Molecular determinants of the CaVbeta-induced plasma membrane targeting of the CaV1.2 channel

Abstract

Ca(V)beta subunits modulate cell surface expression and voltage-dependent gating of high voltage-activated (HVA) Ca(V)1 and Ca(V)2 alpha1 subunits. High affinity Ca(V)beta binding onto the so-called alpha interaction domain of the I-II linker of the Ca(V)alpha1 subunit is required for Ca(V)beta modulation of HVA channel gating. It has been suggested, however, that Ca(V)beta-mediated plasma membrane targeting could be uncoupled from Ca(V)beta-mediated modulation of channel gating. In addition to Ca(V)beta, Ca(V)alpha2delta and calmodulin have been proposed to play important roles in HVA channel targeting. Indeed we show that co-expression of Ca(V)alpha2delta caused a 5-fold stimulation of the whole cell currents measured with Ca(V)1.2 and Ca(V)beta3. To gauge the synergetic role of auxiliary subunits in the steady-state plasma membrane expression of Ca(V)1.2, extracellularly tagged Ca(V)1.2 proteins were quantified using fluorescence-activated cell sorting analysis. Co-expression of Ca(V)1.2 with either Ca(V)alpha2delta, calmodulin wild type, or apocalmodulin (alone or in combination) failed to promote the detection of fluorescently labeled Ca(V)1.2 subunits. In contrast, co-expression with Ca(V)beta3 stimulated plasma membrane expression of Ca(V)1.2 by a 10-fold factor. Mutations within the alpha interaction domain of Ca(V)1.2 or within the nucleotide kinase domain of Ca(V)beta3 disrupted the Ca(V)beta3-induced plasma membrane targeting of Ca(V)1.2. Altogether, these data support a model where high affinity binding of Ca(V)beta to the I-II linker of Ca(V)alpha1 largely accounts for Ca(V)beta-induced plasma membrane targeting of Ca(V)1.2.

FIGURE 1.

Ca_Vα2bδ stimulated Ca_V1.2 whole cell currents. A, panel a, whole cell current traces recorded after the transient expression of the Ca_V1.2 wt channel in the stable Ca_Vβ3 cell line. The charge carrier was 2 mm Ca²⁺. Panel b, whole cell current traces recorded after the transient expression of the Ca_V1.2-HA channel in the stable Ca_Vβ3 cell line. Panel c, whole cell current traces recorded after the transient expression of the Ca_V1.2-HA channel and the Ca_Vα2bδ in the stable Ca_Vβ3 cell line. B, panel a, current-voltage relationships of Ca_V1.2 wt + Ca_Vβ3 ± Ca_Vα2bδ show a typical voltage-dependent activation with a mean current density of −7 ± 2 pA/pF (n = 6) for the wild type Ca_V1.2 channel in the stable Ca_Vβ3 stable cell line as compared with a current density of −41 ± 9 pA/pF (n = 7) for the wild type Ca_V1.2 channel measured in the same cell line after transient transfection with Ca_Vα2bδ subunit. The activation potential of 3 ± 3 mV for the Ca_V1.2 wt + Ca_Vβ was shifted to −10 ± 2 mV in the presence of Ca_Vα2bδ. Panel b, current-voltage relationships of Ca_V1.2-HA/Ca_Vβ3 ± Ca_Vα2bδ show a typical voltage-dependent activation with a mean current density of −9 ± 2 pA/pF (n = 5) for the Ca_V1.2-HA channel in the stable Ca_Vβ3 stable cell line as compared with a current density of −36 ± 8 pA/pF (n = 7) for the Ca_V1.2-HA channel measured in the same cell line after transient transfection with Ca_Vα2bδ subunit. The activation potential of 2 ± 3 mV for the Ca_V1.2-HA/Ca_Vβ was shifted to −12 ± 2 mV in the presence of Ca_Vα2bδ. Patch clamp experiments were carried out in the whole cell configuration in the presence of a 2 mm Ca²⁺ saline solution.

FIGURE 2.

