A Quartet of Leucine Residues in the Guanylate Kinase Domain of Ca_Vβ Determines the Plasma Membrane Density of the Ca_V2.3 Channel

Background: Ca_Vβ subunits stimulate cell surface expression of Ca_V2.3 channels.

Results: Of 33 positions and domains tested, leucine mutants in the guanylate kinase domain of Ca_Vβ3 decreased significantly the surface protein density of Ca_V2.3.

Conclusion: Leucine residues are responsible for the functional modulation by Ca_Vβ.

Significance: A quartet of leucine residues forms the hydrophobic pocket surrounding the α-interacting domain of Ca_V2.3.

Abstract

Ca_Vβ subunits are formed by a Src homology 3 domain and a guanylate kinase-like (GK) domain connected through a variable HOOK domain. Complete deletion of the Src homology 3 domain (75 residues) as well as deletion of the HOOK domain (47 residues) did not alter plasma membrane density of Ca_V2.3 nor its typical activation gating. In contrast, six-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of Ca_V2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of Ca_Vβ (P175A, P179A, M195A, M196A, K198A, S295A, R302G, R307A, E339G, N340G, and A345G) did not significantly alter cell surface targeting or gating modulation of Ca_V2.3. Nonetheless, mutations of a quartet of leucine residues (either single or multiple mutants) in the α3, α6, β10, and α9 regions of the GK domain were found to significantly impair cell surface density of Ca_V2.3 channels. Furthermore, the normalized protein density of Ca_V2.3 was nearly abolished with the quadruple Ca_Vβ3 Leu mutant L200G/L303G/L337G/L342G. Altogether, our observations suggest that the four leucine residues in Ca_Vβ3 form a hydrophobic pocket surrounding key residues in the α-interacting domain of Ca_V2.3. This interaction appears to play an essential role in conferring Ca_Vβ-induced modulation of the protein density of Ca_Vα1 subunits in Ca_V2 channels.

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. The total Ca²⁺ influx through Ca_V proteins is controlled by the single-channel conductance, the probability that the channel is open at a given time and voltage, what is also referred to as gating, and the number of proteins expressed at the membrane. Modulating any of these parameters could be used to alter Ca²⁺ influx and prevent excitable cells from Ca²⁺ overload.

Voltage-gated Ca²⁺ channels form oligomeric complexes that are classified according to the structural properties of the pore-forming Ca_Vα1 subunit. The primary structures for 10 distinct Ca_Vα1 subunits (1–7) are classified into three main subfamilies according to their high voltage-activated gating (HVA Ca_V1 and Ca_V2) or low voltage-activated gating (LVA Ca_V3). In HVA Ca_V1 and Ca_V2 channels, auxiliary subunits include a cytoplasmic Ca_Vβ subunit, a mostly extracellular Ca_Vα2δ subunit, and calmodulin constitutively bound to the C terminus of Ca_Vα1 (7–12) (for review see Ref. 9).

There are four subfamilies of Cavβs, each with multiple splicing isoforms and unique modulatory functions (14, 15). Auxiliary Ca_Vβ subunits modulate activation gating as well as inactivation kinetics of the main Ca_Vα1 subunits of HVA Ca_V1 and Ca_V2 channels (16–23). They also promote cell surface expression in part by preventing protein ubiquitination and degradation of the protein by the ERAD complex (20, 24).

The principal Ca_Vα1-Ca_Vβ interaction site on the pore-forming α1 subunit is a conserved 18-residue sequence in the I-II loop called the α-interacting domain (AID)³ (25). The nanomolar affinity between the AID helix and the Ca_Vβ protein has been thoroughly investigated (26–29). It is primarily secured by the projection of the conserved tryptophan and isoleucine (WI) pair of residues onto the Ca_Vβ fold (27–29). Single point mutations of the C-terminal WI residues on the AID decreased by 1000-fold the affinity of the Ca_Vβ2 for Ca_V1.2 (26) and decreased its cell surface density (17).

Less is known in regard to the molecular determinants in Ca_Vβ that control protein density at the plasma membrane and modulate the activation gating of the channel. It is understood that the N terminus plays a predominant role in modulating inactivation kinetics, either directly by interacting with the Ca_Vα1 (30, 31) and/or indirectly through palmitoylation for Ca_Vβ2a (32). High resolution three-dimensional crystal structures have shown that Ca_Vβ subunits consist of five distinct domains: the N terminus, a Src homology 3 (SH3) domain, a HOOK region, a guanylate kinase (GK) domain, and the C terminus (see Fig. 5A) (27–29). The GK core module appears to be sufficient to confer the activation properties (33–35). The latter includes the structural determinants of the α-binding pocket that are distributed among α3, α6, α7, and α9 helices and β9 and β10 sheets (26–29).

FIGURE 5.

A, schematic diagram of the domain organization of the Ca_Vβ3 subunit based on the crystal structure adapted from Ref. 27 and used in (17). B, typical whole cell current traces were obtained for Ca_V2.3 WT in the presence of a 2 mm Ca²⁺ solution. Ca_Vα2δ-1 was omitted. From left to right are shown no Ca_Vβ3, Ca_Vβ3 WT, and Ca_Vβ3 fragment 58–362. C, peak current densities are plotted as a function of applied voltage. Ca_V2.3 WT + Ca_Vβ3 WT and Ca_V2.3 WT + Ca_Vβ3 58–362 showed a similar typical voltage-dependent activation with a >5-fold increase in peak current densities as compared with Ca_V2.3 WT in the absence of Ca_Vβ3. The numerical values are shown in Table 1. D, the r₅₀ values are shown as means ± S.E. from −15 to +5 mV for Ca_V2.3 WT + Ca_Vβ3 and Ca_V2.3 WT + Ca_Vβ3 58–362 (from left to right on the bar graph). The r₅₀ values of Ca_V2.3 WT + Ca_Vβ3 and Ca_V2.3 WT + Ca_Vβ3 58–362 were significantly not different at p > 0.01 at all voltages.

With some notable exceptions (23, 26, 36), few studies have systematically attempted to correlate Ca_Vβ high affinity binding with the modulation of channel gating and trafficking. Whereas interaction between Ca_Vβ and Ca_Vα1 is required for the trafficking of Ca_Vα1 to the plasma membrane, it remains to be seen whether nanomolar binding of Ca_Vβ to the AID motif of Ca_Vα1 is required to carry both roles. Furthermore, whereas the binding of Ca_Vβ on the AID of Ca_Vα1 has been well characterized (17, 22, 26, 33, 37, 38), few concur on the GK residues in Ca_Vβ that determine protein density at the plasma membrane and contribute to channel modulation.

Complications have arisen in functional studies performed with the Xenopus recombinant system (26, 27, 33, 39–41), including our own (37, 38), because endogenous Ca_Vβs (42) are likely to boost expression of noninteracting mutants. The presence of the Ca_Vα2δ subunit in some experiments could complicate data interpretation. In addition, the Ca_Vβ2a subunit used in most studies is an atypical subunit with an N-terminal palmitoylation site (43) that anchors it to the membrane and could offset the disruption of the high affinity interaction site. Indeed, the palmitoylated Ca_Vβ2a was still able to modulate the biophysical properties of Ca_V2.2 W391A in Xenopus oocytes, indicating that the plasma membrane anchoring afforded by its palmitoylation can substitute in some cases for high affinity interaction with the I-II linker (22).

In this work we have systematically addressed the role of the structural domains of Ca_Vβ3 in regard to protein density and channel function after recombinant expression of HVA Ca_V2.3 in HEKT cells. Selective deletion of the SH3 and HOOK structural domains did not significantly alter the cell surface density or the channel functional modulation by Ca_Vβ. More surprisingly, mutations of residues in the GK domain that were previously identified as underlying the affinity to HVA Ca_Vα1 (26) caused little change in the cell surface density of Ca_V2.3 or in the channel activation gating. Nonetheless, point mutations within a quartet of leucine residues in the α3, α6, β10, and α9 regions of the GK domain significantly decreased cell surface density. Altogether our results suggest that affinity interaction in the micro- to nanomolar range is sufficient to carry the typical Ca_Vβ-induced hyperpolarizing shift in the channel activation gating in the presence of overexpressed Ca_Vβ subunits. Furthermore, we have identified a quartet of leucine residues in the GK domain of Ca_Vβ3 that play a critical role in promoting surface plasma density of Ca_V2.3.

