Phosphorylation Sites in the Hook Domain of Ca_Vβ Subunits Differentially Modulate Ca_V1.2 Channel Function

Abstract

Regulation of L-type calcium current is critical for the development, function, and regulation of many cell types. Ca_V1.2 channels that conduct L-type calcium currents are regulated by many protein kinases, but the sites of action of these kinases remain unknown in most cases. We combined mass spectrometry (LC-MS/MS) and whole-cell patch clamp techniques in order to identify sites of phosphorylation of Ca_Vβ subunits in vivo and test the impact of mutations of those sites on Ca_V1.2 channel function in vitro. Using the Ca_V1.1 channel purified from rabbit skeletal muscle as a substrate for phosphoproteomic analysis, we found that Ser¹⁹³ and Thr²⁰⁵ in the HOOK domain of Ca_Vβ_1a subunits were both phosphorylated in vivo. Ser¹⁹³ is located in a potential consensus sequence for casein kinase II, but it was not phosphorylated in vitro by that kinase. In contrast, Thr²⁰⁵ is located in a consensus sequence for cAMP-dependent phosphorylation, and it was robustly phosphorylated in vitro by PKA. These two sites are conserved in multiple Ca_Vβ subunit isoforms, including the principal Ca_Vβ subunit of cardiac Ca_V1.2 channels, Ca_Vβ_2b. In order to assess potential modulatory effects of phosphorylation at these sites separately from effects of phosphorylation of the α₁1.2 subunit, we inserted phosphomimetic or phosphoinhibitory mutations in Ca_Vβ_2b and analyzed their effects on Ca_V1.2 channel function in transfected nonmuscle cells. The phosphomimetic mutation Ca_Vβ_2b^S152E decreased peak channel currents and shifted the voltage dependence of both activation and inactivation to more positive membrane potentials. The phosphoinhibitory mutation Ca_Vβ_2b^S152A had opposite effects. There were no differences in peak Ca_V1.2 currents or voltage dependence between the phosphomimetic mutation Ca_Vβ_2b^T164D and the phosphoinhibitory mutation Ca_Vβ_2b^T164A. However, calcium-dependent inactivation was significantly increased for the phosphomimetic mutation Ca_Vβ_2b^T164D. This effect was subunit-specific, as the corresponding mutation in the palmitoylated isoform, Ca_Vβ_2a, had no effect. Overall, our data identify two sites of conserved phosphorylation of the HOOK domain of Ca_Vβ subunits in vivo and reveal differential modulatory effects of phosphomimetic mutations in these sites. These results reveal a new dimension of regulation of Ca_V1.2 channels through phosphorylation of the HOOK domains of their β subunits.

1. Introduction

Protein kinases regulate L-type calcium currents conducted by voltage-gated (Ca_V) channels to precisely control numerous physiological functions of nerve and muscle cells. Ca_V1.1 and Ca_V1.2 channels are regulated by a number of protein kinases (PKA, CaMKII, AKT/PKB, PKC and PKG) [1, 2]. PKA phosphorylation is required for up-regulating L-type calcium currents through Ca_V1.1 and Ca_V1.2 channels in the fight-or-flight response [2-5], and CaMKII is essential for frequency-dependent potentiation [6]. Dysregulation of protein kinases is implicated in many pathological conditions [7-9]. Despite its importance in physiology and pathophysiology, the molecular mechanisms of regulation of Ca_V1.1 and Ca_V1.2 channels by protein phosphorylation remain incompletely understood.

Ca_V1.1 and Ca_V1.2 channels in nerve and muscle cells consist of a pore-forming α₁ subunit in association with Ca_Vβ, Ca_Vα2δ, and possibly Ca_Vγ subunits [2, 10, 11]. Calmodulin (CaM), a ubiquitous Ca binding protein, is also associated with this protein complex [12]. Ca_Vα₁ subunits are composed of four homologous domains (I-IV) with six transmembrane segments (S1-S6) and a reentrant pore loop in each [2]. Multiple regulatory sites are located in the large C-terminal domain of Ca_V1.1 and Ca_V1.2 channels [13-16], which is subject to in vivo proteolytic processing near its center [13, 17-19]. An IQ motif in the proximal C-terminus is implicated in Ca/calmodulin-dependent inactivation [14, 15]. Noncovalent interaction of the distal C-terminus with the proximal C-terminal domain has an auto-inhibitory effect by reducing coupling efficiency of gating charge movement to channel opening [16, 20, 21], and the proximal C-terminus EF-hand is required to mediate the auto-inhibitory effect of the distal C-terminus [22]. Recently, it was shown that this autoinhibitory Ca_V1.2 signaling complex with an A Kinase Anchoring Protein bound is sufficient to recapitulate the stimulatory actions of PKA on Ca_V1.2 channels in a non-muscle cell system [23]. This reconstituted regulatory system has allowed functional tests of the role of phosphorylation sites in the α1 subunits in calcium channel regulation.