Ca_Vβ stimulated Ca_V1.2 membrane expression in HEKT cells. A, HA-tagged Ca_V1.2 wt was co-expressed transiently either in the stable Ca_Vβ3 or the stable Ca_Vα2bδ cell line. Cell surface expression of Ca_V1.2 wt was determined in intact cells by flow cytometry using the anti-HA FITC conjugate antibody (Ab). The histogram shows the number of fluorescent cells as a function of the experimental conditions. Cell autofluorescence (HEKT no Ab) was <1% throughout, and the addition of the FITC did not significantly increase the level of fluorescence in HEKT cells not transfected with the HA-tagged Ca_V1.2 (HEKT with Ab). As seen, only co-expression of Ca_V1.2 with Ca_Vβ3 significantly promoted membrane expression of Ca_V1.2 (p < 0.001). Co-expression of Ca_V1.2 with Ca_Vα2bδ did not alter the number of Ca_V1.2 channels at the membrane (p > 0.1). Co-expression with both auxiliary subunits did not further improve the membrane expression of Ca_V1.2. The Ca_V1.2 + Ca_Vβ3 + Ca_Vα2bδ condition (either Ca_V1.2 + Ca_Vβ3 expressed transiently in the stable Ca_Vα2bδ cell line or Ca_V1.2 + Ca_Vα2bδ expressed transiently in the Ca_Vβ3 cell line) was not significantly different from the Ca_V1.2 + Ca_Vβ3 condition (p > 0.1). Similar results were obtained with transient expression systems. The numerical values can be found in Table 1. B, Western blot analyses of HEKT cells transiently or stably transfected with Ca_Vα2bδ or Ca_Vβ3, using Ca_Vα2δ-1 (1:200) and Ca_Vβ3 (1:500) antibodies. Each lane was loaded with 50 μg of protein. Panel a, HEKT cells were transiently or stably transfected with Ca_Vβ3. Lane 1, control nontransfected cells. Lane 2, transient transfection of Ca_Vβ3. Lane 3, control nontransfected cells. Lane 4, stable Ca_Vβ3 cell line. Lane 5, transient co-transfection of Ca_Vβ3 and Ca_Vα2bδ. Panel b, HEKT cells were transiently or stably transfected with Ca_Vα2bδ. Lane 1, control nontransfected cells. Lane 2, transient transfection of Ca_Vα2bδ. Lane 3, control nontransfected cells. Lane 4, stable Ca_Vα2bδ cell line.

FIGURE 3.

Point mutations within the C-terminal residues on the AID helix disrupted the Ca_Vβ stimulation of Ca_V1.2 plasma membrane targeting. A, HA-tagged Ca_V1.2 wt and mutants were expressed transiently either in the HEKT cells or in the stable Ca_Vβ3 cell line. Cell surface expression of the Ca_V1.2 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 Ca_V1.2. The number of fluorescent cells decreased in the order Ca_V1.2-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 Ca_V1.2 wt or mutants in stable Ca_Vβ3 cells using HA (1:500) and Ca_Vβ3 (1:500) antibodies. Lane 1, control nontransfected cells. Lane 2, Ca_V1.2-HA. Lane 3, Ca_V1.2-HA + Ca_Vβ3. Lane 4, Ca_V1.2-HA W470A. Lane 5, Ca_V1.2-HA W470A + Ca_Vβ3. Lane 6, Ca_V1.2-HA G466A/Y467A. Lane 7, Ca_V1.2-HA G466A/Y467A + Ca_Vβ3. 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 Ca_V1.2 wt and mutants were expressed transiently either in the HEKT cells or in the stable Ca_Vβ3 cell line. Cell surface expression of the Ca_V1.2 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 Ca_V1.2 shown in A. The number of fluorescent cells decreased in the order Ca_V1.2-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 Ca_V1.2 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, Ca_V1.2-HA W470A. Lane 3, Ca_V1.2-HA I471A. Lane 4, Ca_V1.2-HA I471L. Lane 5, Ca_V1.2-HA I471R.

FIGURE 4.

The NK domain of Ca_Vβ3 is critical for the plasma membrane targeting of Ca_V1.2. A, schematic diagram of the domain organization of the Ca_Vβ3 subunit based on the crystal structure and adapted from (21). B, HA-tagged Ca_V1.2 wt and Ca_Vβ3 mutants were expressed transiently in the HEKT cells. Cell surface expression of the Ca_V1.2 protein was quantified as described for supplemental Fig. S2. The residues targeted in these experiments are identified in the primary sequence of Ca_Vβ3. The Δ57–123 deletion removed the SH3 domain; the Δ170–175 removed the PYDVVP sequence; and the Δ195–200 removed the MMQKAL sequence, also termed the ABP domain. The numerical values are provided in supplemental Table SIII. C, Western blot analyses of HEKT cell lysates transiently transfected with Ca_Vβ3 constructs using anti-Ca_Vβ3 (1:500). Each lane was loaded with 10 μg of protein except for Ca_Vβ3 Δ54–362 loaded with 50 μg of protein. Lane 1, control nontransfected cells. Lane 2, Ca_Vβ3 wt. Lane 3, Ca_Vβ3 M196A. Lane 4, Ca_Vβ3 Δ170–175. Lane 5, Ca_Vβ3 Δ195–200. Lane 6, Ca_Vβ3 Δ57–123. Lane 7, Ca_Vβ3 Δ57–180. Lane 8, Ca_Vβ3 Δ180–364. Lane 9, Ca_Vβ3 Δ54–362. Western blot analyses confirmed that the Ca_Vβ3 deleted proteins were detected in total cell lysates with the expected molecular weight. D, HA-tagged Ca_V1.2 wt and Ca_Vβ3 fragments were expressed transiently in the HEKT cells. Cell surface expression of the Ca_V1.2 protein was quantified as described for supplemental Fig. S2. The residues targeted in these experiments are identified in the primary sequence of the Ca_Vβ3. The 181–362 fragment is equivalent to the NK domain. The numerical values are provided in supplemental Table SIII. E, Western blot analyses of HEKT cell lysates transiently transfected with the Ca_Vβ3 fragments using anti-c-Myc (1:500). Each lane was loaded with 50 μg of protein. Lane 1, control nontransfected cells. Lane 2, Ca_Vβ3 58–120. Lane 3, Ca_Vβ3 58–362. Lane 4, empty lane. Lane 5, Ca_Vβ3 181–362. The Ca_Vβ3 58–120 fragment formed a 7.4-kDa protein that cannot be seen in this figure. Western blot analyses confirmed that the Ca_Vβ3 fragments were detected in total cell lysates with the expected molecular weight.