EXPERIMENTAL PROCEDURES

Recombinant DNA Techniques

The human Ca_V2.3 (Genbank^TM accession number L27745) (44), the rat Ca_Vβ3 (GenBank^TM accession number M88751) (45), and the rat brain Ca_Vα2bδ-1 (GenBank^TM accession number NM_000722) (46) were used. All subunits were subcloned in commercial vectors under the control of the CMV promoter as explained earlier (17). Some electrophysiological experiments were performed with pGFP-Ca_Vα2δ1 peGFP-C2 vector (Clontech).

The HA epitope tag (YPYDVPDYA) was inserted in the first extracytoplasmic predicted loop in Domain I at position 367 (nucleotides) for Ca_V2.3. The biophysical properties of the HA-tagged Ca_Vα1 subunit of Ca_V2.3 expressed in HEKT cells with the auxiliary Ca_Vβ3 subunit were found not to be significantly different from the wild-type Ca_V2.3 channel expressed under the same conditions (see Table 1). Ca_Vβ3 deletion mutants were produced as described elsewhere (17). The Ca_Vβ3 58–362 fragment was used previously in (17). The boundaries of the five domains/regions of the rat Ca_Vβ3 are: N terminus, Met¹–Pro⁵⁹; SH3 domain, Val⁶⁰–Ser¹²³ and Pro¹⁷⁰–Pro¹⁷⁵; HOOK region, Pro¹²⁰–Pro¹⁶⁹; GK domain, Ser¹⁷⁶–Thr³⁶⁰; and C terminus, His³⁶¹–Tyr⁴⁸⁴ (see Fig. 5A).

TABLE 1.

Biophysical properties of Ca_V2.3 channels in the absence of Ca_Vα2δ-1

Ca_V2.3 wild-type or HA-Ca_V2.3 channels were expressed in HEKT cells with Ca_Vβ3 WT or constructs. Biophysical parameters were measured in the presence of a physiological saline containing 2 mm Ca²⁺ as described elsewhere (17). Ca_Vα2δ-1 was omitted. Activation properties (E_0.5,act and ΔG_act) were estimated from the mean I-V relationships and fitted to a Boltzmann equation. The data are shown as the means ± S.E. of the individual experiments, and the number of experiments appears in parentheses.

CaV2.3 WT in HEKT cells no CaVα2δ-1	Electrophysiological properties
	E0.5,act	ΔGact	Peak density	r50 at +10 mV
	mV	kcal mol−1	pA/pF
No auxiliary subunit	−5 ± 1 (18)	−0.6 ± 0.1 (18)	−6 ± 1 (18)	ND
+ CaVβ3	−10.7 ± 0.3 (49)	−1.32 ± 0.05 (49)	−18 ± 2 (49)	0.38 ± 0.01 (49)
+ CaVβ1a	−10.5 ± 0.5 (6)	−1.2 ± 0.1 (6)	−35 ± 4 (6)	0.51 ± 0.01 (6)
+ CaVβ1b	−12.2 ± 0.6 (12)	−1.6 ± 0.2 (12)	−68 ± 14 (12)	0.54 ± 0.01 (12)
+ CaVβ2a	−11.4 ± 0.6 (6)	−1.3 ± 0.1 (6)	−33 ± 4 (6)	0.85 ± 0.02 (6)
+ CaVβ4	−12.1 ± 0.6 (7)	−1.5 ± 0.1 (7)	−21 ± 2 (7)	0.64 ± 0.02 (7)
+ CaVβ3 M196A	−7.9 ± 0.3 (22)	−0.90 ± 0.04 (22)	−14 ± 1 (22)	0.43 ± 0.01 (22)
+ CaVβ3 M195A	−12 ± 1 (9)	−1.5 ± 0.1 (9)	−24 ± 5 (9)	0.53 ± 0.01 (9)
+ CaVβ3 K198A	−11.6 ± 0.6 (10)	−1.4 ± 0.1 (10)	−13 ± 2 (10)	0.30 ± 0.01 (10)
+ CaVβ3 L200G	−9.8 ± 0.6 (7)	−1.16 ± 0.08 (7)	−18 ± 2 (7)	0.44 ± 0.02 (7)
+ CaVβ3 L303G	−7.2 ± 0.5 (7)	−0.82 ± 0.06 (7)	−17 ± 2 (7)	0.40 ± 0.02 (7)
+ CaVβ3 L337G	−9.3 ± 0.5 (8)	−1.2 ± 0.1 (8)	−19 ± 6 (8)	0.46 ± 0.01 (8)
+ CaVβ3 L342G	−10.1 ± 0.5 (8)	−1.27 ± 0.07 (8)	−15 ± 3 (8)	0.49 ± 0.01 (8)
+ CaVβ3 L200G/L303G	−7.8 ± 0.6 (10)	−1.0 ± 0.1 (10)	−9 ± 1 (10)	0.40 ± 0.03 (10)
+ CaVβ3 L200G/L303G/L337G	−7.9 ± 0.5 (12)	−1.0 ± 0.1 (12)	−6.2 ± 0.8 (12)	0.48 ± 0.03 (12)
+ CaVβ3 L200G/L303G/L337G/L342G	−9.1 ± 0.7 (11)	−1.1 ± 0.1 (11)	−5.7 ± 0.5 (11)	0.45 ± 0.02 (11)
+ CaVβ3 R307G	−15 ± 1 (10)	−2.0 ± 0.1 (10)	−66 ± 11 (10)	0.53 ± 0.01 (10)
+ CaVβ3 Δ57–123	−10 ± 1 (12)	−1.2 ± 0.1 (12)	−38 ± 9 (12)	0.36 ± 0.03 (12)
+ CaVβ3 Δ57–180	−9 ± 1 (6)	−1.0 ± 0.1 (6)	−7 ± 1 (6)	0.37 ± 0.06 (6)
+ CaVβ3 Δ170–175 (ΔPYDVVP)	−10 ± 1 (14)	−1.1 ± 0.1 (14)	−22 ± 6 (14)	0.48 ± 0.02 (14)
+ CaVβ3 Δ57–123 Δ170–175 (ΔSH3)	−8 ± 1 (12)	−0.9 ± 0.1 (12)	−24 ± 3 (12)	0.40 ± 0.03 (12)
+ CaVβ3 Δ175–180	−7.7 ± 0.5 (12)	−1.0 ± 0.1 (12)	−7.1 ± 0.8 (12)	0.42 ± 0.04 (12)
+ CaVβ3 Δ122–169	−9.5 ± 0.5 (11)	−0.95 ± 0.07 (11)	−19 ± 3 (11)	0.35 ± 0.03 (11)
+ CaVβ3 Δ122–175	−7.6 ± 0.5 (11)	−0.85 ± 0.07 (11)	−19 ± 4 (11)	0.33 ± 0.01 (11)
+ CaVβ3 Δ58–362	−13 ± 1 (10)	−1.4 ± 0.1 (10)	−13 ± 4 (10)	0.31 ± 0.03 (10)
+ CaVβ3 Δ180–364	−8.6 ± 0.5 (9)	−1.1 ± 0.1 (9)	−7 ± 1 (9)	0.42 ± 0.05 (9)
HA-CaV2.3 WT + CaVβ3 WT	−12 ± 1 (5)	−1.3 ± 0.1 (5)	−14 ± 2 (5)	0.46 ± 0.03 (9)
HA-Y383F + CaVβ3 WT	−9 ± 1 (9)	−1.0 ± 0.1 (9)	−21 ± 4 (9)	0.47 ± 0.01 (9)
HA-Y383A + CaVβ3 WT	−7.8 ± 0.3 (11)	−0.75 ± 0.05 (11)	−9 ± 1 (11)	0.53 ± 0.02 (11)
HA-R384L + CaVβ3 WT	−12 ± 1 (5)	−1.7 ± 0.2 (4)	−13 ± 2 (4)	0.36 ± 0.03 (5)
HA-R384 M + CaVβ3WT	−16 ± 2 (5)	−1.4 ± 0.1 (5)	−22 ± 2 (5)	0.41 ± 0.04 (5)

Cell Culture and Transfections

HEK293T or HEKT were grown in high glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C under 5% CO₂ atmosphere as described elsewhere (17). RT-PCR conducted in these cells failed to highlight the presence of Ca_Vβ and Ca_Vα2δ auxiliary subunits (47). HEKT cells (80% confluence) were transiently transfected with similar amounts of DNA (4 μg each or 12 μg total): HA-Ca_V2.3, Ca_Vβ3, Ca_Vα2δ-1, or empty vector pCMVTag5 in 10 μl of Lipofectamine 2000 (Qiagen) using a DNA:lipid ratio of 1:2.5 as described elsewhere (17). Transfection rate of the control peGFP plasmid was estimated to be 66 ± 2% (n = 8) as assessed by flow cytometry from the fluorescence of the GFP. Preliminary tests showed that Ca_V2.3 protein expression peaked 24–30 h after transfection.

