Pmal c2e

Expression, purification, and pull-down assays using the Ca_V1.2 α₁1.2 subunit-GST fusion proteins were performed essentially as before (32 (link), 50 (link), 51 (link)). Wild Type (WT) and mutant (K1647A, Y1649A, I1654A) rabbit α₁1.2 AAs 1576-1733 were expressed as GST-fusion proteins using the expression vector pGEX-4T1. Human α-actinin-1 (AAs 391-892)-MBP fusion protein was produced from the expression vector, pMAL-c2e (New England Biolabs, NEB). Expression and purification of MBP-tagged α-actinin-1 was performed according to the manufacturer’s protocol (NEB). For pull-down assays, GST-tagged WT and mutant α₁1.2 AAs 1576-1733 were expressed in E. coli before extraction and adsorption of 150 μg onto glutathione-Sepharose, washing, and incubation with 100 μg of MBP-purified α-actinin-1. Resins were washed three times in ice-cold TBS plus 0.1% Triton X-100 and extracted with 1.5x SDS-PAGE sample buffer at 90 °C for 5 min. Extracted proteins were loaded onto 7.5% polyacrylamide gels, fractionated by SDS-PAGE and transferred to PVDF membranes for immunoblot detection with anti-GST antibodies to assess relative amounts of immobilized α₁1.2 bait and anti-MBP antibodies to determine relative amounts of α-actinin pulled down by the resin.

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).