Skip to main content
. 2010 May 17;285(30):22853–22863. doi: 10.1074/jbc.M110.111062

FIGURE 3.

FIGURE 3.

Point mutations within the C-terminal residues on the AID helix disrupted the CaVβ stimulation of CaV1.2 plasma membrane targeting. A, HA-tagged CaV1.2 wt and mutants were expressed transiently either in the 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 residues targeted in these experiments are underlined within the primary sequence of the AID region of CaV1.2. The number of fluorescent cells decreased in the order CaV1.2-HA wt ≈ L464A, G466A, G466F, Y467G > Y467A, Y467F, G466A/Y467A > Y467S, G466Y/Y467G ≫ G466A/Y467F. The numerical values can be found in supplemental Table SI. B, Western blot analyses of HEKT cells transiently transfected with CaV1.2 wt or mutants in stable CaVβ3 cells using HA (1:500) and CaVβ3 (1:500) antibodies. Lane 1, control nontransfected cells. Lane 2, CaV1.2-HA. Lane 3, CaV1.2-HA + CaVβ3. Lane 4, CaV1.2-HA W470A. Lane 5, CaV1.2-HA W470A + CaVβ3. Lane 6, CaV1.2-HA G466A/Y467A. Lane 7, CaV1.2-HA G466A/Y467A + CaVβ3. Western blot analyses confirmed that the W470A mutant was expressed in total cell lysates and recognized by the anti-HA (1:500). Each lane was loaded with 50 μg of protein. C, HA-tagged CaV1.2 wt and mutants were expressed transiently either in the 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 residues targeted in these experiments are underlined within the primary sequence of the AID region of CaV1.2 shown in A. The number of fluorescent cells decreased in the order CaV1.2-HA wt ≈ I471L > I471F > I471A > I471S > I471R ≫ I471G, W470A, W470F, W470G, W470Y. The numerical values can be found in supplemental Table SI. D, Western blot analyses confirmed that the CaV1.2 W470A, I471A, I471L, and I471R mutants expressed with the expected molecular weight in total cell lysates and were recognized by the anti-HA (1:500). Each lane was loaded with 50 μg of protein. Lane 1, control nontransfected cells. Lane 2, CaV1.2-HA W470A. Lane 3, CaV1.2-HA I471A. Lane 4, CaV1.2-HA I471L. Lane 5, CaV1.2-HA I471R.