Quantification of Ca_V2.3 Surface Expression with FACS

FACS experiments were conducted and analyzed as described elsewhere. Briefly, 24–28 h after transfection, the cells were harvested and stained with the anti-HA FITC conjugate (10 μg/ml) at room temperature for 45 min. A maximum of 10,000 cells resuspended in standard PBS were counted using a FACScalibur® flow cytometer (BD Biosciences) with a FITC filter (530 nm) at the flow cytometry facility located in the Department of Microbiology of the Université de Montréal. The level of background fluorescence was adjusted with cells incubated in the absence of the fluorophore. The parameters of the M1 region were set from Gaussian distribution of the fluorescence intensity obtained in the absence of the FITC antibody. The M2 region was calculated from the Gaussian distribution observed in the higher decades of the fluorescence log scale in the presence of the FITC antibody. The cell fluorescence intensity and, by extension, the cell surface expression of the HA tag were estimated from the M2 peak value (see supplemental Fig. S2 of Ref. 17). Three control conditions were always carried out for each series of experiments: (a) nontransfected HEKT cells with anti-HA FITC antibody; (b) HEKT cells transfected with HA-Ca_V2.3 without Ca_Vβ3 WT; and (c) HEKT cells transfected with HA-Ca_V2.3 WT and Ca_Vβ3 WT. Peak fluorescence values obtained for the control conditions were compared with previous measures for internal consistency. Each novel mutant and/or experimental condition was conducted in triplicate and sometimes repeated over 2 weeks. The experiments performed in the 24–28-h range after transfection yielded M2 values with an experimental variation lower than 10% between samples and between series of experiments. In any case, a maximum of 10% variation in the measure of fluorescence was tolerated. All control conditions were pooled and reported in Tables 3 and 4.

TABLE 3.

Fluorescent-activated cell sorting analysis of Ca_V2.3

FACS results obtained after the transient transfection of HA-Ca_V2.3 WT in either HEKT control cells or stable Ca_Vβ3 cells. One day (24–28 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 (BD Biosciences), and fluorescence was quantified using CellQuest software (BD Biosciences).The results are reported as percentage values of cells in higher quadrant. The data were pooled from experiments carried out over a period of 12 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.

Constructs transient expression	Fluorescence in HEKT	Fluorescence in CaVβ3 stable
	%	%
Cells no Ab	0.22 ± 0.01 (33)	0.21 ± 0.02 (28)
Cells with Ab	0.5 ± 0.1 (35)	0.6 ± 0.1 (30)
HA-CaV1.2 WT	3.8 ± 0.5 (15)	27 ± 1 (18)
HA-CaV1.2 WT + CaVβ3	26 ± 1 (56)	ND
HA-CaV2.3 WT	1.7 ± 0.1 (31)	20 ± 1 (30)
HA-CaV2.3 WT + CaVβ3	36.3 ± 0.08 (46)	ND
HA-CaV2.3 G382A	35 ± 1 (3)	19 ± 1 (7)
HA-CaV2.3 Y383A	27 ± 1 (3)	4.9 ± 0.6 (7)
HA-CaV2.3 Y383F	37 ± 1 (3)	13 ± 1 (7)
HA-CaV2.3 Y383G	12 ± 1 (3)	4.0 ± 0.3 (7)
HA-CaV2.3 R384M	32 ± 2 (3)	19 ± 1 (6)
HA-CaV2.3 R384L	31 ± 2 (3)	18 ± 1 (3)
HA-CaV2.3 W386A	1.2 ± 0.2 (3)	1.5 ± 0.3 (7)
HA-CaV2.3 WT + CaVβ1a	45 ± 2 (3)	ND
HA-CaV2.3 WT + CaVβ1b	41 ± 2 (3)	ND
HA-CaV2.3 WT + CaVβ2a	9 ± 1 (5)	ND
HA-CaV2.3 WT + CaVβ4	40 ± 2 (3)	ND
HA-CaV2.3 WT + CaVβ3 + CaVα2δ-1	34 ± 2 (6)	ND
HA-CaV2.3 WT + CaVα2δ-1	1.2 ± 0.2 (7)	21 ± 1 (5)
HA-CaV2.3 WT + CaM	1.5 ± 0.2 (9)	ND
HA-CaV2.3 WT + CaM1234	1.2 ± 0.2 (9)	ND

TABLE 4.

Fluorescent-activated cell sorting analysis of Ca_Vβ3 constructs

FACS results obtained after the transient transfection of Ca_V2.3-HA WT in HEKT cells. The results are reported as percentage values of cells in the higher quadrant of fluorescence. The data were pooled from experiments carried out over a period of 12 months. The data are shown as the means ± S.E. of the individual experiments, and the number of experiments (n) appears in the last column.

CaVβ constructs	Fluorescence
Transient expression HA-CaV2.3 HEKT cells	Mean	S.E.	n
	%	%
CaVβ3 WT	36.3	0.8	46
Δ57–123 (ΔNSH3)	26	1	6
Δ57–180 (ΔSH3)	2.9	0.4	3
Δ180–364 (ΔGK)	1.4	0.4	5
Δ54–362 (ΔSH3-GK)	3	1	3
Δ170–175 (ΔPYDVVP)	23	2	7
Δ170–175 Δ180–364 (ΔPYDVVPΔGK)	3	1	3
Δ170–175 Δ57–123 (ΔPYDVVPΔNSH3)	25	2	8
Δ195–200 (ΔMMQKAL)	1.6	0.6	5
ΔHOOK (Δ122–169)	24	2	4
ΔHOOKΔPYDVVP (Δ122–175)	22	1	4
ΔPSMRPV (Δ175–180)	3	0.8	3
P175A	33	1.5	3
P175G	32	2.4	3
P179A	29	0.7	3
P179G	30	0.7	3
M195A	37	0.3	5
M196A	22	0.7	9
K198A	41	0.4	4
L200G	18	0.7	3
S295A	37	1.5	4
R302G	43	1.1	4
L303G	8.0	0.2	7
R307A	27	1.1	3
R307G	33	1.6	3
R307K	33	0.6	3
L337G	16	0.7	6
E339G	21	1.3	3
N340G	33	1.0	3
L342G	16	0.5	6
A345G	20.2	0.7	3
L200G/L303G	1.7	0.1	3
L200G/L303G/L337G	2.1	0.1	3
L200G/L303G/L337G/L342G	2.5	0.1	3