In our previous studies, we took advantage of the ease of purification of Ca_V1.1 channels from rabbit skeletal muscle to identify sites of in vivo phosphorylation of the α1 subunits [24]. We then used our reconstituted regulatory system to analyze the functional effects of mutations in the homologous sites in the Ca_V1.2 channel, which are highly conserved. We found that two conserved sites located at the interface between the distal and proximal C-terminal domains were required for regulation of basal and PKA-stimulated channel activity [23]. Both a PKA site at Ser¹⁷⁰⁰ and a casein kinase II site at Thr¹⁷⁰⁴ were required for normal regulation of basal channel activity, whereas only Ser¹⁷⁰⁰ was required for stimulation of channel activity by PKA [23]. These results suggest that PKA phosphorylation of Ca_V1.2 at Ser¹⁷⁰⁰ relieves the autoinhibition of the distal C-terminal on Ca_V1.2 channel function allowing the PKA-dependent increase in current amplitude. Mice with mutations in Ser¹⁷⁰⁰ and Thr¹⁷⁰⁴ have greatly reduced basal L-type calcium currents and much reduced response to β-adrenergic stimulation [25, 26], as expected from these studies in transfected nonmuscle cells.

Phosphorylation sites in Ca_Vβ subunits were identified previously by a variety of biochemical and proteomic techniques [18, 27-29], but the level of phosphorylation in vivo, and the physiological significance of these phosphorylation sites remain uncertain. In the experiments described here, we have used mass spectrometry (LC-MS/MS) of purified skeletal muscle Ca_V1.1 channels for phosphoproteomic analysis and whole-cell patch clamp studies of the expressed Ca_V1.2 channel for functional analysis. With this approach, we identified two in-vivo phosphorylation sites in the Hook domain of Ca_Vβ subunits. Phosphomimetic and phosphoinhibitory mutations in the analogous sites differentially modulate the function of full-length Ca_V1.2 channels expressed in nonmuscle cells.

2. Experimental Procedures

2.1. Protein purification, and sample preparation

All animal procedures were conducted in compliance with the recommendations of the Institutional Animal Care and Use Committee of the University of Washington. Rabbit skeletal muscle Ca_V1.1 channels were purified as previously described [30]. Male New Zealand White rabbits (2.5 kg, Western Oregon Rabbit Co) were euthanized by lethal injection of pentobarbital, skeletal muscle was rapidly harvested, rinsed briefly in phosphate buffered saline (PBS, pH 7.4), snap frozen in liquid nitrogen, and stored at −80°C. All purification steps were carried out at 4°C in the presence of the following protease and phosphatase inhibitors: 1 μM aprotinin, 1 μM pepstatin, 10 μM leupeptin, 100 μM benzamidine, 1 mM phenanthroline, 10 μM E-64, 20 μg/μL soybean trypsin inhibitor, 0.5 mM NaVO₄, 100 nM microcystin, 10 nM cyclosporin A. Ca_V1.1 was purified using wheat germ agglutinin linked agarose (Vector Labs) and anion exchange DEAE Sephadex A-25 (GE Healthcare), chromatography [30, 31]. Pure protein was snap frozen in liquid nitrogen and stored at −80°C in single-use aliquots. For mass spectrometric analysis, 1-2.5 μg of pure protein was separated by SDS-PAGE, the band of interest was excised and subjected to in-gel trypsin (Gold, Promega) digestion by standard procedures (http://donatello.ucsf.edu/ingel.html); 1-3 pmol of tryptic peptides were analyzed by mass spectrometry.

