In vitro protein-protein interaction assay

For in vitro pull-down assays, we used the GST-CT1 plasmid, which covers rabbit α₁1.2 AAs 1513-1733 in the GST-fusion protein expression vector pGEX-4T1 (GE healthcare) as described (32 (link)). The site-specific mutations K1647A, Y1649A, and I1654A were generated via QuikChange II as above (see Supporting Information, Table S4). A cDNA fragment encoding AAs 391-892 of human α-actinin-1 was sub-cloned into KpnI/EcoRI digested plasmid DNA of the MBP fusion protein expression vector, pMAL-c2e (NEB). For identification of Ca_V1.2 expressing HEK293T/17 transfectants in electrophysiological, flow cytometry and surface biotinylation studies, we used a rat α₁1.2 (GenBank ID: M67515.1) construct that carried an HA tag in the S5-H5 extracellular loop of domain II, which only modestly reduces current density in heterologous expression systems (25 (link), 49 (link)). This α₁1.2 cDNA was subcloned into a SalI/SacII restriction endonuclease digested pECFP-C1 vector (Clontech). The site-specific mutations K1647A, Y1649A, and I1654A in the rat α₁1.2 IQ domain were again generated via QuikChange II with rat-appropriate oligonucleotide primer sets (see Supporting Information, Table S5).