Patch Clamp Experiments in HEKT Cells

Whole cell voltage clamp recordings were performed 30 h after transfection using the methods described above in the presence of the peGFP vector (0.2 μg) as a control for transfection. Patch clamp experiments were carried out with the Axopatch 200-B amplifier (Molecular Devices, Union City, CA). Electrodes were filled with a solution containing 140 mm CsCl, 0.6 mm NaGTP, 3 mm MgATP, 10 mm EGTA, 10 mm Hepes titrated to pH 7.3 with NaOH. Pipette resistance ranged from 2 to 4 mΩ. The cells were bathed in a modified Earle's saline solution 135 mm NaCl, 20 mm TEACl, 2 mm CaCl₂, 1 mm MgCl₂, 10 mm Hepes titrated to pH 7.3 with KOH. PClamp software Clampex 10.2 coupled to a Digidata 1440A acquisition system (Molecular Devices) was used for on-line data acquisition and analysis. Pipette and cell capacitance cancellation and series resistance compensation were applied (up to 80%) using the cancellation feature of the amplifier. Cellular capacitance was estimated by measuring the time constant of current decay evoked by a 10-mV depolarizing pulse applied to the cell from a holding potential of −100 mV. A series of 150-ms voltage pulses were applied from a holding potential of −100 mV at a frequency of 0.2 Hz, from −80 to +60 mV at 5-mV intervals. Unless stated otherwise, the data were sampled at 5 kHz and filtered at 1 kHz. Experiments were performed at room temperature (20–22 °C). Activation parameters were estimated from the peak I-V curves obtained for each channel combination and are reported as the means of individual measurements ± S.E. as described elsewhere (48). Briefly, the I-V relationships were normalized to the maximum amplitude and were fitted to a Boltzmann equation with E_0.5,act being the midpotential of activation. The free energy of activation was calculated using the midactivation potential with Equation 1,

where z is the effective charge displacement during activation, and T, F, and R have their usual meaning (49). The r₅₀ ratio is defined as the ratio of peak whole cell currents remaining 50 ms later (I_{50 ms}/I_Peak). It has been used elsewhere to estimate inactivation kinetics of Ca_V2.3 (48, 50, 51).

For each novel mutant tested, the biophysical parameters of Ca_V2.3 WT + Ca_Vβ3 WT were measured the same day under the same experimental conditions from the transfection protocol up to the bath solutions. Experiments performed under the same conditions yielded peak current densities ± 15% between samples and between series of experiments. All of the experiments were pooled and are reported in Tables 1 and 2.

TABLE 2.

Biophysical properties of Ca_V2.3 channels in the presence of Ca_Vα2δ-1

Ca_V2.3 wild-type channels were expressed in HEKT cells with Ca_Vβ3 WT or constructs with Ca_Vα2δ-1. Biophysical parameters were measured in the presence of a physiological saline containing 2 mm Ca²⁺ as described elsewhere (17). Activation properties (E_0.5,act and ΔG_act) were estimated from the mean I-V relationships and fitted to a Boltzmann equation. The data are shown as the means ± S.E. of the individual experiments, and the number of experiments appears in parentheses.

CaV2.3 in HEKT cells 2 mm Ca2+	Electrophysiological properties
	E0.5,act	ΔGact	Peak density	r50 at +10 mV
	mV	kcal mol−1	pA/pF
+ CaVα2δ-1	−4.0 ± 0.7 (10)	−0.44 ± 0.07 (10)	−12 ± 2 (10)	0.43 ± 0.02 (9)
+ CaVβ3 WT + CaVα2δ-1	−9.1 ± 0.2 (95)	−1.3 ± 0.1 (95)	−65 ± 5 (95)	0.25 ± 0.01 (95)
+ CaVβ3 P175A + CaVα2δ-1	−9.5 ± 0.9 (8)	−1.6 ± 0.3 (8)	−82 ± 21 (8)	0.30 ± 0.01 (8)
+ CaVβ3 M196A + CaVα2δ-1	−8.6 ± 0.8 (9)	−1.1 ± 0.1 (9)	−41 ± 9 (9)	0.39 ± 0.02 (9)
+ CaVβ3 M195A + CaVα2δ-1	−10 ± 1 (8)	−1.6 ± 0.3 (8)	−157 ± 25 (8)	0.41 ± 0.01 (8)
+ CaVβ3 K198A + CaVα2δ-1	−7.7 ± 0.2 (9)	−0.97 ± 0.04 (9)	−56 ± 9 (9)	0.26 ± 0.01 (9)
+ CaVβ3 L200G + CaVα2δ-1	−8.6 ± 0.5 (11)	−1.02 ± 0.07 (11)	−43 ± 6 (11)	0.29 ± 0.02 (11)
+ CaVβ3 S295A + CaVα2δ-1	−8.1 ± 0.5 (10)	−1.6 ± 0.3 (10)	−71 ± 12 (10)	0.33 ± 0.01 (10)
+ CaVβ3 R302G + CaVα2δ-1	−8.1 ± 0.3 (11)	−1.1 ± 0.1 (11)	−67 ± 11 (11)	0.36 ± 0.01 (11)
+ CaVβ3 L303G + CaVα2δ-1	−5.3 ± 0.5 (8)	−0.60 ± 0.06 (8)	−47 ± 10 (8)	0.34 ± 0.03 (8)
+ CaVβ3 R307G + CaVα2δ-1	−10.6 ± 0.6 (9)	−1.5 ± 0.1 (9)	−128 ± 20 (9)	0.44 ± 0.01 (9)
+ CaVβ3 R307A + CaVα2δ-1	−13 ± 1 (10)	−1.7 ± 0.2 (10)	−98 ± 21 (10)	0.40 ± 0.02 (10)
+ CaVβ3 R307K + CaVα2δ-1	−12.1 ± 0.7 (10)	−1.7 ± 0.2 (10)	−130 ± 28 (10)	0.38 ± 0.01 (10)
+ CaVβ3 L337G + CaVα2δ-1	−6.5 ± 0.6 (8)	−0.80 ± 0.09 (8)	−82 ± 10 (8)	0.33 ± 0.02 (8)
+ CaVβ3 E339G + CaVα2δ-1	−6.3 ± 0.6 (10)	−0.78 ± 0.08 (10)	−67 ± 12 (10)	0.43 ± 0.01 (10)
+ CaVβ3 L342G + CaVα2δ-1	−7.2 ± 0.4 (8)	−0.90 ± 0.07 (8)	−78 ± 19 (8)	0.39 ± 0.01 (8)
+ CaVβ3 L200G/L303G + CaVα2δ-1	−7.1 ± 0.7 (11)	−0.9 ± 0.1 (11)	−10 ± 2 (11)	0.34 ± 0.04 (11)
+ CaVβ3 L200G/L303G/L337G + CaVα2δ-1	−6.6 ± 0.7 (11)	−0.81 ± 0.09 (11)	−10 ± 2 (11)	0.41 ± 0.03 (11)
+ CaVβ3 L200G/L303G/L337G/L342G + CaVα2δ-1	−8.2 ± 0.5 (12)	−0.94 ± 0.07 (12)	−9 ± 1 (12)	0.37 ± 0.04 (12)
+ CaVβ3 Δ57–123 (ΔNSH3) + CaVα2δ−1	−8.4 ± 0.5 (9)	−1.1 ± 0.1 (9)	−117 ± 16 (9)	0.24 ± 0.01 (9)
+ CaVβ3 Δ57–180 + CaVα2δ-1	−5.3 ± 0.7 (9)	−0.6 ± 0.1 (9)	−9 ± 1 (9)	0.43 ± 0.03 (9)
+ CaVβ3 Δ122–169 + CaVα2δ-1	−5 ± 1 (9)	−0.6 ± 0.1 (9)	−38 ± 7 (9)	0.19 ± 0.01 (9)
+ CaVβ3 Δ122–175 + CaVα2δ-1	−5.6 ± 0.7 (10)	−0.6 ± 0.1 (10)	−32 ± 7 (10)	0.22 ± 0.01 (10)
+ CaVβ3 Δ175–180 + CaVα2δ-1	−3.4 ± 0.5 (9)	−0.36 ± 0.05 (9)	−14 ± 2 (9)	0.45 ± 0.02 (9)
+ CaVβ3 Δ57–123 Δ170–175 (ΔSH3) + CaVα2δ-1	−6.6 ± 0.7 (13)	−0.9 ± 0.2 (13)	−74 ± 15 (13)	0.27 ± 0.01 (13)

Western Blots

Protein expression of all constructs was confirmed by Western blotting in total cell lysates as previously described (17) using the following primary antibodies: anti-Ca_V2.3 (Alomone; 1:250), anti-HA (Covance Biotechnology, Québec, Canada) (1:500), anti-Ca_Vβ3 (Alomone; 1:5000), and anti-GAPDH (Sigma 1:10000).

