein was as Hoenderop J.G. D. D. J. V. Nilius B. Bindels R.J. J. 2003; PubMed Scopus Google Scholar). REA protein was with a and to on of with mm mm The with on and by and of TRPM6 α-kinase domain was on the of α-kinase domain H. M. J. Mol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and and by and and as Hoenderop J.G. D. D. J. V. Nilius B. Bindels R.J. J. 2003; PubMed Scopus Google Scholar). REA and by and by in the an by the and and A of to was a of by the current to the cell membrane The of current was determined by the current and the The and of data The mm mm and mm The solutions mm mm and mm the effect of on TRPM6-mediated to the and thereby via the for the cells with REA and α-kinase with 17β-estradiol for and for on in mm mm mm the and of the with in the and by The was the with cells with in cells with 17β-estradiol for with The was as D. Hoenderop J.G. Bindels R.J. Am. J. Physiol. Renal Physiol. 2006; PubMed Scopus Google Scholar). The TRPM6 expression was by for the membrane and for the cell the by for in and to with with and the was as J.G. M. Kemp A.W. R. Bindels R.J. J. 2002; PubMed Scopus Google Scholar). kidney for with and (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). TRPM6, was used with secondary with a In was by the The in a of of kinase mm mm mm and of for The was by with mm mm mm was by are as between was determined by of In the of between the of by was REA with interacting with the TRPM6 α-kinase domain, the of a with the α-kinase domain of TRPM6 in kidney by was REA K. R. P. S. A. PubMed Scopus Google was identified as an interacting protein of the TRPM6 α-kinase domain. binding used to the interaction between REA and TRPM6 α-kinase domain. REA protein was with and on REA specifically with the but not with the between TRPM6 and REA was in cells by studies of and in HEK293 cells. REA with the but not with REA to the 6th, 7th, and 8th in the TRPM6 the REA binding within TRPM6, a of in the α-kinase domain was TRPM6 α-kinase as and for interaction with REA protein the interaction between the whereas no effect on the interaction with Moreover, a protein the between the and of the TRPM6 α-kinase domain to REA the REA binding within TRPM6 is to the between the and The and of the was and by of The of to binding of to the the REA binding in the TRPM6 α-kinase domain, the of this domain was by on the between TRPM6 and α-kinase (15Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, H. M. J. Mol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of the between the TRPM6 and α-kinase these a secondary The and of the REA binding 7th, and 8th suggested the of the REA binding are the of the TRPM6 α-kinase domain. RACK1 also binds to this we RACK1 can the interaction between TRPM6 and shown in REA in with α-kinase in the and of REA with TRPM6 in the of REA, was on The of for REA was in most as in The was in the REA is in kidney where TRPM6 is (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). study the between REA and TRPM6 in was on kidney This for REA in the DCT cells that been in active Mg2+ reabsorption (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar) REA TRPM6 in a role of REA on TRPM6 channel activity was by in HEK293 cells. rectifying currents by in HEK293 cells TRPM6 and A and These currents significantly inhibited in cells TRPM6 and REA A and the inhibitory effect of REA was for TRPM6, REA not the current in HEK293 cells C and the inhibitory effect of REA on TRPM6, and in HEK293 cells and These and into HEK293 cells via the shown in and REA significantly inhibited the TRPM6-mediated but the protein not the of the not the TRPM6-mediated the phosphotransferase-deficient mutant was used to the role of TRPM6 activity in this REA was to inhibit the as and Importantly, the showed that the not the binding between TRPM6 and REA Importantly, in demonstrated of TRPM6 with REA we the effect of activation by phorbol 12-myristate 13-acetate-PMA on the regulatory effect of REA, since it has been shown that RACK1 inhibition can by of of (15Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). of cells TRPM6 and REA significantly TRPM6 channel activity. the current in the of was not in cells TRPM6 Finally, we REA is a of The in showed that REA is not by TRPM6 inhibits TRPM6 channel