J Biol Chem. 2009 May 29;284(22):14788-95. doi: 10.1074/jbc.M808752200. Epub 2009 Mar 26.

Regulation of the epithelial Mg2+ channel TRPM6 by estrogen and the associated repressor protein of estrogen receptor activity (REA).External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Cao, G., van der Wijst, J., van der Kemp, A., van Zeeland, F., Bindels, R. J., Hoenderop, J. G.,
["Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Institute for Genetic and Metabolic Disease, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands."]
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.
PMID: 19329436External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Screening Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPM6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 REA Affinity purification-mass spectrometry Mouse Full-length Mouse Kidney Tissue lysates 19329436
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vitro validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with recombinant proteins Reference
TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Expression system Species Region Expression system
TRPM6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 REA Fusion protein-pull down assay Mouse Full-length E. coli Mouse Full-length In vitro translation 19329436
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vivo validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with endogenous proteins Assay with overexpressed proteins Reference
Cell or tissue Cell or tissue TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Species Region
TRPM6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 REA Co-immunoprecipitation HEK293 Human Full-length Mouse Full-length 19329436
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Characterization Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Binding region mapping Stoichiometry Affinity (Kd) Reference
TRP channel Interactor
TRP channel Interactor Method Species Region Species Region
TRPM6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 REA Fusion protein-pull down assay Mouse 1857-1885 Mouse Not determined 19329436
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Functional consequence Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
TRP channel Interactor Method Post-translational modification Subcellular trafficking Activity Reference
TRPM6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 REA Patch clamp Inhibition 19329436
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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