Homology Modeling

The primary sequence of the AID region from Ca_V2.3 was aligned with the similar regions in Ca_V1.2 (58% identity) by using T-Coffee (52). The computer-based molecular model of the AID from Ca_V2.3 in complex with Ca_Vβ3 was obtained with Modeler 9v4 using the molecular coordinates of Ca_V1.2 with Ca_Vβ3 (Protein Data Bank code 1VYT) (27). Fifty models were generated. Violations of spatial restraints were minimized using the molecular protein density function algorithm in Modeler. The model with the lowest molecular protein density function was used for this study.

RESULTS

α-Interacting Domain and Cell Surface Expression of Ca_V2.3

Co-expression of Ca_V2.3 with Ca_Vβ3 increased whole cell currents in HEKT cells from −6 ± 1 pA/pF (n = 18) (Table 1) for the wild-type Ca_V2.3 channel expressed alone to a current density of −18 ± 2 pA/pF (n = 49) for Ca_V2.3 + Ca_Vβ3. Similar results were obtained for the HA-tagged Ca_V2.3 channels (Table 1). Co-expression with Ca_Vα2δ-1 subunit further increased whole cell currents to −65 ± 5 pA/pF (n = 95) (Fig. 1, A and B, and Table 2), confirming that Ca_Vα2δ-1 increases whole cell current density of HVA Ca_V1 and Ca_V2 channels (16, 17). The increase in cell current density was accompanied by an apparent acceleration of the inactivation kinetics with r₅₀ values decreasing from 0.38 ± 0.01 (n = 49) to 0.25 ± 0.01 (n = 95) at +10 mV in the presence of Ca_Vα2δ-1 (Fig. 1C) but failed to significantly shift the midpotential of activation (E_0.5,act) toward negative potentials (Tables 1 and 2), an observation that was also reported elsewhere (47).

FIGURE 1.

Ca_Vβ3 stimulated whole cell currents and cell surface density of Ca_V2.3. A, whole cell current traces recorded after the transient expression of the Ca_V2.3 WT channel in HEKT cells. The charge carrier was 2 mm Ca²⁺. From left to right are shown Ca_V2.3 WT alone, Ca_V2.3 WT + Ca_Vα2δ-1, Ca_V2.3 WT + Ca_Vβ3, and Ca_V2.3 WT + Ca_Vβ3 + Ca_Vα2δ-1. A series of 150-ms voltage pulses were applied between −80 mV and +60 mV (5-mV steps) from a holding potential of −100 mV. B, peak current densities are plotted as a function of applied voltage. Channels Ca_V2.3 WT + Ca_Vβ3 ± Ca_Vα2δ-1 showed a typical voltage-dependent activation with a mean current density of −6 ± 1 pA/pF (n = 18) for Ca_V2.3 WT alone as compared with a current density of −18 ± 2 pA/pF (n = 49) obtained after transient overexpression of Ca_V2.3 WT + Ca_Vβ3. Overexpression of Ca_V2.3 WT + Ca_Vβ3 + Ca_Vα2δ-1 increased the mean current density to −65 ± 5 pA/pF (n = 95). Ca_Vβ3 hyperpolarized the activation potential of Ca_V2.3 from E_{0.5, act} = −5 ± 1 mV (n = 18) for Ca_V2.3 alone to E_{0.5, act} = −10.7 ± 0.3 mV (n = 49) for Ca_V2.3 + Ca_Vβ3 channels. The numerical values can be found in Table 1. C, the r₅₀ values (the fraction of peak whole cell currents remaining after a 50-ms pulse) are shown as the means ± S.E. from −10 to +20 mV for Ca_V2.3 WT + Ca_Vβ3 and Ca_V2.3 WT + Ca_Vβ3 + Ca_Vα2δ-1 (from left to right on the bar graph). The r₅₀ values of Ca_V2.3 WT + Ca_Vβ3 ± Ca_Vα2δ-1 were significantly different at p < 0.0001 at all voltages. D, HA-tagged Ca_V2.3 WT was expressed transiently either in the HEKT cell line or in the stable Ca_Vβ3 cell line. Cell surface expression of HA-Ca_V2.3 WT was determined in intact cells by flow cytometry using the anti-HA FITC conjugate antibody. 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 shown). As seen, only co-expression with Ca_Vβ3 significantly promoted membrane expression of Ca_V2.3 (p < 0.001). Co-expression of Ca_V2.3 with Ca_Vα2δ-1 did not alter the number of Ca_V2.3 at the membrane as compared with control conditions (p > 0.1). Co-expression with both auxiliary subunits did not further improve the membrane expression of Ca_V2.3 as compared with the Ca_V2.3 + Ca_Vβ3 condition. The numerical values can be found in Tables 1–3.

Flow cytometry assays carried out with the HA-tagged Ca_V2.3 in the stable Ca_Vβ3 confirmed that Ca_Vβ is the critical auxiliary subunit in stimulating the plasma membrane density of Ca_V2.3 (Fig. 1D). Roughly 40% of the cells transfected with HA-Ca_V2.3 and Ca_Vβ3 were fluorescent, suggesting that a substantial fraction of Ca_V2.3 proteins remained in cytoplasmic compartments as noted for Ca_V1.2 (17, 20). Ca_Vβ1a, Ca_Vβ1b, and Ca_Vβ4 isoforms were as proficient as Ca_Vβ3 in chaperoning Ca_V2.3 to the membrane, but the palmitoylated Ca_Vβ2a did not appear to be as powerful as the other Ca_Vβ subunits (Fig. 2 and Tables 1 and 3).

FIGURE 2.

Ca_Vβ isoforms stimulated whole cell currents and cell surface density of Ca_V2.3. A, Ca_V2.3 WT was expressed transiently in the presence of Ca_Vβ isoforms. Whole cell current traces recorded after the transient expression of the Ca_V2.3 WT channel in the presence of Ca_Vβ1a, Ca_Vβ1b, Ca_Vβ2a, and Ca_Vβ4 isoforms in HEKT cells. The charge carrier was 2 mm Ca²⁺. Ca_Vα2δ was omitted. B, cell surface expression of HA-Ca_V2.3 WT was determined in intact cells by flow cytometry using the anti-HA FITC conjugate antibody. The histogram shows the number of fluorescent cells as a function of the Ca_Vβ isoforms. C, the r₅₀ values indicate that inactivation kinetics were significantly slower for Ca_V2.3 + Ca_Vβ2a. The numerical values are shown in Tables 1–3.

There was no significant increase in cell fluorescence when overexpressing Ca_Vα2δ-1 or calmodulin wild type alone with Ca_V2.3 (Table 3). Because the whole cell current density results from the NP_oΔ_i product, where N is the number of proteins at the membrane, P_o is the open channel probability, and Δ_i is the single channel conductance, these data suggest that Ca_Vα2δ-1 improves the open channel probability rather than increasing the number of channels at the membrane as shown for Ca_V1.2 (17, 20). The strong functional modulation by Ca_Vα2δ-1 contrasts with its milder effects on the total Ca_V2.3 protein density, especially when compared with the robust increase conferred by Ca_Vβ3 (Fig. 3).

FIGURE 3.