2.2. In vitro phosphorylation

Pure Ca_V1.1 was first dephosphorylated with 5 U Calf Intestinal-alkaline Phosphatase (CIP, NEB) for 2 h at 30°C in assay buffer (20 mM Tris pH7.5, 10 mM MgCl₂, 2 mM DTT, 10 mM NaCl, 200 mM ATP) and phosphatase was quenched with 10 mM NaVO₄. Then Ca_V1.1 was phosphorylated with 100 U PKA (Sigma, from Bovine heart), 100 U CaMKII (NEB), or 25 U CK2 (NEB) for 2.5 h at 30°C in assay buffer (20 mM Tris pH 7.5, 10 mM MgCl₂, 2 mM DTT, 10 mM NaCl, 200 mM ATP). Reactions were quenched, proteins were resolved by SDS-PAGE, subjected to in-gel digestion, and phosphate incorporation was assessed by LC-MS and LC-MS/MS.

2.3 Mass spectrometry

Mass spectrometry was performed using an Agilent 1100 series HPLC (Agilent Technologies) coupled to an LCQ Classic ion trap mass spectrometer (ThermoElectron). Peptides were loaded on to a Paradigm Platinum Peptide Nanotrap (Microm) then separated on a reverse-phase capillary column (10 cm × 75 μm, Jupiter Proteo C12, Phenomenex) with a linear gradient from 2%-40% acetonitrile in 40 min. One full mass scan was acquired (300-2000 Da) and then the four most intense peaks were selected for MS/MS analysis. The dynamic exclusion limit was set to exclude a given m/z after it had been sequenced 2x during a 90 sec interval. Mass spectra were analyzed using TurboSequest configured with the following parameters: a peptide mass tolerance of 2.5 Da (avg), a fragment ion mass tolerance of 1.0 Da (avg), differential modification on S/T/Y +80 Da, and allowance of two incomplete cleavages. All MS/MS peak assignments were manually validated. The relative abundance of the modified forms for each peptide was determined using the ICIS peak detection method (Xcalibur) for the indicated extract ions. The percentages were calculated based on the sum of integrated peak areas for all detected modified forms of the peptide.

2.4. cDNA constructs

To construct mutants Ca_Vβ_2b^S152A/E and Ca_Vβ_2b^T164A/D mutagenic primers were designed that contained an internal HindIII restriction site. Ca_Vβ_2b^S152A/E and Ca_Vβ_2b^T164A/D were all amplified in a two-phase manner, using a SacII-XhoI cDNA fragment of Ca_Vβ_2b or Ca_V1.2 in pBluescript SK+. Phase I generated the 5′ and the 3′ arms using the WT DNA template. PCR products were gel purified. Phase II combined the 5′ and 3′ arms to serve as both initial primers and template. PCR products were precipitated, washed, dried, and resuspended. PCR products and vector were cut with Sac II and Xho I and isolated by ethanol precipitation. The digested vector was treated with calf alkaline phosphatase (CIP, NEB). Digested DNA was run out on a 1.25% agarose gel. Fragment bands were excised and purified either by Spin-X column (Fisher Scientific) or QIAQuick (QIAGEN). Fragments were ligated (Fast-Link, Epicentre). A negative control of vector and no insert was run at the same time. Ligation mixtures were then transformed into competent DH5 cells and samples were plated onto LB plates overnight. DNA was extracted from mutant colonies by basic SDS/sodium acetate method. Samples were screened by cutting with restriction enzymes whose recognition sites were silently built into the mutation primer. Positive samples were further purified (Quantum miniprep kit, Bio-Rad Laboratories) and sequenced (BigDye, ABI). When the sequences were confirmed to be correct, DNA from the original mini-prep was retransformed, amplified, and extracted (QUANTUM maxiprep kit, Bio-Rad Laboratories). Each mutant DNA in pBluescript SK+ (Stratagene) and the full-length WT Ca_Vα1.2 subunit in pCDNA3 (Stratagene) were digested with Sac II and Xho I, ligated, and subcloned. Samples were sequenced again to confirm the sequence of the final construct.

2.5. Cell culture

TsA-201 cells were grown to 80% confluence and transfected with an equimolar ratio of cDNA encoding Ca_V1.2 full-length (FL), Ca_Vβ_2b or mutated Ca_Vβ_2b (Ca_Vβ_2b^S152A/E and Ca_Vβ_2b^T164A/D, Ca_Vα₂δ₁, and CD8 as a cell surface marker (EBO-pCD-Leu2; American Type Culture Collection) using Fugene (Roche). 15–24 h after transfection, cells were suspended, plated at low density in 35-mm dishes, and incubated at 37°C in 10% CO₂ for at least 17 h before recording using the whole-cell configuration of the patch clamp technique. Transiently transected cells were visualized with latex beads conjugated to an anti-CD8 antibody (Invitrogen).

