Molecular Determinants of the Ca_Vβ-induced Plasma Membrane Targeting of the Ca_V1.2 Channel

Abstract

Ca_Vβ subunits modulate cell surface expression and voltage-dependent gating of high voltage-activated (HVA) Ca_V1 and Ca_V2 α1 subunits. High affinity Ca_Vβ binding onto the so-called α interaction domain of the I-II linker of the Ca_Vα1 subunit is required for Ca_Vβ modulation of HVA channel gating. It has been suggested, however, that Ca_Vβ-mediated plasma membrane targeting could be uncoupled from Ca_Vβ-mediated modulation of channel gating. In addition to Ca_Vβ, Ca_Vα2δ and calmodulin have been proposed to play important roles in HVA channel targeting. Indeed we show that co-expression of Ca_Vα2δ caused a 5-fold stimulation of the whole cell currents measured with Ca_V1.2 and Ca_Vβ3. To gauge the synergetic role of auxiliary subunits in the steady-state plasma membrane expression of Ca_V1.2, extracellularly tagged Ca_V1.2 proteins were quantified using fluorescence-activated cell sorting analysis. Co-expression of Ca_V1.2 with either Ca_Vα2δ, calmodulin wild type, or apocalmodulin (alone or in combination) failed to promote the detection of fluorescently labeled Ca_V1.2 subunits. In contrast, co-expression with Ca_Vβ3 stimulated plasma membrane expression of Ca_V1.2 by a 10-fold factor. Mutations within the α interaction domain of Ca_V1.2 or within the nucleotide kinase domain of Ca_Vβ3 disrupted the Ca_Vβ3-induced plasma membrane targeting of Ca_V1.2. Altogether, these data support a model where high affinity binding of Ca_Vβ to the I-II linker of Ca_Vα1 largely accounts for Ca_Vβ-induced plasma membrane targeting of Ca_V1.2.

Introduction

Voltage-dependent Ca²⁺ channels (Ca_V) are membrane proteins that play a key role in promoting Ca²⁺ influx in response to membrane depolarization in excitable cells. To this date, molecular cloning has identified the primary structures for 10 distinct calcium channel Ca_Vα₁ subunits (1–7) that are classified into three main subfamilies according to their high voltage-activated (HVA)² gating (Ca_V1 and Ca_V2) or low voltage-activated gating (Ca_V3). In addition to the transmembrane pore-forming Ca_Vα1 subunit, Ca_V1 and Ca_V2 channels arise from the multimerization of three other proteins (7): a cytoplasmic Ca_Vβ subunit, a mostly extracellular Ca_Vα2δ subunit, and calmodulin constitutively bound to the C terminus of Ca_Vα1 (8–12).

A considerable body of work documents the interaction and modulation of the Ca_Vα1 subunit of Ca_V1 and Ca_V2 channels (13–18) by the auxiliary Ca_Vβ. The high affinity Ca_Vα1-Ca_Vβ interaction site on the pore-forming Ca_Vα1 subunit is a conserved 18-residue sequence in the I-II linker called the α interaction domain (AID) (19, 20) that has been structurally resolved by high resolution x-ray crystallography (21–23). Structural work showed that the AID forms a α-helix that binds to the α binding pocket (ABP) in the Ca_Vβ nucleotide kinase (NK) domain. It has been proposed that the MMQKAL cluster of residues within the latter determines the high affinity nanomolar interaction between the two proteins (24–29). Numerous mutational analyses of the AID residues have correlated the Ca_Vβ-induced biophysical modulation with the high affinity binding of Ca_Vβ to the AID peptide in a variety of Ca_Vα1 isoforms for Ca_V1 and Ca_V2 channels (25, 29–32).

The association of Ca_Vα1 and Ca_Vβ subunits is also critical for proper channel maturation and cell surface expression of Ca_V2.2 (17), Ca_V1.2 (33, 34), and Ca_V2.3 (35). In Ca_V2.2, the I-II linker is presumed to play a role in this process (17, 18), and mutations within the AID motif eliminated its cell surface expression and biophysical modulation by Ca_Vβ1b and Ca_Vβ3 (32). In addition, the Ca_Vβ2-induced increase in Ca_V1.2 whole cell currents was abolished with the AID-defective YWI/AAA mutant (29), suggesting that high affinity binding of Ca_Vβ onto AID is required to traffic Ca_Vα1 to the plasma membrane. Nonetheless, the unique character of the high affinity AID-ABP interface in the membrane targeting of Ca_Vα1 has been questioned (27, 36–40). In particular, it has been suggested that Ca_Vβ-mediated plasma membrane targeting could be uncoupled from Ca_Vβ-mediated modulation of channel gating (26, 41) with important contributions from other intracellular regions (33, 39, 42–44).

