*MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.
†Department of Physiology, Radboud university medical center, 6500 HB Nijmegen, The Netherlands.
Biochem J. 2014 Jun 1;460(2):165-75. doi: 10.1042/BJ20131639.
Mutations in the gene that encodes the atypical channel-kinase TRPM6 (transient receptor potential melastatin 6) cause HSH (hypomagnesaemia with secondary hypocalcaemia), a disorder characterized by defective intestinal Mg2+ transport and impaired renal Mg2+ reabsorption. TRPM6, together with its homologue TRPM7, are unique proteins as they combine an ion channel domain with a C-terminally fused protein kinase domain. How TRPM6 channel and kinase activity are linked is unknown. Previous structural analysis revealed that TRPM7 possesses a non-catalytic dimerization motif preceding the kinase domain. This interacts with a dimerization pocket lying within the kinase domain. In the present study, we provide evidence that the dimerization motif in TRPM6 plays a critical role in regulating kinase activity as well as ion channel activity. We identify mutations within the TRPM6 dimerization motif (Leu1718 and Leu1721) or dimerization pocket (L1743A, Q1832K, A1836N, L1840A and L1919Q) that abolish dimerization and establish that these mutations inhibit protein kinase activity. We also demonstrate that kinase activity of a dimerization motif mutant can be restored by addition of a peptide encompassing the dimerization motif. Moreover, we observe that mutations that disrupt the dimerization motif and dimerization pocket interaction greatly diminish TRPM6 ion channel activity, in a manner that is independent of kinase activity. Finally, we analyse the impact on kinase activity of ten disease-causing missense mutations that lie outwith the protein kinase domain of TRPM6. This revealed that one mutation lying nearby the dimerization motif (S1754N), found previously to inhibit channel activity, abolished kinase activity. These results provide the first evidence that there is structural co-ordination between channel and kinase activity, which is mediated by the dimerization motif and pocket interaction. We discuss that modulation of this interaction could comprise a major regulatory mechanism by which TRPM6 function is controlled.
TRPM6（瞬时受体电位 melastatin 6）编码基因的突变导致 HSH（低镁血症伴继发性低钙血症），这是一种特征为肠道镁转运缺陷和肾脏镁重吸收受损的疾病。TRPM6 与其同源物 TRPM7 是独特的蛋白质，因为它们将离子通道结构域与 C 端融合的蛋白激酶结构域结合在一起。TRPM6 通道和激酶活性如何连接尚不清楚。先前的结构分析表明，TRPM7 具有位于激酶结构域之前的非催化二聚化基序。该基序与位于激酶结构域内的二聚化口袋相互作用。在本研究中，我们提供了证据表明，TRPM6 中的二聚化基序在调节激酶活性以及离子通道活性方面起着关键作用。我们鉴定了 TRPM6 二聚化基序（Leu1718 和 Leu1721）或二聚化口袋（L1743A、Q1832K、A1836N、L1840A 和 L1919Q）内的突变，这些突变会破坏二聚化并确定这些突变会抑制蛋白激酶活性。我们还证明，包含二聚化基序的肽可恢复二聚化基序突变体的激酶活性。此外，我们观察到，破坏二聚化基序和二聚化口袋相互作用的突变极大地降低了 TRPM6 离子通道活性，这种方式与激酶活性无关。最后，我们分析了位于 TRPM6 蛋白激酶结构域外的十个致病错义突变对激酶活性的影响。结果表明，先前发现的位于二聚化基序附近的一个突变（S1754N），可抑制通道活性，同时也可抑制激酶活性。这些结果首次表明，通道和激酶活性之间存在结构协调，这种协调是由二聚化基序和口袋相互作用介导的。我们讨论了这种相互作用的调节可能是 TRPM6 功能受控制的主要调节机制。
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