2.6. Electrophysiology

Patch pipettes (2.5–3.5 MΩ) were pulled from micropipette glass (VWR Scientific) and fire-polished. Currents were recorded with an Axopatch 200B amplifier (MDS Analytical Technologies) and sampled at 5 kHz after anti-alias filtering at 2 kHz. Data acquisition and command potentials were controlled by Pulse (Pulse 8.50; HEKA), and data were stored for off-line analysis. Voltage protocols were delivered at 10s intervals unless otherwise noted, and leak and capacitive transients were subtracted using a P/4 protocol. Approximately 80% of series resistance was compensated with the voltage clamp circuitry.

For whole-cell voltage clamp recordings of Ca_V1.2 current in tsA-201 cells with Ba²⁺ or Ca²⁺ as a charge carrier (I_CaV1.2(Ba/Ca)), the extracellular bath solution contained (in mM): 10 BaCl₂ (or 1.8 mM CaCl₂), 140 Tris, 2 MgCl₂, and 10 D-glucose, titrated to pH 7.3 with MeSO₄. The intracellular solution contained (in mM): 130 CsCl, 10 HEPES, 4 MgATP, 1 MgCl₂, and 10 EGTA titrated to pH 7.3 with CsOH [32]. When Na⁺ was used as charge carrier (I_CaV1.2(Na)), the extracellular solution contained (in mM): 150 NaCl, 10 HEPES, 0.2 MgCl₂, 0.25 µM EDTA, and 10 D-glucose titrated to pH 7.3 with MeSO₄. The intracellular solution contained (in mM): 150 CsOH, 110 glutamate, 20 HCl, 10 HEPES, 5 TrisATP, 4.3 MgCl₂, and 10 EGTA titrated to pH 7.6 with CsOH [32].

2.7. Data analysis

Voltage-clamp data were compiled and analyzed using Igor (IGOR Pro version 5.0, Wavemetrics Inc.) and Excel (Excel 97, Microsoft). For measurement of Ca_V1.2 I-V relationship and voltage dependence of activation, peak step currents were measured from a 20 ms depolarization to potentials between −50 and 80 mV, while tail currents were measured during the subsequent repolarization to −40 mV. For the measurement of the voltage dependence of inactivation, 4-s depolarizations to potentials from −80 to 30 mV were applied to inactivate a fraction of Ca_v1.2 channels. A standard test pulse of 30 ms to 30 mV was applied, and peak tail currents were measured during repolarization to −40 mV immediately following the test pulse. Activation and inactivation data were fit to a Boltzmann function (Prism 5.0d, GraphPad Software Inc.). To normalize for variation in transfection efficiency currents were normalized to the gating charge (Q_on)[33]. For Ca_V1.2 inactivation, currents were elicited by test pulses between −80 mV to 30 mV of 1000 ms duration. To quantify inactivation, peak currents elicited by 1000 ms depolarizations to 0 mV were normalized to 1.0, and the fraction of peak current remaining at the end of the voltage pulse (r₁₀₀₀) was measured. Pulses were applied every 30 s.

All data are presented as mean ± SEM. The statistical significance of differences between the various experimental groups was evaluated using the Student’s t test or one-way ANOVA, followed by the Newman-Keuls post-test; p-values are presented in the text.

Results

3.1. Sites of phosphorylation of Ca_Vβ_1a subunits

Ca_V1.1 channels in partially purified preparations of skeletal muscle transverse tubule membranes were labeled with [³H]isradipine, solubilized with digitonin, and purified by chromatography on wheat germ agglutinin-Sepharose and DEAE-Sepharose [30, 31, 34]. The purified protein was concentrated by re-chromatography on wheat germ agglutinin-Sepharose, and the subunits were separated by SDS-PAGE (Fig. 1A). The band at approximately 55 kDa containing the Ca_Vβ_1a subunit was excised, extracted, and prepared for mass spectrometry as described under Experimental Procedures (Fig. 1A). Analysis of the mass spectra revealed phosphorylation of Ser¹⁹³ in the peptide Ser¹⁸⁶-Arg²⁰², which has a molecular mass of 820.4 without phosphorylation and 860.4 after phosphorylation (Fig. 2). In addition, we observed phosphorylation of Thr²⁰⁵ in the peptide Arg²⁰³-Lys²⁴⁰ (Fig. 3). Both of these sites are located in the HOOK domain, which connects the SH3 domain and the guanylate kinase domain (Fig. 1B). The functional role of the HOOK domain is unknown.