In addition to Ca_Vβ, the ancillary subunit Ca_Vα2δ and the ubiquitous calmodulin (CaM) protein have also been proposed to modulate HVA channel maturation and targeting (9). For instance, co-expression of Ca_Vα2δ promoted the trafficking of the Ca_Vα1 subunit of Ca_V2.2 in COS-7 cells (45), suggesting that Ca_Vα2δ could promote targeting of all HVA Ca_Vα1 subunits. CaM is a soluble, 17-kDa Ca²⁺-binding protein that serves as a critical Ca²⁺ sensor for Ca²⁺-dependent inactivation and facilitation upon Ca²⁺ binding in many Ca_V1 and Ca_V2 channels (46), of which Ca_V1.2 and Ca_V2.1 have been best characterized (8, 47). Constitutive apocalmodulin binding was reported on multiple sites in the Ca_Vα1 subunit of Ca_V1.2 (48) of which the C-terminal pre-IQ and IQ domains are best characterized (49). Mutations (TLF/AAA and I/E) in the pre-IQ and the IQ CaM-binding domains of the C terminus decreased the whole cell current density of Ca_V1.2, suggesting that Ca²⁺/CaM could modulate channel trafficking through its interaction with the C terminus (50) as it has been shown for small activated potassium channels (51).

To gauge the synergetic role of intracellular domains and auxiliary subunits in the steady-state plasma membrane expression of Ca_V1.2, we used a flow cytometry assay with an extracellularly HA-tagged Ca_V1.2 protein. Co-expression with Ca_Vβ3 produced a robust enhancement in the plasma membrane targeting of the Ca_Vα1 subunit of Ca_V1.2. The WI residues in the AID helix of the I-II linker of Ca_V1.2 were critical for Ca_Vβ-stimulated plasma membrane targeting of Ca_V1.2. No other combination with or without the auxiliary calmodulin and/or the Ca_Vα2bδ subunit produced any significant increase in the plasma membrane targeting of Ca_V1.2. Hence, Ca_Vβ appears to be the most potent determinant in the plasma membrane targeting of Ca_V1.2. Altogether, our data support a model where high affinity binding of the ABP of Ca_Vβ to the AID helix of Ca_Vα1 largely accounts for Ca_Vβ-induced plasma membrane targeting of Ca_V1.2.

EXPERIMENTAL PROCEDURES

Recombinant DNA Techniques

The rabbit Ca_V1.2 (GenBank^TM accession number X15539), the rat Ca_Vβ3 (GenBank^TM accession number M88751) (52), the rat brain Ca_Vα2bδ (GenBank^TM accession number NM_000722) (53), and the human CaM (GenBank^TM accession number M27319) were used. All of the subunits were subcloned in commercial vectors under the control of the cytomegalovirus promoter (see supplemental text for details).

For the Ca_Vβ3 deletion mutants, flanking NotI sites were inserted around the region(s) to be deleted. Following restriction digest of the NotI fragment and religation of the cohesive ends, the resulting NotI site was mutated back to the wild type amino acids. The Ca_Vβ3 fragments (numbered from their deduced amino acid sequence) were subcloned into the NotI sites of the pCMV-Tag5a vector (see supplemental text for details) that is a C-terminal c-Myc tagging vector. A Kozak sequence and an ATG initiation codon were inserted at the 5′-end of the nucleotide sequence.

The calmodulin wild type cDNA was subcloned in the pMT21 vector (54). The dominant negative mutant of CaM (CaM_1,2,3,4) that impaired high affinity Ca²⁺ binding is D20A/D56A/D93A/D129A, which has been described elsewhere (55).

Insertion of the HA Tag in the Ca_Vα1 Subunit

The hemagglutinin (HA) epitope tag (YPYDVPDYA) was inserted in the first extracytoplasmic predicted loop in Domain I at position 574 (nucleotide) for Ca_V1.2. The biophysical properties of the HA-tagged Ca_Vα1 subunit of Ca_V1.2 expressed in HEKT cells with the auxiliary Ca_Vβ3 subunit were found not to be significantly different from the wild type Ca_V1.2 channel expressed under the same conditions (see Fig. 1). In addition, cDNA injection of Ca_V1.2-HA constructions in concert with Ca_Vα2bδ and Ca_Vβ3 subunits in Xenopus oocytes yielded a biophysical profile not significantly different from that reported previously for the Ca_V1.2 (56) channels expressed under the same conditions. Hence, the HA-tagged version of the Ca_Vα1 subunit of Ca_V1.2 will be referred to as Ca_V1.2 wt throughout the text.

