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J. Biol. Chem., Vol. 283, Issue 29, 19999-20007, July 18, 2008

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Role of the -Kinase Domain in Transient Receptor Potential Melastatin 6 Channel and Regulation by Intracellular ATP^*

Stéphanie Thébault¹, Gang Cao¹, Hanka Venselaar, Qi Xi, René J. M. Bindels, and Joost G. J. Hoenderop²

From the Department of Physiology and the Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands

Transient receptor potential melastatin 6 (TRPM6) plays an essential role in epithelial Mg²⁺ transport. TRPM6 and its closest homologue, TRPM7, both combine a cation channel with an -kinase domain. However, the role of this -kinase domain in TRPM6 channel activity remains elusive. The aim of this study was to investigate the regulation of TRPM6 channel activity by intracellular ATP and the involvement of its -kinase domain. We demonstrated that intracellular Na- and Mg-ATP decreased the TRPM6 current in HEK293 cells heterogeneously expressing the channel, whereas Na-CTP or Na-GTP had no effect on channel activity. Whole cell recordings in TRPM6-expressing HEK293 cells showed that deletion of the -kinase domain prevented the inhibitory effect of intracellular ATP without abrogating channel activity. Mutation of the conserved putative ATP-binding motif GXG(A)XXG (G1955D) in the -kinase domain of TRPM6 inhibited the ATP action, whereas this effect remained preserved in the TRPM6 phosphotransferase-deficient mutant K1804R. Mutation of the TRPM6 autophosphorylation site, Thr¹⁸⁵¹, into either an alanine or an aspartate, resulted in functional channels that could still be inhibited by ATP. In conclusion, intracellular ATP regulates TRPM6 channel activity via its -kinase domain independently of -kinase activity.

Received for publication, January 8, 2008 , and in revised form, May 16, 2008.

^* This work was supported by an EURYI 2006 award from the European Science Foundation, The Netherlands Organization for Scientific Research Grants ZonMw 9120.6110 and NWO-CW 700.55.302, Human Frontiers Science Program Grant RGP32/2004, Dutch Kidney Foundation Grant C03.6017, and long term EMBO Fellowship ALTF 727-2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: 286 Physiology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: 31-24-3610580; Fax: 31-24-3616413; E-mail: j.hoenderop{at}ncmls.ru.nl.

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