The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics.

Vladimir Chubanov,Astrid Kollewe,Annette Nicke,Fong Tsuen Tseung,Bernd Fakler,Wolfgang Bildl,Catrin Swantje Müller,Thomas Gudermann,Uwe Schulte,Susanna Zierler,Leonor Correia,Alexander Haupt,Eva Schmidt,Anna Rössig

doi:10.7554/elife.68544

Abstract

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+, and Ca2+, and thus shapes cellular excitability, plasticity, and metabolic activity. The molecular appearance and operation of TRPM7 channels in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative mass spectrometry (MS) analysis. We found that native TRPM7 channels are high-molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ADP-ribosylation factor-like protein 15 (ARL15). Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that complex formation effectively and specifically impacts TRPM7 activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.

Highlights

transient receptor potential melastatin-subfamily member 7 (TRPM7) encodes a bi-functional protein with a transient receptor potential (TRP) ion channel domain fused to a C-terminal -type serine/threonine-protein kinase
To see whether this large molecular size is a peculiarity of HEK293 cells, we recapitulated the analysis for TRPM7 channels expressed in mouse brain using a recently developed technique that combines BN-PAGE with cryo-slicing and quantitative mass spectrometry (csBN-MS, [31])
We show that TRPM7 forms macromolecular complexes by assembling with CNNM proteins 1-4 and ADP-ribosylation factor-like protein 15 (ARL15)

Summary

Introduction

TRPM7 encodes a bi-functional protein with a transient receptor potential (TRP) ion channel domain fused to a C-terminal -type serine/threonine-protein kinase (reviewed in [1,2,3]). TRPM7 is involved in various cellular processes such as homeostatic balance, cell motility, proliferation, differentiation and regulation of immune responses [1,2,3]. Free Mg2+, the Mg·ATP complex, and phosphatidylinositol-4,5-bisphosphate (PIP2) were described as physiological regulators of the channel activity of TRPM7 [19, 22]. While Mg2+ or Mg·ATP act as negative regulators, PIP2 appears to be a crucial co-factor of the active channel [19, 22]. The effects of Mg2+, Mg·ATP, or PIP2 on TRPM7 activity are poorly understood, and most likely, there are additional regulators of TRPM7 function with hitherto unknown molecular identity

Methods

Results

Conclusion