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.

Cell Culture and Transfections

tsA-201 (HEK293T or HEKT), a subclone of the human embryonic kidney cell line HEK-293 that expresses the simian virus 40 T-antigen, and COS1 cells were grown in Dulbecco's high glucose minimum essential medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin at 37 °C under 5% CO₂ atmosphere. COS1, HEKT, stable Ca_Vβ3, and Ca_Vα2bδ cells lines were transiently transfected with HA-tagged Ca_V1.2 cDNA using Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions. Protein expression of the auxiliary subunits in the stable and transient cell lines were confirmed routinely by Western blotting (see Fig. 2B). Transfection rate of the control pEGFP plasmid was estimated to be 66 ± 2% (n = 4) as assessed by flow cytometry from the fluorescence of the green fluorescent protein. Preliminary tests showed that Ca_V1.2 protein expression peaked 24–36 h after transfection.

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.

Western Blots

Protein expression of all constructs was confirmed by Western blotting in total cell lysates. HA-tagged Ca_V1.2 constructs were detected with anti-HA. The procedures are detailed in the supplemental text. Briefly, the membranes were incubated with anti-HA (1:500) (Covance Biotechnology, Québec, Canada) and revealed with an anti-mouse horseradish peroxidase secondary antibody (1:10000; Jackson Immunoresearch).

Fluorescence-activated Cell Sorting (FACS) Experiments

Cell surface expression of the Ca_V1.2 subunits was determined by flow cytometry using a FACScalibur® flow cytometer (Becton Dickinson) at the flow cytometry facility of the Department of Microbiology of the Université de Montréal. The cells expressing the extracellular HA tag were detected using an anti-HA-conjugated FITC fluorophore with a FITC filter (530 nm). The relative intensity of staining provided a metric to quantify cell surface expression of the HA-tagged Ca_V1.2 proteins (see supplemental Figs. S1 and S2 and supplemental text for details). The HA-tagged Ca_V1.2 construct was systematically tested as a control with the mutant channels.

Immunofluorescence

For fluorescence microscopy, Ca_Vβ3 stable cells were grown on sterile poly-d-lysine-coated coverslips. The cells were fixed 24 h after transfection in 4% paraformaldehyde, permeabilized with 0.075% saponin for 10 min at room temperature, washed in phosphate-buffered saline, and blocked in IgG-free 2% bovine serum albumin in phosphate-buffered saline for 20 min. The cells were incubated with FITC-conjugated anti-HA antibody (1:100) for 1 h at room temperature prior to the cells being mounted (Prolong antifade kit; Invitrogen) on glass microscope slides. HA-tagged Ca_V1.2 channels (wild type and mutant) were visualized (×60) using an Olympus microscope IX-81 microscope along with Image-Pro Plus 5.0 software.

Statistical Analysis

Statistical analyses were performed using the built-in one-way analysis of variance fitting routine for two independent populations of Origin 7.0. The data were considered statistically significant at p < 0.01.

RESULTS

Ca_Vα2δ Increases Whole Cell Currents of Ca_V1.2

Co-expression of Ca_V1.2 and Ca_V2.1 with the auxiliary Ca_Vα2δ subunit was shown to stimulate whole cell currents (45) in COS-7 cells, suggesting that Ca_Vα2δ could promote plasma membrane targeting of HVA Ca_Vα1 subunits. In Ca_V1.2, the gating charge appears to be unaffected by co-expression with Ca_Vα2δ, suggesting that Ca_Vα2δ stimulates channel facilitation by setting Ca_V1.2 channels in a conformational state very close to the open state without increasing protein density (57). To evaluate the functional role of Ca_Vα2δ, Ca_V1.2 wt and HA-tagged Ca_V1.2 α1 subunits were transiently transfected in the Ca_Vβ3 stable HEKT cell line in the absence and in the presence of Ca_Vα2bδ. As shown in Fig. 1A, whole cell currents, recorded in the presence of a physiological solution containing 2 mm Ca²⁺ (see the supplemental text), were significantly larger when measured in the presence of the Ca_Va2δ, confirming that Ca_Vα2δ stimulates whole cell currents of Ca_V1.2 (9). As shown in Fig. 1B, average whole cell current density increased from −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. Similar results were obtained for the HA-tagged Ca_V1.2 channels (Fig. 1B).

