Expression and Mutagenesis of Mouse TRPM7

For structural experiments and planar lipid bilayer recordings, cDNA for mouse Trpm7 (NM_021450) truncated C-terminally (the corresponding protein residues 1–1280)^{42 (link)} was introduced into the pEG-BacMam vector for protein expression in mammalian cells^{57 (link)}, with an N-terminal region coding for the streptavidin affinity tag (residues WSHPQFEK), followed by the green fluorescent protein (GFP) and thrombin cleavage site (residues LVPRG), as described before^{58 ,59} . For functional experiments, mouse Trpm7 was introduced into the pIRES2-eGFP expression vector reported previously^{36 (link)}. Point mutations in TRPM7 were introduced using the QuikChange system (Thermo Fisher Scientific) according to the manufacturer’s protocol and verified by sequencing (Eurofins, Germany).