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. 2017 Sep 1;292(42):17236–17249. doi: 10.1074/jbc.M117.806893

Figure 8.

Figure 8.

Extracellular loop S5S6 in repeat III has a modest impact on the interaction between CaVα2δ1 and CaV1. 2/CaVβ3 proteins. A, 3D model of IIIS5S6 in CaV1.2 (residues 1059–1204) is shown in cyan, and the cache1 domain (residues 104–223) in CaVα2δ1 is shown in deep blue. The carboxyl group on the side chain of Arg-1119 in CaV1.2 is located close enough (< 3 Å) to the main-chain atoms of Glu-171 in CaVα2δ1 to potentially contribute to the formation of hydrogen bonds. In contrast, the 3D model does not predict a favorable interaction between Lys-1100 in CaV1.2 and Glu-174 in CaVα2δ1 with a minimum distance estimated to be at 4.6 Å. Modeling was achieved with Modeler 9.17. The figure was produced using PyMOL. B–D, HEKT cells were transiently transfected with pmCherry–CaVα2δ1–HA WT and pCMV–CaVβ3–c-Myc and either pCMV–CaV1.2 WT, pCMV–CaV1.2 R1119A, or pCMV–CaV1.2 K1100A. Cell lysates were immunoprecipitated overnight with anti-c-Myc magnetic beads to capture CaVβ3, eluted in a Laemmli buffer, and fractionated by SDS-PAGE using 8% gels. B, immunoblotting (IB) was carried out on total proteins (20 μg) collected from the cell lysates for each of the three proteins (CaV1.2, CaVα2δ1, and CaVβ3) before the immunoprecipitation assay (Input) to confirm that each protein was translated at the expected molecular mass. Each experimental condition is identified by the specific CaV1.2 construct. C, immunoblotting was carried out as detailed earlier. Images for CaVα2δ1 were captured after 1, 20, and 200 s of exposure. The signal for the anti-CaVα2δ1 was detected only after a 200-s exposure when probed in the presence of CaV1.2 R1119A/CaVβ3. D, protein lysates that ran through without binding to the antibody-bead complex (flow-through fraction) were collected, diluted in a Laemmli buffer, and fractionated by SDS-PAGE using an 8% gel and revealed with the anti-CaVα2δ1. As seen, mCherry–CaVα2δ1–HA is present in all flow-through fractions at the expected molecular mass (175 kDa) confirming that the proteins were appropriately translated and were present in the preparation in detectable quantities throughout.