Ca_Vβ Promotes Membrane Targeting of Ca_V1.2

To determine whether Ca_Vα2bδ stimulates plasma membrane targeting of Ca_V1.2 channels, protein density of the extracellularly HA-tagged Ca_V1.2 channel was quantified with an anti-HA-conjugated FITC fluorophore. Fig. 2A shows the histogram of the fluorescent signal measured after transient expression of the Ca_Vα1 and the auxiliary subunit (either Ca_Vβ3 or Ca_Vα2bδ) in nonpermeabilized cells. Protein expression was confirmed by Western blotting (Fig. 2B). As seen, less than 0.5% of the cell population produced autofluorescence, whereas only 1% of the cells were fluorescent after the addition of the FITC antibody to control nontransfected cells (see raw data in supplemental Figs. S1 and S2). Transient co-expression of the HA-tagged Ca_V1.2 subunit in the stable Ca_Vβ3 cell line increased the number of proteins detected at the membrane from a value of 4.5 ± 0.5% (n = 25) in the nontransfected cell line to 23 ± 1% (n = 29) with Ca_Vβ3 (p < 0.001) (Table 1).

TABLE 1.

Fluorescence-activated cell sorting analysis of Ca_V1.2 ± auxiliary subunits

FACS results obtained after the transient transfection of Ca_V1.2-HA wt in either HEKT control cells, stable Ca_Vβ3 cells, or stable Ca_Vα2bδ cells. One day (24 h) after transfection, the cells were incubated with anti-HA FITC conjugate (10 μg/ml) at room temperature for 45 min. FACS separation of FITC-positive cells was performed on a FACScalibur® flow cytometer (Becton Dickinson), and fluorescence was quantified using CellQuest software (Becton Dickinson). The results are reported as percentage values of cells in M2. The data were pooled from experiments carried out over a period of 8 months. The data are shown as the means ± S.E. of the individual experiments, and the number of experiments appears in parentheses. ND, not determined.

Construct transient expression	Cell lines
	HEKT	HEKT CaVβ3 stable	HEKT CaVα2bδ stable	HEKT CaVβ3 + CaVα2bδ stable
	%	%	%	%
Cells without antibody	0.3 ± 0.1 (32)	0.18 ± 0.04 (20)	0.54 ± 0.07 (3)	2.8 ± 0.2 (3)
Cells with antibody	1.1 ± 0.3 (19)	1.0 ± 0.4 (19)	0.93 ± 0.05 (3)	4.2 ± 0.4 (3)
CaV1.2-HA wt	4.5 ± 0.5 (25)	23 ± 1 (29)	6 ± 1 (3)	19 ± 2 (3)
CaV1.2-HA wt + CaVβ3	25 ± 2 (3)	ND	21 ± 2 (3)	ND
CaV1.2-HA wt + CaVβ2a	14 ± 1 (3)	ND	ND	ND
CaV1.2-HA wt + CaVβ4	22 ± 3 (3)	ND	ND	ND
CaV1.2-HA wt + CaVα2bδ + CaM wt	2 ± 1 (3)	ND	ND	ND
CaV1.2-HA wt + CaVα2bδ	8 ± 3 (5)	26 ± 1 (3)	ND	ND
CaV1.2-HA wt + CaM wt	1.5 ± 0.3 (3)	19.1 ± 0.3 (3)	2.7 ± 0.4 (3)	ND
CaV1.2-HA wt + CaM1,2,3,4	1.4 ± 0.1 (3)	19.1 ± 0.6 (3)	ND	ND
CaV1.2-HA wt + CaVβ3 + CaVα2bδ	23 ± 2 (5)	ND	ND	ND

The results obtained with Ca_Vβ3 contrast with the effect observed when co-expressing HA-tagged Ca_V1.2 with Ca_Vα2bδ (p > 0.1). No further increase in the fluorescent signal was observed in the combined presence of the two auxiliary subunits. Similar results were obtained when Ca_V1.2 was transiently expressed, either in a background of stably transfected Ca_Vβ3 or in a background of stably transfected Ca_Vα2bδ cells (see Table 1 for numerical values). The maximum fluorescence obtained with Ca_Vβ3 confirms that Ca_Vα2bδ has little effect by itself on the Ca_V1.2 protein density at the plasma membrane. Among Ca_Vβ subunits, transient co-expression of Ca_V1.2-HA with Ca_Vβ4 caused a similar boost in plasma membrane expression, whereas Ca_Vβ2a was found to be slightly less potent for a Ca_Vβ3 ≈ Ca_Vβ4 > Ca_Vβ2a ranking (Table 1). Altogether, these results validated the fluorescence sorting analysis of HA-tagged Ca_Vα1 proteins to evaluate steady-state protein level in intact cells independently of channel gating.

