These mutants were largely detected at the cell surface with the relative fluorescence signals being 60C80% of the signals measured for mCherry-CaV21-HA WT (Table 2). modulation was partially preserved in CaV21 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured Nerolidol immunoblots of CaV21 G1060I and CaV21 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, CaV21 1059C1063, but not CaV21 G1060A, failed to co-immunoprecipitate with CaV1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the CaV21 subunit at the predicted membrane interface and further suggest that preventing GPI anchoring of CaV21 averts its cell-surface expression, its interaction with CaV1, and modulation of CaV1.2 currents. three-dimensional cryo-electron microscopy Nerolidol (3D cryo-EM) structure of the rabbit CaV21 protein. Schematic representation of the rabbit CaV21 protein in complex with the pore-forming subunit of CaV1.1 (and the von Willebrand factor type A domain (residues 251C443) is shown in and putative disulfide bonds are identified by primary sequence Nerolidol alignment of the rabbit and the rat CaV21 protein. The rat CaV21 (GenBankTM “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_012919″,”term_id”:”402744513″NM_012919) was subcloned in the pmCherry-N1 vector to Nerolidol express the fluorophore at the C-terminal end of the protein. The hemagglutinin (were used to represent the amino acids. Herein, we show that the native CaV21 protein from rat cardiomyocytes is a substrate for prokaryotic phosphatidylinositol-phospholipase C (PI-PLC). Deletion of the last 24 C-terminal residues, including the hydrophobic domain of the rat CaV21, had little impact on CaV1.2 currents demonstrating that the C-terminally cleaved CaV21 proteins achieve the conformation congruent with the modulation of CaV1.2 channels. In contrast, deletion of four residues surrounding Cys-1059 (rat isoform), which was the last amino acid identified in the 3D structure, impaired up-regulation of CaV1.2 currents. The migration profile of the C-terminal fragments was also significantly altered by single mutations in the 1059C1061 region, suggesting that the proteolytic cleavage was influenced by the chemical nature of the side chain in the site. More importantly, mutations of the predicted GPI-anchor sites markedly reduced the plasma membrane localization of CaV21 proteins and prevented its co-immunoprecipitation with CaV1.2. Altogether, our data are compatible with a model where GPI-anchored CaV21 proteins are preferentially assembled and trafficked to the cell surface with the CaV1.2 channel complex. Results Residues at the membrane interface in CaV21 are essential for the functional modulation of CaV1.2 currents The high-resolution 3D cryo-EM structure of the purified rabbit CaV21 subunit did not resolve the last 31 C-terminal residues, deduced from the nucleotide sequence suggesting that these residues are cleaved in the mature protein and replaced by a GPI anchor in the skeletal L-type Ca2+ channel (Fig. 1= 3) but not completely eradicated (Fig. 2), suggesting that transmembrane and GPI-anchored forms of CaV21 may co-exist in native tissues as shown for other proteins (53). To evaluate the functional impact of the C-terminal residues, deletion mutants of the rat mCherry-CaV21-HA construct (Fig. 1ventricular myocytes were isolated from adult rats. Cell lysates were incubated for 2 h either with 5 units/ml phosphatidylinositol-phospholipase C or the vehicle solution. Protein fractions (total cell lysates, cytosolic, total membrane, and plasma membrane fraction) were electrophoresed on an Sav1 8% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and Nerolidol probed with an anti-CaV21 (Alomone, 1:1000) and anti-pan-cadherin (Invitrogen, 1:5000). Each lane was loaded with 20 g of protein. The plasma membrane fraction was identified as the cadherin-enriched fraction. Cadherin was used as a loading control. expression of the native CaV21 was normalized to the density of cadherin ([CaV21]/[cadherin]) in all fractions in the absence and presence of PLC. The relative intensity of [CaV21]/[cadherin] at the plasma membrane fraction was then estimated as the ratio of [CaV21]/[cadherin] over the sum of the [CaV21]/[cadherin] protein density signals measured in all fractions and normalized by the relative intensity of the [CaV21]/[cadherin] in the plasma membrane fraction in the absence of PLC. As seen, there was a 41 5% (= 3) decrease in the relative intensity of the CaV21 proteins found in the PLC-treated plasma membrane fraction..