Figure 1. Sites of phosphorylation of Ca_Vβ_1a in Ca_V1.1 channels in skeletal muscle.

A. Ca_V1.1 channels were isolated from rabbit skeletal muscle as described in Experimental Procedures. Subunits were separated by SDS-PAGE and stained with Commassie blue. The protein band containing Ca_Vβ_1a was excised as illustrated and used for analysis by mass spectrometry. B. Alignment of the amino acid sequences of the Ca_Vβ subunits with secondary structure motifs indicated. Stars, Ser¹⁹³ and Thr²⁰⁵.

Figure 2. Identification of phosphorylated Ser¹⁹³ by mass spectrometry.

Ca_Vβ_1a was isolated and analyzed as described in Experimental Procedures. The MS/MS spectrum of the phosphorylated tryptic peptides of Ca_Vβ_1a (Parent MH²⁺ 860.4) is illustrated. For clarity, only the relevant portion of the spectrum is shown.

Figure 3. Identification of phosphorylated Thr²⁰⁵ by mass spectrometry.

Ca_Vβ_1a was isolated and analyzed as described in Experimental Procedures. The MS/MS spectrum of the phosphorylated tryptic peptides of Ca_Vβ_1a (Parent MH³⁺ 1385.3) is illustrated. For clarity, only the relevant portion of the spectrum is shown.

Ser¹⁹³ is in the amino acid sequence -SSLGD/E-, which is conserved in all four Ca_Vβ subunits (Fig. 1B). The analogous sequence in Ca_Vβ_2b, the most abundant isoform in mammalian heart [35], contains the potential phosphorylation target Ser¹⁵². The consensus sequence SXXD/E is a potential site for phosphorylation by casein kinase II (www.phosphosite.org). However, the level of phosphorylation of this site in vivo was low, and casein kinase II treatment did not increase phosphorylation of this peptide in purified Ca_V1.1 channels in vitro (Fig. 4). This site may be phosphorylated at a low level by another kinase in vivo, or it may only be phosphorylated by casein kinase II under specific physiological conditions in vivo.

Figure 4. Phosphorylation of Ser¹⁹³ by casein kinase II.

Purified Ca_V1.1 channel was phosphorylated by purified casein kinase II as described in Experimental Procedures. The Ca_Vβ_1a subunit was isolated by SDS-PAGE and analyzed by MS/MS. Integrated intensities of the peptide peaks representing unphosphoryated Ca_Vβ_1a (0P) and phosphorylated Ca_Vβ_1a (1P) are illustrated.

Thr²⁰⁵ is in the amino acid sequence -RRTP-, which is a consensus for phosphorylation by PKA (Fig. 5). The Hook domain of Ca_Vβ₁ was previously shown to be phosphorylated by PKA at Thr²⁰⁵ in vitro in purified preparations of skeletal muscle calcium channels [18], and bioinformatic analysis suggests that Ca_Vβ₁^T205 is within a strong consensus sequence for PKA/CaMKII/PKC phosphorylation [18, 27]. This site is conserved in Ca_Vβ1, Ca_Vβ2, and Ca_Vβ4, although the consensus is less optimal for phosphorylation in Ca_Vβ4 because one of the two Arg residues is replaced by Phe (Fig. 1B). Incubation of purified Ca_V1.1 channels with PKA in vitro substantially increased the phosphorylation of Thr²⁰⁵ (Fig. 5), consistent with a significant physiological role of phosphorylation of this site. This sequence is conserved as -RKST- in Ca_Vβ_2b with Thr¹⁶⁴ as the phosphorylated residue.

Figure 5. Phosphorylation of Thr²⁰⁵ by PKA.

Purified Ca_V1.1 channel was phosphorylated by purified PKA as described in Experimental Procedures. The Ca_Vβ_1a subunit was isolated by SDS-PAGE and analyzed by MS/MS. Integrated intensities of the peptide peaks representing unphosphoryated Ca_Vβ_1a (0P) and phosphorylated Ca_Vβ_1a (1P) are illustrated.