α Interaction Domain: the Role of the WI Pair

Crystallographic analyses have shown that the AID-Ca_Vβ interaction is anchored through a set of six residues, Asp, Leu, Gly, Tyr, Trp, and Ile, distributed among three α-helical turns of the I-II linker of Ca_V1.2 (21–23), with the WI pair of residues being most critical for the AID-Ca_Vβ protein interaction (29). To evaluate whether the AID-Ca_Vβ interaction controls the plasma membrane targeting of Ca_V1.2, the HA-tagged Ca_V1.2 subunit was transiently co-expressed in HEKT cells or in Ca_Vβ3 stable cells. Ca_Vβ3 stimulated the plasma membrane targeting of N-terminal mutants: L464A, G466A, G466F, Y467G, Y467A, Y467S, and Y467F (Fig. 3A, supplemental Fig. S4, and supplemental Table SII). When compared with the control situation (±Ca_Vβ3), there was a 4–8-fold stimulation in the plasma membrane targeting mutations in the order G466A ≈ G466F > Y467F > Y467G > L464A ≈ Y467A > Y467S. Hence, no single point mutation in the N-terminal region of the AID completely abolished the Ca_Vβ3-induced stimulation in the plasma membrane targeting of Ca_V1.2. In contrast, the Ca_Vβ3 stimulation effect was completely eradicated in the double mutant G466A/Y467F, even though each individual mutation behaved like the wild type channel, suggesting that each residue contributes to the high affinity interaction with Ca_Vβ3.

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.

Point mutations in the C-terminal WI pair yielded a different picture. I471L was the only mutant that was detected at the membrane to the same extent as the wild type channel in the presence of Ca_Vβ3. However, Ca_Vβ3 stimulated significantly the plasma membrane targeting of I471A and I471F mutants. W470Y, W470F, W470A, W470G, I417G, and I471R were not significantly different in the presence or in the absence of Ca_Vβ3 (Fig. 3C and supplemental Table SII). Western blots carried out in total cell lysates with the anti-HA confirmed that all of the Ca_V1.2 mutants tested produced proteins with the expected molecular weight (Fig. 3, B and D). Immunofluorescence microscopy confirmed that W470A disrupted the plasma membrane targeting of Ca_V1.2 in the presence of Ca_Vβ3 (supplemental Fig. S5). Membrane expression of Ca_V1.2 in cultured hippocampal neurons was also disrupted after mutation of the key tryptophan residue to alanine (58).

Furthermore, double mutations in the same region completely eradicated the Ca_Vβ3 stimulation of Ca_V1.2 plasma membrane targeting (supplemental Fig. S6 and Table SI). Partial (Δ458–463) or complete (Δ458–475) removal of the AID-binding site within the I-II loop yielded similar results, confirming that no other low affinity Ca_Vβ site within the Ca_Vα1 subunit could promote the plasma membrane targeting of Ca_V1.2 in the absence of the AID region. Isothermal titration calorimetry assays have substantiated that the affinity of Ca_Vβ2a for the AID region of Ca_V1.2 decreased after single-point mutations of these residues. There was an >1000-fold increase in the K_d with the W/A and I/A mutants, whereas alanine mutation of the Leu and Gly residues imparted a smaller 5–10-fold decrease in the Ca_Vβ2a affinity (29). Substitution of the tryptophan residue by either tyrosine or phenylalanine only partly compensated for the mutation, confirming the requirement of a residue containing a double aromatic ring at this position. For the neighboring isoleucine position, mutation with the conserved leucine residue was found to preserve the Ca_Vβ3-induced membrane targeting of Ca_V1.2. For comparison, I387L in Ca_V2.3 was the only mutant tested in the WI pair that supported Ca_Vβ3 binding and Ca_Vβ3 modulation of gating (31). In contrast, none of the Ca_V1.2 mutations identified in the short QT syndrome, an inherited form of cardiac arrhythmia (59), was shown to affect the Ca_Vβ3 plasma membrane targeting of Ca_V1.2 (supplemental Fig. S7 and Table SII). Altogether our results support a strong correlation between Ca_Vβ3 binding affinity to the AID region as determined from fusion proteins and from isothermal titration calorimetry assays (29) and its role in promoting the targeting of Ca_V1.2 proteins at the plasma membrane. More importantly our results suggest that the molecular determinants that account for Ca_Vβ3 binding to the AID region are also responsible for the Ca_Vβ3-induced stimulation of the plasma membrane targeting of Ca_V1.2 proteins.