FIGURE 5.

Ca_Vβ3 Δ57–123 stimulated Ca_V1.2 whole cell currents. A, whole cell current traces recorded after the transient expression of the Ca_V1.2-HA channel with Ca_Vα2bδ and Ca_Vβ3 wt (left panel) or with Ca_Vα2bδ and Ca_Vβ3 Δ57–123 (right panel) in HEKT cells. All of the subunits were transiently expressed. B, current-voltage relationships of Ca_V1.2-HA + Ca_Vα2bδ + Ca_Vβ3 wt (filled circles) and Ca_V1.2-HA + Ca_Vα2bδ + Ca_Vβ3 Δ57–123 (open triangles) show a typical voltage-dependent activation with a mean current density of −21 ± 2 pA/pF (n = 5) for Ca_V1.2-HA + Ca_Vα2bδ + Ca_Vβ3 wt as compared with a current density of −23 ± 4 pA/pF (n = 4) for Ca_V1.2-HA + Ca_Vα2bδ + Ca_Vβ3 Δ57–123 measured under the same conditions. Patch clamp experiments were carried out in the whole cell configuration in the presence of a 2 mm Ca²⁺ saline solution.

FIGURE 6.

Overexpressing CaM wt and CaM_1,2,3,4 did not alter the pattern of Ca_Vβ stimulation of Ca_V1.2 plasma membrane targeting. A, HA-tagged Ca_V1.2 wt was expressed transiently either in the HEKT cells or in the stable Ca_Vβ3 cell line. Cell surface expression of the Ca_V1.2 protein was quantified as described for supplemental Fig. S2. The number of fluorescent cells was not significantly influenced by overexpressing the wild type CaM or the dominant negative mutant CaM_1,2,3,4. The numerical values can be found in Table 1 and supplemental Table SIV. B, HA-tagged Ca_V1.2 wt and the double W470A/I471A mutant were expressed transiently either in the stable Ca_Vβ3 or in the stable Ca_Vα2bδ cell line. Cell surface expression of the Ca_V1.2 protein was quantified as described for supplemental Fig. S2. The number of fluorescent cells was not significantly increased by overexpressing CaM wt in any cell line as compared with the control Ca_V1.2 cells (p > 0.05). Similar data were obtained with the W470A mutant that abrogated Ca_Vβ3 binding and stimulation of Ca_V1.2 plasma membrane targeting. The numerical values can be found in Table 1 and supplemental Table SIV.

FIGURE 7.

Mutations within the high affinity CaM binding motif did not alter the Ca_Vβ stimulation of Ca_V1.2 plasma membrane targeting. A, flow cytometry data. HA-tagged Ca_V1.2 wt and mutants were expressed transiently either in HEKT cells or in the stable Ca_Vβ3 cell line. Cell surface expression of the Ca_V1.2 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 Ca_V1.2-HA wt protein under the same conditions. From left to right, the channels were Ca_V1.2-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 Ca_V1.2 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, Ca_V1.2-HA. Lane 3, Ca_V1.2-HA I1654A. Lane 4, Ca_V1.2-HA I1654A/Q1655A. Lane 5, Ca_V1.2-HA ΔC1643. Lane 6, Ca_V1.2-HA ΔC1644–1666. Lane 7, Ca_V1.2-HA ΔC1623–1666. Each lane was loaded with 50 μg of protein. C, Western blot analyses confirmed that the Ca_V1.2 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, Ca_V1.2-HA wt. Lane 3, Ca_V1.2-HA T1591A/L1592A/F1593A. Each lane was loaded with 50 μg of protein.