FIGURE 7.
Mutations within the high affinity CaM binding motif did not alter the CaVβ stimulation of CaV1.2 plasma membrane targeting. A, flow cytometry data. HA-tagged CaV1.2 wt and mutants were expressed transiently either in HEKT cells or in the stable CaVβ3 cell line. Cell surface expression of the CaV1.2 protein was quantified as described for supplemental Fig. S2. The number of fluorescent cells decreased significantly for the mutants ΔC1623–1666, I1654A, I1654A/Q1655A, and T1591A/L1592A/F1593A (p < 0.01) as compared with the CaV1.2-HA wt protein under the same conditions. From left to right, the channels were CaV1.2-HA wt, ΔC1623–1666, ΔC1643–1666, I1654A, Q1655A, I1654A/Q1655A, Y1657A, F1658A, Y1657A/F1658A, and T1591A/L1592A/F1593A. The numerical values can be found in supplemental Table SIV. B, Western blot analyses confirmed that the CaV1.2 mutant proteins were detected in total cell lysates by the anti-HA (1:500) with the expected molecular weight. Lane 1, nontransfected cells. Lane 2, CaV1.2-HA. Lane 3, CaV1.2-HA I1654A. Lane 4, CaV1.2-HA I1654A/Q1655A. Lane 5, CaV1.2-HA ΔC1643. Lane 6, CaV1.2-HA ΔC1644–1666. Lane 7, CaV1.2-HA ΔC1623–1666. Each lane was loaded with 50 μg of protein. C, Western blot analyses confirmed that the CaV1.2 mutant proteins were detected in total cell lysates by the anti-HA (1:500) with the expected molecular weight. Lane 1, nontransfected cells. Lane 2, CaV1.2-HA wt. Lane 3, CaV1.2-HA T1591A/L1592A/F1593A. Each lane was loaded with 50 μg of protein.