The NK Domain of Ca_Vβ Is Essential for Plasma Membrane Targeting of Ca_V1.2

The observation that different Ca_Vβs, which all share a conserved core containing the SH3 and NK domains, cause different biophysical effects on Ca_Vα1 subunits suggests that other regions besides the conserved AID-ABP interaction, could influence channel conformational changes (13). The SH3 domain of Ca_Vβ2a was found to bind to the I-II linker of Ca_V2.1 channels, suggesting that low affinity interactions outside of the AID-ABP interface could contribute to the full functional effects of the Ca_Vβ subunit (40). A few years later, however, the conserved AID-NK domain interaction was found to be necessary for Ca_Vβ-stimulated Ca_V2.1 channel surface expression (60). To evaluate whether the AID-NK interaction controls the plasma membrane targeting of Ca_V1.2, the HA-tagged Ca_V1.2 subunit was transiently co-expressed in HEKT cells in the presence of Ca_Vβ3 full-length or deleted constructs as well as with Ca_Vβ3 fragments.

We found that the NK domain of Ca_Vβ3 (180–364) (Fig. 4A) was required for the plasma membrane targeting of Ca_V1.2 (Fig. 4B and supplemental Table SIII). Targeted deletion of the SH3 domain between residues 57 and 123 preserved 80% of the Ca_V1.2 protein detected at the membrane (Fig. 4B). The deleted Ca_Vβ3 Δ57–123 construct preserved the typical Ca_Vβ3 modulation of channel gating and inactivation current kinetics. Peak whole cell current density was not significantly affected (Fig. 5).

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.

The Ca_Vα1-Ca_Vβ interaction appears to require the MMKQAL motif in the α3 helix of the NK domain and was identified in the crystal structure (21–23) as critical for the high affinity AID-ABP interaction. Indeed deletion of the 195–200 residue region of Ca_Vβ3 completely abolished plasma membrane targeting of Ca_V1.2 (Fig. 4B), and the single point mutation M196A in Ca_Vβ3, equivalent to M245 in Ca_Vβ2a (29), significantly decreased plasma membrane targeting with only 13 ± 1% (n = 3) fluorescent cells (supplemental Table SIII). Nonetheless, the NK domain (181–362 fragment) alone was not sufficient for targeting Ca_V1.2 to the membrane (Fig. 4D). Only the larger fragment (58–362) that includes part of the SH3 domain was found to stimulate significantly the plasma membrane targeting of Ca_V1.2. The integrity of the constructions was verified by Western blot (Fig. 4, C and E).

Calmodulin in the Plasma Membrane Targeting of Ca_V1.2

CaM interacts with multiple sites in the Ca_Vα1 subunit of Ca_V1.2 (48, 61), of which the C-terminal pre-IQ and IQ domains are best characterized. Constitutive CaM binding to the N terminus has also been reported (62). To determine whether low affinity binding of CaM to intracellular regions contributes to trafficking of Ca_V1.2 channels (50, 63), Ca_V1.2 was co-expressed with CaM wt or the dominant negative mutant of CaM (CaM_1,2,3,4) in HEKT control cells (supplemental Fig. S8) and in Ca_Vβ3 stable cells. Overexpression of CaM wt or its negative dominant mutant in Ca_Vβ3 stable cells did not significantly alter whole cell currents measured in the presence of 2 mm Ca²⁺ with peak current densities of −8 ± 2 pA/pF (n = 9) (CaM wt) and of −9 ± 3 pA/pF (n = 9) (CaM_1,2,3,4), whereas co-expression with the latter significantly decreased calcium-dependent inactivation kinetics (supplemental Fig. S9). Cytometry flux assays also failed to show a change in the plasma membrane expression of Ca_V1.2 with or without Ca_Vβ3 (Fig. 6 and supplemental Table SIV). These data contrast with previous reports that CaM_1,2,3,4 co-expression reduced peak Ca_V1.2 current amplitude in HEK cells compared with CaM co-expression (8). It suggests that Ca_Vβ3 is the dominant subunit to promote plasma membrane targeting of Ca_V1.2 and that CaM does not act synergistically with Ca_Vβ3 under these conditions.

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.

Overexpression of CaM wt was reported to promote the plasma membrane targeting of Ca_V1.2 proteins in COS1 cells, provided there was a complete absence of Ca_Vβ (63). To test the hypothesis that CaM could chaperone Ca_V1.2 to the membrane in the presence of Ca_Vα2bδ in our expression system, FACS experiments were carried out in the stable Ca_Vα2bδ cell line. As seen in Fig. 6B, CaM was unable to increase the number of Ca_V1.2 proteins at the membrane in the absence of Ca_Vβ3 under these conditions. Overexpression of CaM wt with the double mutant W470A/I471A (supplemental Table SIV) also failed to promote plasma membrane targeting of Ca_V1.2, thus ruling out a mechanism whereby low affinity binding of Ca_Vβ subunit to the AID region could mask the CaM effect.

