Curr Biol. 2008 Feb 12;18(3):168-76. doi: 10.1016/j.cub.2007.12.058.
RACK1 inhibits TRPM6 activity via phosphorylation of the fused alpha-kinase domain.
Cao, G., Thebault, S., van der Wijst, J., van der Kemp, A., Lasonder, E., Bindels, R. J., Hoenderop, J. G.,
["Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands."]
["Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands."]
BACKGROUND: The maintenance of the body's Mg(2+) balance is of great importance because of its involvement in numerous enzymatic systems and its intervention in neuromuscular excitability, protein synthesis, and nucleic acid stability. Recently, the transient receptor potential melastatin 6 (TRPM6) was identified as the gatekeeper of active Mg(2+) transport and therefore plays a crucial role in the regulation of Mg(2+) homeostasis. Remarkably, TRPM6 combines a Mg(2+) channel with an alpha-kinase domain whose function remains elusive. RESULTS: Here, we identify the receptor for activated C-kinase 1 (RACK1) as the first regulatory protein of TRPM6 that associates with the alpha-kinase domain. RACK1 and TRPM6 are both present in renal Mg(2+)-transporting distal convoluted tubules. We demonstrate that RACK1 inhibits channel activity in an alpha-kinase activity-dependent manner, whereas small interference (si) RNA-mediated knockdown of RACK1 increases the current. Moreover, threonine(1851) in the alpha-kinase domain was identified as an autophosphorylation site of which the phosphorylation state is essential for the inhibitory effect of RACK1. Importantly, threonine(1851) was crucial for the Mg(2+) sensitivity of TRPM6 autophosphorylation and channel activity. TRPM6 channel activity was less sensitive to Mg(2+) when RACK1 was knocked down by siRNA. Finally, activation of protein kinase C by phorbol 12-myristate 13-acetate-PMA prohibited the inhibitory effect of RACK1 on TRPM6 channel activity. CONCLUSIONS: 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.
PMID: 18258429

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Screening
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Experimental screening | Non-experimental screening | Reference | ||||||||
TRP channel construct | Interactor source | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Organ/tissue | Sample type | |||
TRPM6 |
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RacK1 | Yeast two-hybrid | Human | 1075-2022 | Mouse | Kidney | cDNA library | 18258429 | |
TRPM7 |
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RacK1 | Inference | Prediction | 18258429 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Validation: In vitro validation
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Assay with recombinant proteins | Reference | |||||||||
TRP channel construct | Interactor construct | |||||||||
TRP channel | Interactor | Method | Species | Region | Expression system | Species | Region | Expression system | ||
TRPM6 |
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RacK1 | Yeast two-hybrid | Human | 1075-2022 | Yeast | Mouse | Full-length | Yeast | 18258429 |
TRPM7 |
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RacK1 | Fusion protein-pull down assay | Mouse | ơ-kinase domain | E. coli | Mouse | Full-length | In vitro translation | 18258429 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Validation: In vivo validation
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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 |
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RacK1 | Co-immunoprecipitation | HEK293 | Human | Full-length | Not specified | Not specified | 18258429 | |
TRPM6 |
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RacK1 | Co-immunofluorescence staining | Mouse kidney section | 18258429 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Characterization
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Binding region mapping | Stoichiometry | Affinity (Kd) | Reference | |||||||
TRP channel | Interactor | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPM6 |
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RacK1 | Fusion protein-pull down assay | Mouse | 1857-1885 | Mouse | Not determined | 18258429 | ||
TRPM7 |
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RacK1 | Fusion protein-pull down assay | Mouse | ơ-kinase domain | Mouse | Not determined | 18258429 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Functional consequence
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TRP channel | Interactor | Method | Post-translational modification | Subcellular trafficking | Activity | Reference | ||||||
TRPM6 |
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RacK1 | Patch clamp | Inhibition | 18258429 | |||||||
TRPM7 |
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RacK1 | Patch clamp | Inhibition | 18258429 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