Ca_Vα2δ-1 mildly improves half-life of Ca_V2.3 upon arrest of cellular protein synthesis. HEKT cells were transiently transfected simultaneously with the indicated constructions. Exactly 24 h after transfection, subconfluent cells were incubated with cycloheximide (100 μg/ml) for up to 48 h, to block de novo protein synthesis. At the indicated time points (0 h or no cycloheximide, 6 h, 12 h, 24 h, 36 h, and 48 h), cell lysates were prepared and fractionated by SDS-PAGE (10%), followed by immunoblotting to visualize Ca_V2.3 (Alomone; 1:250), Ca_Vβ3 (Invitrogen; 1:1000), Ca_Vα2δ-1 (Alomone; 1:250), and GAPDH (Sigma; 1:10,000). The protein density of Ca_V2.3 in total membrane lysates was expressed relative to GAPDH and normalized to the protein density measured at time 0. The transient increase in Ca_V2.3 protein density measured at 6 and 12 h in the presence of Ca_Vβ3 was not statistically significant (p > 0.01). As seen, Ca_Vβ3 was more effective than Ca_Vα2δ-1 in preventing the degradation of Ca_V2.3, but Ca_Vα2δ-1 increased the lifetime of Ca_V2.3 as compared with the same protein expressed alone. The histograms were produced using values obtained from two different series of experiments and estimated with Image J. A, HA-Ca_V2.3 with the mock vector. B, HA-Ca_V2.3 with Ca_Vβ3. C, HA-Ca_V2.3 with Ca_Vα2δ-1. D, HA-Ca_V2.3 with Ca_Vβ3 and Ca_Vα2δ-1. The molecular mass of Ca_V2.3 is 250 kDa; that of Ca_Vα2δ-1 is 175 kDa; that of Ca_Vβ3 is 60 kDa; and that of GAPDH is 40 kDa.

To examine which AID residues (Fig. 4A) are required to promote cell surface expression of Ca_V2.3, conserved and nonconserved residues in the AID were mutated and functionally expressed in HEKT cells. Cell surface expression were maintained with Ca_V2.3 G382A, Y383A, and Y383F (Fig. 4, B and C), although the cell surface density decreased significantly for Y383G. The Ca_Vβ3 modulation of activation gating and inactivation kinetics of Y383F and Y383A mutants was not significantly different from Ca_V2.3 WT (Fig. 4D and Table 1). Nonetheless, W386A failed to migrate to the plasma membrane. Hence, only the most severe reduction in the binding affinity as seen with W386A (26) was found to disrupt plasma membrane density.

FIGURE 4.

A, primary sequence of the AID locus in the I-II linker of Ca_V2.3. The residues that were mutated are underlined. B, HA-tagged Ca_V2.3 WT and mutants were expressed transiently with Ca_Vβ3 WT + Ca_Vα2δ-1. Whole cell currents were recorded in a 2 mm Ca²⁺ solution. From left to right are shown the typical whole cell current traces obtained for HA-Ca_V2.3 WT, HA-Ca_V2.3 Y383F, HA-Ca_V2.3 Y383A, and HA-Ca_V2.3 R384M. C, HA-tagged Ca_V2.3 was expressed transiently in the HEKT cell line (hatched bars) or in the stable Ca_Vβ3 cell line (dark gray bars). Cell surface expression of Ca_V2.3 WT and mutants was determined in intact cells by flow cytometry using the anti-HA FITC conjugate antibody. The pattern of cell surface expression decreased in the order HA-Ca_V2.3 WT ≈ G382A ≈ Y383F > R384M ≈ R384L ≈ Y383A > Y383G ≫ W386A for the transient overexpressed Ca_Vβ3. D, the r₅₀ values for Ca_V2.3 mutants expressed with Ca_Vβ3 WT and alternatively the Ca_Vβ3 fragment 58–362 (SH3-HOOK-GK). The r₅₀ values measured for the channel combinations displayed in B were not significantly different between 0 and +10 mV (p > 0.05).

Isothermal titration calorimetry assays (26) showed that the mutation of the nonconserved arginine residue (Arg³⁸⁴) located between the GY and WI in the AID helix increased the binding affinity of the Ca_V2.3 peptide for Ca_Vβ2a with a K_d decreasing from 54 to 8.6 nm. Ca_V2.3 R384M and R384L mutations, however, did not significantly influence the number of Ca_V2.3 proteins at the membrane, nor did they alter the Ca_Vβ3 modulation of channel gating (Fig. 4 and Table 1).

The SH3 and HOOK Domains of Ca_Vβ Are Not Essential for Ca_Vβ-mediated Modulation

We next addressed the importance of the different structural domains of Ca_Vβ in the Ca_Vβ-induced modulation of cell surface density and channel gating of Ca_V2.3. Co-expression with the SH3-HOOK-GK fragment of Ca_Vβ3 (in amino acids 58–362) boosted cell surface expression and generated whole cell currents with biophysical parameters not significantly different from the wild-type version of Ca_Vβ3 (Fig. 5 and Table 1), suggesting that the C-terminal does not contribute significantly to functional modulation in contrast to results obtained with Ca_Vβ2a (19).

Dissection of the SH3-HOOK-GK fragment (amino acids 57–362) along structural boundaries yielded truncated constructs Ca_Vβ ΔNSH3 (Δ57–123), Ca_Vβ ΔHOOK (Δ122–169), Ca_Vβ Δβ5 sheet (Δ170–175) (formally part of the SH3), and Ca_Vβ ΔGK (Δ180–364) (Fig. 6). Except for the latter construct, each Ca_Vβ construct increased surface density of Ca_V2.3 (Fig. 7 and Table 4) and produced channels with typical voltage-dependent activation gating (Table 1). The integrity of the Ca_Vβ deletion constructs were confirmed by Western blots (data not shown).

FIGURE 6.

A, schematic diagram of the domain organization of the Ca_Vβ3 subunit based on the crystal structure and adapted from (27) and used in (17). B, homology model of AID from Ca_V2.3 in complex with Ca_Vβ3 highlighting structural regions in Ca_Vβ3 and the number of amino acids marking the boundaries of the regions herein studied. The AID from Ca_V2.3 is shown as a yellow helix with the side chain of Trp³⁸⁶ protruding in red. In each panel, the region of interest is shown in violet. Panel a, Ca_Vβ3 HOOK (123–170). Panel b, Ca_Vβ3 β5 sheet (amino acids 170–175). Panel c, Ca_Vβ3 β6 sheet (amino acids 175–180). Panel d, Ca_Vβ3 α3 helix (amino acids 191–207). Panel e, Ca_Vβ3 α6 helix (amino acids 297–306). Panel f, Ca_Vβ3 α7 helix (amino acids 307–312). Panel g, Ca_Vβ3 β10 sheet (amino acids 334–337). Panel h, Ca_Vβ3 α9 helix (amino acids 343–360). The figure was produced using PyMOL (DeLano Scientific).

FIGURE 7.

A, typical whole cell current traces were recorded in a 2 mm Ca²⁺ solution for Ca_V2.3 WT in the presence of Ca_Vβ3 deleted mutants. Ca_Vα2δ was omitted. From left to right are shown Ca_Vβ3 Δ57–123 (ΔNSH3), Ca_Vβ3 Δ57–123 Δ170–175 (ΔSH3), Ca_Vβ3 Δ122–169 (ΔHOOK), Ca_Vβ3 Δ122–175 (ΔHOOK + Δβ5 sheet), and Ca_Vβ3 Δ170–175 (Δβ5 sheet). B, cell surface density of HA-tagged Ca_V2.3 WT + Ca_Vβ3 WT or mutants was estimated by a flow cytometry assay using the anti-HA FITC conjugate antibody. Co-expression with Ca_Vβ3 deleted mutants Δ57–123, Δ122–169, and Δ170–175, as well as point mutations P175G and P175A in the β5 sheet of Ca_Vβ3, promoted the cell surface expression of Ca_V2.3 to >70–90% of the level obtained with Ca_Vβ3 WT. In contrast, there was no significant increase in the surface expression with Ca_Vβ3 Δ175–180 and Ca_Vβ3 Δ195–200 as compared with Ca_V2.3 in the absence of Ca_Vβ3 (Table 4). C, mean peak current densities as a function of voltage are shown for Ca_V2.3 with either Ca_Vβ3 Δ57–123 Δ170–175 (ΔSH3), Ca_Vβ3 Δ122–169 (ΔHOOK), Ca_Vβ3 Δ122–175 (ΔHOOK + Δβ5 sheet), or Ca_Vβ3 Δ170–175 (Δβ5 sheet). Ca_Vβ3 deleted constructs successfully increased peak current densities without any significant change in the activation potential as compared with Ca_V2.3 WT + Ca_Vβ3 WT. D, the r₅₀ values are shown as means ± S.E. from −5 to +15 mV for Ca_V2.3 WT + Ca_Vβ3 WT and Ca_Vβ3 Δ57–123, Ca_Vβ3 Δ122–169, Ca_Vβ3 Δ122–175, Ca_Vβ3 Δ170–175, and Ca_Vβ3 ΔSH3 (Δ57–123 Δ170–175). Inactivation kinetics were significantly slower for the Δ170–175 (p < 0.0001) at all voltages as compared with Ca_V2.3 WT + Ca_Vβ3 WT. The numerical values are shown in Table 1.