3.2. Modulation of Ca_V1.2 channel activity by mutation of Ser¹⁵² in Ca_Vβ_2b

Ca_V1.2 channels have multiple sites of phosphorylation on their pore-forming α₁ subunits, which complicates interpretation of experiments that manipulate phosphorylation of Ca_Vβ subunits by activation of protein kinases. Moreover, the kinase(s) that phosphorylate Ser¹⁹³/Ser¹⁵² in vivo are unknown. Therefore, to probe potential functional effects for phosphorylation of this site in Ca_V1.2 channel regulation, we created phosphomimetic and phosphoinhibitory mutants by substitution of Glu and Ala, respectively, for Ser¹⁵². We expressed Ca_V1.2 channels with the Ca_Vβ_2b mutants Ca_Vβ_2b^S152A and Ca_Vβ_2b^S152E in human embryonic kidney tsA-201 cells and processed them for electrophysiological analysis. The phosphoinhibitory mutation, Ca_Vβ_2b^S152A, increased Ca_V1.2 channel current amplitude, whereas phosphomimetic mutation, Ca_Vβ_2b^S152E, decreased Ca_V1.2 current amplitude relative to Ca_Vβ_2b^WT (Fig. 7A). The Ca_V1.2 current amplitudes were −6.49 ± 0.9 pA/fC (n = 8) and −2.95 ± 0.88 pA/fC (n = 6) (P < 0.05), for Ca_Vβ_2b^S152A and Ca_Vβ_2b^S152E, respectively. The phosphomimetic mutation Ca_Vβ_2b^S152E also shifted the I/V relationship and the voltage dependence of activation and inactivation to more positive membrane potentials, whereas the phosphoinhibitory mutation Ca_Vβ_2b^S152A had the opposite effects (Fig. 6A-C). The activation V_1/2 values were −11.1 ± 2.2 mV (n = 8) and 7.5 ± 5.2 mV (n = 6) (P < 0.05), for Ca_Vβ_2b^S152A and Ca_Vβ_2b^S152E, respectively, whereas the activation V_1/2 for Ca_Vβ_2b^WT was intermediate at −1.0 ± 2.7 mV (n = 12). Similar results were observed with these phosphomimetic mutations inserted into Ca_Vβ_1b. Overall, these results suggest that introduction of negative charge at this novel phosphorylation site in the HOOK domain of Ca_Vβ subunits acts as a gating modifier to reduce opening probability of Ca_V1.2 channels and to impede the voltage-induced activation and inactivation gating of Ca_V1.2 channels.

Figure 7. Ca_Vβ_2b Hook-domain phosphorylation at Thr¹⁶⁴ does not alter current amplitude, voltage dependence of activation or inactivation.

A. Effect of “phosphorylation mimetics” of Ca_Vβ_2b^T164 on Ca_V1.2 I-V relationship. Ca²⁺ was the charge carrier and in order to normalize for Ca_V1.2 channel expression we divided the current amplitude by gating current (Q_on)[32]. B. Ca_Vβ_2b^T164D and Ca_Vβ_2b^T164A did not alter the voltage dependence ofactivation of Ca_V1.2 channels, or C. voltage dependence of inactivation of Ca_V1.2 channels. Ca_Vβ_2b^WT (n = 8, 8, 4), Ca_Vβ_2b^T164A (n = 6, 6, 4) and Ca_Vβ_2b^T164D (n = 9, 9, 6) for panel A, B and C, respectively. Activation (Act.) and inactivation (Inact.).

Figure 6. Ca_Vβ_2b Hook-domain phosphorylation at Ser¹⁵² attenuates Ca_V1.2 current and shifts to the right the voltage dependence of activation of Ca_V1.2 channels.