The 1643–1666 fragment in the C terminus forms the high affinity (K_d > 3 nm) IQ-binding domain that co-crystallized with CaM (49). This high affinity binding site overlaps with the C-terminal “targeting domain” identified previously (42, 64). To test the hypothesis that constitutive calmodulin binding to the IQ motif is required for plasma membrane targeting, FACS experiments were carried out after mutations of the aromatic residues responsible for the high affinity (K_d ≈ 3 nm) CaM binding (49). Complete deletion of the 1643–1666 fragment did not alter surface labeling, whereas the W470A mutation in the ΔIQ channel eliminated plasma membrane targeting of Ca_V1.2 (supplemental Table SIV), suggesting that the IQ domain is not likely to act as a retention signal. Furthermore, point mutations Q1655A, Y1657A, and F1658A, as well as multiple mutations I1654A/F1658A and Y1657A/F1658A, and I1654A/Y1657A/F1658A did not alter plasma membrane targeting of Ca_V1.2. Plasma membrane targeting was not affected by a triple mutation in the pre-IQ domain (Ca_V1.2 T1651A/F1652A/L1653A) and was modestly supported in the I1654A and I1654A/Q1655A mutants (Fig. 7 and supplemental Table SIV). It should be remembered that the I/A mutation only moderately affected Ca²⁺/CaM binding to the C-terminal peptide of Ca_V1.2 as compared with the I/E mutant (65). Deleting the larger 1623–1666 region, identified as an important targeting domain (42), completely eradicated the plasma membrane expression of Ca_V1.2 both in the presence and in the absence of Ca_Vβ3 (Fig. 7 and supplemental Table SIV). Furthermore, as shown by others before (50), the Ca_V1.2 protein could not be detected at the membrane in the presence of the triple T1591A/L1592A/F1593A mutation. The three TLF residues are located in a pre-IQ apocalmodulin-binding site (peptide A) (48, 66), but overexpression with CaM wt or CaM_1,2,3,4 did not rescue plasma membrane targeting (supplemental Table SIV). Altogether, these data highlight the role of the C terminus in the plasma membrane targeting of Ca_V1.2 and suggest that high affinity Ca²⁺/CaM binding is not critical for the plasma membrane targeting of Ca_V1.2.

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.

DISCUSSION

To exhibit functional activity, ion channels must be targeted to the plasma membrane. Co-expression of Ca_V1.2 with either Ca_Vα2bδ or CaM (alone or in combination) failed to promote significantly the detection of fluorescently labeled Ca_V1.2-HA channels in intact cells by flow cytometry. Co-expression of Ca_V1.2 in the presence of Ca_Vβ3 with either Ca_Vα2δ or CaM failed to further increase the number of Ca_V1.2 proteins detected at the plasma membrane. Furthermore, plasma membrane targeting of AID-disrupted Ca_V1.2 mutants (thus in the absence of high affinity Ca_Vβ binding) could not be recovered by overexpressing the calmodulin protein alone or in combination with the auxiliary Ca_Vα2bδ subunit, suggesting that Ca_Vβ is the critical auxiliary subunit in the plasma membrane targeting of Ca_V1.2.

Plasma membrane targeting of Ca_V1.2 was decreased but not abolished in the double I1654A/Q1655A mutant in the presence of Ca_Vβ3 and was not altered in the absence of Ca_Vβ3, thus ruling out molecular models whereby the IQ motif, containing a polybasic motif, could serve as an endoplasmic reticulum retention signal (67). However, Ca_Vβ-induced membrane expression was unaffected by the complete deletion of the high affinity Ca²⁺/CaM IQ-binding site (ΔC1643–1666), suggesting that high affinity Ca²⁺/CaM binding to this domain (1643–1666) is not required. In contrast, deletion of the larger 1623–1666 region in the C terminus and mutation of the TLF site, located in a pre-IQ apocalmodulin-binding site, abolished plasma membrane targeting of Ca_V1.2 in agreement with previous reports (43, 44, 50). Clearly the C terminus of Ca_V1.2 harbors key site targeting signals, but our current data do not support a model whereby high affinity Ca²⁺/CaM binding to the IQ domain (1643–1666) is required. Whether the C terminus is required for proper protein folding or for anchoring apocalmodulin onto low affinity CaM-binding sites on the C terminus (50, 63) as in SK channels (51, 68) will await further structural studies.