activity in a rapid and phosphorylation-dependent of and of cells with of the current of and cells. of TRPM6 cells with of the current of and cells. studies of GST, and in HEK293 cells REA expression was by in protein kinase of TRPM6 and TRPM6 and REA for expression by effect of activation by on the current of TRPM6 and and TRPM6 and REA with TRPM6 and and with TRPM6 and REA the and the role of REA in the estrogen pathway K. R. P. S. A. PubMed Scopus Google Scholar, J. A. L. Mol. Biol. 2005; PubMed Scopus Google Scholar, B. Q. A. Mol. 2008; PubMed Scopus Google the of estrogen on the TRPM6 and REA interaction was by this and with REA in HEK293 cells, to 17β-estradiol for and The interaction between REA and TRPM6 α-kinase domain was significantly by 17β-estradiol treatment whereas REA was in the of estrogen on TRPM6 channel activity was this HEK293 cells TRPM6 for with 17β-estradiol and by shown in and the TRPM6 current was significantly with 17β-estradiol with the of 17β-estradiol treatment on the of TRPM6 the membrane was by cell HEK293 cells. shown in treatment with 17β-estradiol not the of TRPM6 the TRPM6 was in as determined in the cell In the we identified REA as a protein and demonstrated that this protein inhibits the channel activity in a REA binds to the TRPM6 α-kinase domain and with the Mg2+ channel in the renal REA specifically inhibits channel activity of TRPM6, but not this inhibition on TRPM6 REA has no effect on the TRPM6 phosphotransferase-deficient mutant. the between REA and TRPM6 is a dynamic process, which is regulated by 17β-estradiol Finally, 17β-estradiol treatment stimulates TRPM6 activity via a rapid TRPM6 functions as the gatekeeper of active transepithelial Mg2+ (6Walder R.Y. Landau D. Meyer P. Shalev H. Tsolia M. Borochowitz Z. Boettger M.B. Beck G.E. Englehardt R.K. Carmi R. Sheffield V.C. Nat. Genet. 2002; 31: 171-174Crossref PubMed Scopus (459) Google Scholar, 7Schlingmann K.P. Weber S. Peters M. Niemann Nejsum L. Vitzthum H. Klingel K. Kratz M. Haddad E. Ristoff E. Dinour D. Syrrou M. Nielsen S. Sassen M. Waldegger S. Seyberth H.W. Konrad M. Nat. Genet. 2002; 31: 166-170Crossref PubMed Scopus (630) Google Scholar, 8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Here, REA was identified as a dynamic of TRPM6 channel activity a molecular in Mg2+ REA binds to the α-kinase domain of TRPM6, which was identified by the of a with the α-kinase domain of TRPM6 in kidney by This interaction was by between REA and TRPM6 α-kinase domain in HEK293 cells. and suggest that REA specifically binds to the 6th, 7th, and 8th β-sheets in the α-kinase domain, which has been demonstrated as the binding for RACK1 as (15Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). This to the that most in this are on the of the of the TRPM6 α-kinase domain, thereby the interaction with its regulatory proteins. Importantly, we demonstrated that RACK1 has no effect on the binding between REA and the α-kinase domain. This by binding within this by a binding for REA Moreover, we showed that REA is in tubules in The of REA and TRPM6 in DCT the of the interaction between proteins. REA was identified as an interacting protein of the estrogen receptor where it functions as a the transcriptional activity of K. R. P. S. A. PubMed Scopus Google Scholar). of REA significantly estrogen in J. A. L. Mol. Biol. 2005; PubMed Scopus Google Scholar). et al. B. Q. A. Mol. 2008; PubMed Scopus Google Scholar) showed that REA a with and acts as a transcriptional for study demonstrated a role of REA in TRPM6 regulation by that the inhibitory effect of REA on TRPM6 channel activity is not via the transcriptional This is by the that rapid of the into cells a inhibition of channel activity. Moreover, this inhibitory effect is on the kinase activity of the TRPM6 α-kinase domain since the TRPM6 phosphotransferase-deficient mutant is to REA can still to the TRPM6 mutant. we demonstrated that activation by potentiated the inhibitory effect of these data suggest that the activity of TRPM6 α-kinase domain plays a role in the REA effect. the activity of the TRPM6 and α-kinase has been (15Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, K. M. van B. L. Ryazanov A.G. K. van J. 2006; PubMed Scopus Google Scholar, C. Ryazanov A.G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. K. H. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C. Ryazanov A.G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Am. PubMed Scopus Google Scholar, C. K. R. T. A. 2003; Full Text Full Text PDF PubMed Scopus Google the regulatory role of this domain on channel activity remains elusive. it has been shown that these are not essential for channel activation (15Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, M. H. K. H. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C. K. R. T. A. 2003; Full Text Full Text PDF PubMed Scopus Google studies demonstrated an regulatory role in modulation of channel activity (15Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, S. Cao G. Venselaar H. Xi Q. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2008; 283: 19999-20007Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). In this we demonstrated that ATP can TRPM6 channel activity the ATP binding motif within the α-kinase domain, of the TRPM6 α-kinase activity S. Cao G. Venselaar H. Xi Q. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2008; 283: 19999-20007Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, we showed that the inhibitory effect of RACK1 on TRPM6 is on the TRPM6 activity (15Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). It has also been that the mutant is to in agreement with studies that α-kinase activity the inhibition of channel activity C. K. R. T. A. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, P. R. A. J. Physiol. 2006; PubMed Scopus Google Scholar). Remarkably, while REA inhibits TRPM6 channel it has no effect on TRPM7, the closest homologue of on the the REA binding in TRPM6 α-kinase domain is in TRPM7, REA most binds to α-kinase domain as However, it has been reported that the α-kinase of TRPM6 is of of TRPM7, but not the of these α-kinase C. Ryazanov A.G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). is that REA is a of TRPM6, but not TRPM7, and this as a of the REA regulatory activity on However, we demonstrated that REA not by TRPM6 is not in the of the study regulation of TRPM6 channel free TRPM6 but on its α-kinase activity. REA has been as a in the estrogen pathway K. R. P. S. A. PubMed Scopus Google Scholar, J. A. L. Mol. Biol. 2005; PubMed Scopus Google Scholar, B. Q. A. Mol. 2008; PubMed Scopus Google Scholar). data showed that the interaction between REA and TRPM6 is a dynamic process that is regulated by with 17β-estradiol the interaction between TRPM6 and REA, and its inhibitory effect on TRPM6 channel activity. data demonstrated that of 17β-estradiol a of TRPM6-mediated It is that estrogen can Mg2+ and uptake J. Am. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar, P. C. Google Scholar, M. Full Text Full Text PDF PubMed Scopus Google Scholar). In this Groenestege et al. (9Groenestege W.M. Hoenderop J.G. van den Heuvel L. Knoers N. Bindels R.J. J. Am. Soc. Nephrol. 2006; 17: 1035-1043Crossref PubMed Scopus (184) Google Scholar) demonstrated that the renal TRPM6 mRNA level in ovariectomized rats was significantly reduced, whereas 17β-estradiol treatment normalized TRPM6 mRNA that estrogen can Mg2+ homeostasis via a transcriptional evidence showed in addition to the classical transcriptional pathway, estrogen has the to via K. 2006; PubMed Scopus Google Scholar, T. 2007; PubMed Scopus Google Scholar, T. P. N. 2006; PubMed Scopus Google Scholar, Mol. Biol. 2005; PubMed Scopus Google Scholar). current data a rapid regulatory pathway for since the cells with 17β-estradiol for to we demonstrated that 17β-estradiol significantly the interaction between TRPM6 α-kinase domain and In of REA inhibition on TRPM6 channel the molecular mechanism of estrogen to of the TRPM6 and REA interaction and of the REA It of to the pathway in estrogen to of the TRPM6 and REA It is that estrogen an estrogen receptor and its pathway, which to TRPM6 REA and we REA as a negative modulator in active transepithelial Mg2+ via TRPM6 channel activity. estrogen the interaction between TRPM6 and REA and thereby TRPM6-mediated Mg2+ which as a rapid pathway for estrogen to the Mg2+ These new on the molecular regulation of TRPM6 and to a of the molecular of transepithelial Mg2+ (re)absorption. We R. M. D. van den and M. for