The SH3 domain was shown to support channel endocytosis (53) and to promote calpain-mediated Ca_Vβ3 proteolysis through a PEST-like motif (54). Deletion of the N-terminal of SH3 domain (Δ57–123), deletion of the six-residue β5 strand (Δ170–175 or ΔPYDVVP, formerly known as the β-interaction domain or BID (55)), or deletion of both regions (ΔSH3) yielded channels with robust peak current density and typical voltage-dependent activation gating in the absence and in the presence of Ca_Vα2δ-1 as a background subunit (Fig. 7 and Tables 1 and 2). Deletion of the HOOK domain (Δ122–169 and Δ122–175) in Ca_Vβ3 did not significantly alter any of the biophysical parameters (activation potential, peak current density, and inactivation kinetics) (Fig. 7 and Table 1). In contrast, small six-residue deletions in the GK domain with Ca_Vβ3 Δβ6 or Δ175–180 and Ca_Vβ3 Δ195–200 failed to promote cell surface density of Ca_V2.3 channels (Fig. 7B).

Mutations of Leucine Residues in the GK Domain Decrease Plasma Membrane Density

A mutational analysis was thus carried to seek out single residues in the GK domain that are responsible for modulation of gating and cell surface density of Ca_V2.3. We focused on residues of Ca_Vβ3 that were highly likely to interact with Trp³⁸⁶ and Ile³⁸⁷ residues in Ca_V2.3 (38) based upon the changes in the energetics of interaction between the AID peptide from Ca_V1.2 and Ca_Vβ2a (26), as well as from the predictions of the homology model (Fig. 6). Note that the GK domains of Ca_Vβ2a and Ca_Vβ3 are well conserved with more than 87% identity in this stretch of 188 amino acids. Residues in Ca_Vβ3 were either substituted by glycine to minimize side chain interactions or substituted with alanine to preserve the α-helicoidal structure.

Nearly 20 point mutations were tested in the GK domain of Ca_Vβ3, namely in the β6 sheet (P175A, P175G, P179A, and P179G); in the α3 helix (M195A, M196A, K198A, and L200G); in the β9 sheet (S295A); in the α6 helix (R302G, L303G, R307A, R307G, and R307K); in the β10 sheet (E339G, L337G, and N340G); and in the α9 helix (L342G and A345G). All Ca_Vβ3 mutations produced functional Ca_V2.3 channels (Tables 1, 2, and 4). Most notably, Ca_Vβ3 P175A (β6), Ca_Vβ3 M196A (α3), Ca_Vβ3 S295A (β9), Ca_Vβ3 R302G (α6), and Ca_Vβ3 E339G (β10) behaved like Ca_Vβ3 WT. These Ca_Vβ mutants stimulated cell surface density, boosted whole cell peak current density, and imparted the small but significant negative shift in the activation potential of Ca_V2.3 (Tables 1, 2, and 4).

Mutations at position Arg³⁰⁷ (R307A, R307G, and R307K) in the α6 helix significantly increased peak current densities as compared with Ca_Vβ3 WT (Tables 1 and 2). In addition, the Ca_Vβ3 Arg³⁰⁷ mutants decreased the inactivation kinetics, suggesting that the open state was stabilized by these mutants. These effects were observed despite a documented decrease in the affinity of the equivalent R356A mutation in Ca_Vβ2a (equivalent to Arg³⁰⁷ in Ca_Vβ3) with a K_d increasing from 5 to 345 nm (26).

In contrast, point mutations of four leucine residues (L200G, L303G, L337G, and L342G) significantly decreased cell surface density of Ca_V2.3 by ≈50–60% (Fig. 8). The three-dimensional homology model suggests that the four leucine residues in Ca_Vβ3 could form a hydrophobic pocket surrounding Ca_V2.3 Trp³⁸⁶ and Ca_V2.3 Ile³⁸⁷ (Fig. 9). Note that the strongest effects were observed with Ca_Vβ3 L303G, which is predicted to interact with Ile³⁸⁷ in Ca_V2.3. Multiple point mutations were constructed to test the hypothesis that the four leucine residues form a single interaction site. The double, triple, and quadruple Ca_Vβ3 mutants L200G/L303G, L200G/L303G/L347G, and L200G/L303G/L337G/L342G Ca_Vβ3 mutants all ablated cell surface density of Ca_V2.3 (Table 4). Furthermore, the peak current densities measured with these Ca_Vβ3 mutants were not statistically different (p > 0.1) from the densities measured in the absence of Ca_Vβ3 (Tables 1 and 2). Finally, the normalized protein density of Ca_V2.3 measured in membrane lysates was significantly decreased with Ca_Vβ3 L303G as compared with Ca_Vβ3 WT but was nearly abolished with the quadruple Ca_Vβ3 leucine mutant L200G/L303G/L337G/L342G (Fig 10). In the context where the fluorescence level measured in our FACS assay results from the net balance between anterograde and retrograde trafficking, this observation suggests that the decreased protein density at the plasma membrane could result from a decrease in the Ca_V2.3 total protein density.

FIGURE 8.

A, typical whole cell current traces were recorded in a 2 mm Ca²⁺ solution for Ca_V2.3 WT in the presence of the Ca_Vβ3 mutants. Ca_Vα2δ-1 was omitted. From left to right are shown Ca_Vβ3 L200G, Ca_Vβ3 L303G, Ca_Vβ3 L337G, and Ca_Vβ3 L342G. B, HA-tagged Ca_V2.3 was expressed transiently in the HEKT cell line with Ca_Vβ3 mutants. Cell surface expression was determined in intact cells by flow cytometry. Ca_Vβ3 mutants L200G, L303G, L337G, and L342G curtailed membrane protein expression by 50–60%. Multiple mutations L200G/L303G (L1,2G), L200G/L303G/L337G (L1,2,3G), and L200G/L303G/L337G/L342G (L1,2,3,4G) abolished the Ca_Vβ stimulation of cell surface density (Table 4). C, normalized peak current densities are plotted as a function of applied voltage. All of the Ca_Vβ3 mutants produced Ca_V2.3 currents with similar voltage-dependent activation (Table 1). D, the r₅₀ values are shown ± S.E. from −10 to +20 mV for Ca_V2.3 WT in the presence of Ca_Vβ3 WT, Ca_Vβ3 L200G, Ca_Vβ3 L303G, Ca_Vβ3 L337G, and Ca_Vβ3 L342G (from left to right on the bar graph). Inactivation kinetics were significantly slower for Ca_Vβ3 L342G at all voltages (p < 0.01).

FIGURE 9.

Homology model of the 18-residue-long AID region from Ca_V2.3 in complex with Ca_Vβ3. The quartet of leucine residues in Ca_Vβ3 surrounding the AID helix is shown in three different orientations. The Ca_Vβ3 subunit is in green, and the AID helix is shown in yellow with the protruding side chains of Trp³⁸⁶ and Ile³⁸⁷ shown in red and the side chains of the four leucine residues displayed in violet. A, with the AID region shown horizontally and Ca_V2.3 W386A facing down. B, with the AID region shown horizontally and Ca_V2.3 W386A facing up. C, with the AID region shown vertically.

FIGURE 10.