Ca_Vα2δ and Surface Targeting of Ca_V1.2

It is widely acknowledged that Ca_Vα2δ subunits facilitate the voltage dependence of channel gating and potentiate whole cell currents in a number of recombinant HVA Ca_Vα1-Ca_Vβ subunit combinations (69–71). For instance, electrophysiological measurements carried out with Ca_V2.3 in HEK cells showed a small but significant increase in peak current density in the presence of Ca_Vα2δ that arose, at least in part, from an increase in the number of functional channels (72). Mutating the von Willebrand factor-A domain of Ca_Vα2δ-2 was shown to increase the intracellular localization of Ca_V1.2 or Ca_V2.2 throughout the cell (45), suggesting that Ca_Vα2δ could play a role in the plasma membrane targeting of HVA Ca_Vα1 subunits. It can, however, be argued that the 30% decrease of Ca_V1.2 at the cell surface assessed from cell surface biotinylation assays cannot fully account for the 3-fold decrease in channel peak current density observed with the Ca_Vα2δ-2 mutant (45). Indeed, it has been shown elsewhere that the gating charge of Ca_V1.2 is unaffected by co-expression with Ca_Vα2δ, supporting a model where Ca_Vα2δ stimulates peak currents by setting Ca_V1.2 channels in a conformational state very close to the open state without increasing protein density (57). Our current electrophysiological and flow cytometry measurements also support a role for Ca_Vα2δ in the modulation of Ca_V1.2 channel gating rather than plasma membrane protein density. At this point, however, our data do not exclude a role for Ca_Vα2δ at early steps of protein biosynthesis and/or recycling that could not be detected in our steady-state assay.

Ca_Vβ Stimulated Surface Labeling of Ca_V1.2

Co-expression with Ca_Vβ subunits produced a 3–5-fold enhancement in the plasma membrane targeting of the Ca_Vα1 subunit of Ca_V1.2, with Ca_Vβ3 and Ca_Vβ4 being the most potent effects. No other combination with or without the auxiliary CaM and/or the Ca_Vα2bδ subunit produced any significant increase in the plasma membrane targeting of Ca_V1.2. Hence, Ca_Vβ appears to be the most potent determinant in the plasma membrane targeting of Ca_V1.2. Our detailed mutational analysis supports a model where high affinity binding of the ABP of Ca_Vβ to the AID helix of Ca_Vα1 largely accounts for Ca_Vβ-induced plasma membrane targeting of Ca_V1.2.

The mechanism whereby Ca_Vβ antagonizes ER retention of the Ca_Vα1 subunit remains debated (73). In the two-site model, Ca_Vβ stimulation of protein expression and modulation of gating are controlled by distinct sites through a two-to-one stoichiometry. This model opens up the possibility that secondary Ca_Vβ-binding sites could contribute to plasma membrane targeting. Several observations could be suggestive of such a mechanism. Surface expression of Ca_V1.2-HA channels in Xenopus oocytes was not increased by injection of the Ca_Vβ2a protein, and even decreased gating currents and surface expression of the Ca_V1.2-ΔAID-expressing oocytes (36, 74). Covalently linking Ca_Vβ2b to the C terminus of Ca_V1.2 stimulated whole cell currents but failed to modulate channel gating in HEK cells (37, 38). Ca_Vβ2b-induced modulation of trafficking and gating was also uncoupled in N-terminally truncated Ca_V1.2 (39). Deletion of a low affinity interaction site between the SH3 module of Ca_Vβ and the I-II linker of the Ca_V2.1 subunit (outside the AID-GK interaction) did not affect Ca_V2.1 protein trafficking (40). Small fragments of Ca_Vβ2 arising from putative splice variants were also shown to bind to the C terminus of the Ca_V1.2 subunit where they promoted membrane targeting in the absence of the GK/SH3 module of Ca_Vβ subunits (75, 76). It remains, however, difficult to assess the physiological relevance of these findings, given that they result from in vitro interaction studies between isolated peptides.

In the one-site model, Ca_Vβ interacts sequentially with the Ca_Vα1 subunit through a unique binding site in the I-II linker in a 1:1 stoichiometry to dislodge ER retention signals and modulate gating (25). In the intact channel, high affinity binding of Ca_Vβ onto the AID motif would account for both Ca_Vβ-induced modulation of gating and the Ca_Vβ-plasma membrane trafficking of Ca_Vα1 (32, 77, 78). For Ca_V1.2 expressed in a mammalian cell system, the Trp⁴⁷⁰ and Ile⁴⁷¹ residues previously shown to account for the high affinity binding of Ca_Vβ (29) onto the Ca_V1.2 subunit were herein found to account for the Ca_Vβ stimulation of Ca_V1.2 plasma membrane targeting. As mentioned earlier, disruption of these residues alone or in combination had a dominant effect and abrogated cell surface labeling of Ca_V1.2. Our data hence support a model whereby high affinity binding of the MMQKAL motif of Ca_Vβ to the AID helix of the Ca_Vα1 subunit is required for chaperoning and modulating HVA Ca_V channels.