Quikchange system

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).

Mouse Trpm7, mouse Trpm6, and human TRPM6 cDNA in pIRES2-EGFP vector were reported previously (Chubanov et al., 2004 (link); Ferioli et al., 2017 (link)). cDNA encoding C-terminally His-tagged human TRPV1 (NG_029716 Hayes et al., 2000 (link)) was cloned into the pNKS2 vector (Gloor et al., 1995 (link)) using standard restriction enzyme (BamHI/SmaI) cloning techniques. The mouse Trpm7 cDNA in the pOG1 and mouse Trpm7-Myc and Trpm7-HA cDNA variants in pcDNA3.1/V5-His TA-TOPO vector were described earlier (Chubanov et al., 2004 (link); Ferioli et al., 2017 (link)). Expression constructs encoding Myc-Flag-tagged (C-end) mouse Cnnm1-4 and Arl15, and Arl8A cDNAs in the pCMV6-Entry expression vector were acquired from OriGene (MR218318 for Cnnm1, MR218370 for Cnnm2, MR224758 for Cnnm3, MR215721 for Cnnm4, MR218657 for Arl15, and MR201740 for Arl8a) and verified by sequencing. 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).