Ca_V2.3 protein density decreased with Ca_Vβ3 Leu mutants. A, representative Western blot analyses of HEKT cells transiently co-transfected with pCDNA3-Ca_V2.3-HA and pCMV-Tag5-Ca_Vβ3 WT and mutants using anti-Ca_V2.3 (Alomone; 1:500), anti-Ca_Vβ3 (Invitrogen; 1:10,000), and anti-GAPDH (Sigma; 1:10,000) antibodies. Each lane was loaded with 20 μg of protein. Lane 1, Ca_V2.3-HA + Ca_Vβ3 WT. Lane 2, Ca_V2.3-HA + Ca_Vβ3 L303G. Lane 3, Ca_V2.3-HA + Ca_Vβ3 L200G/L303G. Lane 4, Ca_V2.3-HA + Ca_Vβ3 L200G/L303G/L337G. Lane 5, Ca_V2.3-HA + Ca_Vβ3 L200G/L303G/L337G/L342G. B, protein density of Ca_V2.3 was normalized using GAPDH as an internal loading control and estimated using Image J. Maximum density was obtained for Ca_V2.3 and Ca_Vβ3. The histogram was produced using values obtained from two different gels.

DISCUSSION

There is considerable interest in identifying molecules that modulate protein-protein interactions in vivo. In this regard, specifically modulating the interaction of Ca_Vβ with HVA Ca_V channels could be a strategy to design new HVA Ca_V agonists and antagonists. Residues of Ca_Vβ carrying nanomolar affinity binding onto the Ca_Vα1 of Ca_V1.2 have been identified from high resolution three-dimensional crystal structures and isothermal calorimetry assays (26–29). Although mutations of residues in Ca_Vα1 abolishing the protein-protein interaction are incompatible with Ca_V1.2 function (17, 26), there has been some divergence regarding the functional importance of the domains in Ca_Vβ subunits (26, 27, 33, 39–41). The range of approaches and recombinant systems could explain in part the diverse conclusions. We have thus systematically addressed the role of Ca_Vβ in channel function based upon the structural data currently available. Our results show that protein-protein affinity measured in vitro may not be the only predictor of channel modulation especially in the context where Ca_Vβ subunits control surface density and prevent degradation of Ca_Vα1 subunits from Ca_V1 and Ca_V2 channels by the ERAD complex.

Site Occupancy Is Sufficient to Modulate Protein Density and Gating

It is well understood that high affinity binding of Ca_Vβ onto the AID motif appears to be a prerequisite for both Ca_Vβ-induced modulation of gating and Ca_Vβ-stimulated plasma membrane trafficking of Ca_Vα1 (17, 22, 56, 57). In our hands, mutations within the AID of Ca_V2.3 that were shown to moderately increase (R384M and R384L) or decrease (G382A, Y383A, and Y383F) binding affinity (26) did not alter the Ca_Vβ3 modulation of cell surface density and hyperpolarization of gating. Ca_Vβ mutations or Ca_V2.3 mutations (Y383A) that moderately reduced the number of Ca_V2.3 proteins at the membrane produced channels with biophysical properties (peak current density, activation gating, and inactivation kinetics) similar to Ca_V2.3 WT + Ca_Vβ3 WT control channels. This result is remarkably reminiscent of the Y383S mutation in Ca_V2.2 that decreased the affinity of Ca_Vβ1b binding to the I-II linker of Ca_V2.2 from a K_d value of 14 nm to one of 329 nm while preserving its effect on current density and cell surface expression in HEKT cells (23).

Nonetheless, complete disruption of this interaction with Ca_V2.3 W386A, Ca_Vβ3 Δ175–180, or Ca_Vβ3 Δ195–200 abrogated cell surface labeling of Ca_V2.3, which evidently translated in the absence of function. This is a surprising observation given that the reverse observation was reported in the Xenopus expression system. AID-deficient Ca_V2.3 channels migrated to the plasma membrane but were not functionally modulated by Ca_Vβ when expressed in Xenopus oocytes (37, 38). It certainly raises questions about the transposition of data from Xenopus oocytes to mammalian cells.

Partial Deletions of the GK Domain of Ca_Vβ3 Disrupt Plasma Membrane Density of Ca_V2.3

The deletion of the HOOK region and thus the absence of intramolecular coupling between the SH3 and GK in Ca_Vβ did not impact significantly the voltage-dependent activation gating or the inactivation kinetics of Ca_V2.3 despite the report that the variable HOOK domain plays a role in the inactivation kinetics of Ca_Vβ2a (40). Our observation also seems to contradict a previous report that strong SH3-GK intramolecular coupling conferred by short linkers (<3 amino acids) confers fast inactivation kinetics (19, 35). It also disagrees with the observation that the β5 sheet in the SH3 domain of Ca_Vβ2a plays an important role in the modulation of Ca_V2.1 channels (35). Discrepancies can be attributed to the idiosyncrasy of the Ca_Vβ2a subunit and/or the Xenopus expression system used in those studies. In our hands, complete deletion of the SH3 or the HOOK domains in Ca_Vβ3 failed to abrogate either its chaperone function or its modulation of channel gating.

Leucine Residues in the GK Domain of Ca_Vβ3 Determine Cell Surface Density of Ca_V2.3

Close to 20 point mutations were performed in the GK domain of Ca_Vβ3. The majority of the mutations did not alter the Ca_Vβ-induced stimulation of cell surface density and hyperpolarization of gating despite a documented decrease in the binding affinity (26). Among the Ca_Vβ mutations herein tested, four mutations (M196A, L303G, R307A, and L342G) were expected to decrease significantly the binding affinity of Ca_Vβ3 to Ca_V2.3 (26). Ca_Vβ2a M245A (equivalent to Ca_Vβ3 M196A) yielded a 200-fold decrease in the affinity for Ca_V1.2 (26), with the K_d increasing from 5 nm to 1.1 μm. Nonetheless, whole cell currents of Ca_V2.3 obtained in the presence of Ca_Vβ3 M196A were not significantly different from those obtained with Ca_Vβ3 WT both in terms of peak current density, voltage dependence of activation, and inactivation kinetics. In contrast, mutations of leucine residues at positions 200, 303, 337, and 342 (either individually or in combination) significantly reduced the modulation of Ca_V2.3 function and its cell surface density when compared with Ca_Vβ3 WT. Remarkably, the affinities measured between the AID peptide of Ca_V1.2 and the Ca_Vβ2 mutants L352A and L392A (equivalent to Ca_Vβ3 L303 and L342) were ≈20-fold lower than for Ca_Vβ2 WT but still 100-fold higher than with Ca_Vβ2 M245A (26). Our functional screen thus discriminated residues in Ca_Vβ3 that were initially believed to behave similarly based upon their binding energies.

It remains possible that the binding affinities of the Ca_V2.3 + Ca_Vβ3 complex cannot be simply extrapolated from the in vitro binding energies measured with Ca_Vβ2a bound onto the AID peptides from Ca_V1.2 channels (26). A recent study performed with larger peptides (≈30–40 residues) points to important structural differences between the highly conserved I-II linkers of Ca_V1.2 and Ca_V2.2 (13).

With these reservations in mind, our results altogether suggest that nanomolar protein-protein affinity may not be the sole determinant of the modulation of Ca_Vα1 channel function by Ca_Vβ. Given that Ca_V2.3 W386A was not measured at the membrane but that Ca_Vβ3 M196A sustained surface density of Ca_V2.3 proteins, it can be concluded that functional modulation occurs in the nano- to micromolar range. Even with a moderate affinity, “occupancy of the AID site” could be sufficient to carry the Ca_Vβ-induced modulation of channel function in HVA Ca_V1 and Ca_V2 channels (23). In this scheme, occupancy of the AID site by Ca_Vβ could either unmask retention signals for protein targeting or mask ubiquitination sites on the Ca_Vα1 subunit and prevents its degradation by the proteasome.

Conclusion

Mutations of four leucine residues (Leu²⁰⁰, Leu³⁰³, Leu³³⁷, and Leu³⁴²) were each expected to moderately decrease binding affinity, significantly decreasing the cell surface density of Ca_V2.3 protein. Simultaneous mutation of the four leucine residues completely abolished surface and total protein density of Ca_V2.3. We propose that these four leucine residues form a hydrophobic pocket that is required to promote van der Waals interactions with Trp³⁸⁶ and Ile³⁸⁷ (WI pair) in the AID region of Ca_V2.3. Hence, the four leucine residues in the GK domain of Ca_Vβ3 and the WI pair in the AID of Ca_V2.3 appear to be essential determinants in the modulation of high voltage-activated calcium channel function by Ca_Vβ auxiliary subunits.