A. Effect of “phosphorylation mimetics” of a novel CK2 phosphorylation site in Ca_Vβ_2b^S152 on Ca_V1.2 channel I-V relationship. Ba²⁺ was the charge carrier and in order to normalize for Ca_V1.2 channel expression we divided the current amplitude by gating current (Q_on)[32]. Inset, representative normalized typical current traces of Ca_Vβ_2b^S152A, Ca_Vβ_2b^S152E, and Ca_Vβ_2b^WT taken at the peak of the I-V relationship (calibration bar: 5 ms and 2.5 pA/fC. The dotted line represents zero current level). B. Ca_Vβ_2b^S152A shifts to the left the voltage dependence of activation of Ca 1.2 channels while Ca_Vβ_2b^S152E shifts to the right the voltage dependence of activation of Ca 1.2 channels relative to Ca_Vβ_2b^WT. C. Ca_Vβ_2b^S152A shifts to the left the voltage dependence of inactivation of Ca_V1.2 channels while Ca_Vβ_2b^S152E shifted to the right the voltage dependence of inactivation of Ca_V1.2 channels relative to Ca_Vβ_2b^WT. Activation and inactivation parameters were: Ca_V1.2 FL^WT: V_1/2 = −1.0 ± 2.7 mV, k = −12.1 ± 1.0, n = 12 and V_1/2 = −28.0 ± 1.0 mV, k = 8.7 ± 0.6, n = 11; Ca_Vβ_2b^S152A: V_1/2 = − 11.1 ± 2.2 mV, k = −11.5 ± 2.2, n = 8 and V_1/2 = −35.0 ± 2.3 mV, k = 7.8 ± 0.5, n = 8; and Ca_Vβ_2b^S152E: V_1/2 = 7.5 ± 5.2 mV, k = − 11.7 ± 1.8, n = 6 and V_1/2 = −22.7 ± 3.0 mV, k = 12.3 ± 0.8, n = 6 for activation and inactivation parameters, respectively. There is a significant difference in V_1/2 of activation (≈18 mV, P < 0.05) and V_1/2 SS-inactivation (≈12 mV, P < 0.01) between Ca_Vβ_2b^S152A and Ca_Vβ_2b^S152A.

3.3. Modulation of Ca_V1.2 channel activity by mutation of Ca_Vβ_2b Thr¹⁶⁴

To probe the role of phosphorylation of Thr¹⁶⁴ in Ca_V1.2 channel regulation, we expressed Ca_V1.2 channels with the phosphoinhibitory and phosphomimetic mutations Ca_Vβ_2b^T164A and Ca_Vβ_2b^T164D in tsA-201 cells and performed electrophysiological analysis in the presence of the physiological charge carrier Ca²⁺ (1.8 mM). These mutations did not have strong effects on the I/V relationship or the voltage dependence of activation and inactivation (Fig. 7). However, we observed enhanced rate and extent of inactivation of Ca_V1.2 channels with WT and Ca_Vβ_2b^T164D subunits compared to Ca_Vβ_2b^T164A subunits (Fig. 8A and B). Analysis of two-exponential fits of these results showed that these effects were primarily on the second time constant for inactivation (Table 1). The results for WT Ca_Vβ were similar to T164D, suggesting that this site is at least partially phosphorylated when Ca²⁺ currents are measured in normal physiological medium. We also engineered this mutation in Ca_Vβ_2a to test if this effect would be present with a Ca_Vβ subunit that confers decreased inactivation to Ca_V1.2 channels because of its membrane tethering by N-terminal palmitoylation [36]. Ca_V1.2 channels with Ca_Vβ_2a^T164A or Ca_Vβ_2a^T164D mutants had similar inactivation properties (Fig. 8C and D; Table 1). These results suggest that negative charge at Ca_Vβ_2b^T164 enhances the transition of Ca_V1.2 channels from open to inactivated state by accelerating the second rate constant governing this inactivation transition and that N-terminal palmitoylation of the Ca_Vβ_2a subunit [36] overrides this effect.

Figure 8. Ca_Vβ_2b Hook-domain phosphorylation at Thr¹⁶⁴ enhanced Ca²⁺-dependent inactivation (CDI) of Ca_V1.2 channels.

A. “Phosphorylation mimetics” Ca_Vβ_2b^T164D enhances Ca_V1.2 channel inactivation. Currents were elicited by 1000 ms steps to 0 mV from a holding potential of −80 mV with Ca²⁺ as charge carrier. Average traces are represented. The dotted line in panels A, C, and E represent zero current level. Ca_Vβ_2b^T164D: n = 12 and Ca_Vβ_2b^T164A: n = 10. B. Bar graph of mean r₁₀₀₀ values measured with Ca_Vβ_2b^T164D and Ca_Vβ_2b^T164A were significantly different (* P < 0.01). C. Effects of Ca_Vβ_2aT164D and Ca_Vβ_2a^T164A on Ca_V1.2 channel inactivation. Ca_Vβ_2a^T164D: n = 4; Ca_Vβ_2a^T164A: n = 4. D. Bar graph of mean r₁₀₀₀ values measured with Ca_Vβ_2a^T164D and Ca_Vβ_2a^T164A were not significantly different (NS). E. Effect of “phosphorylation mimetics” of Ca_Vβ_2a^T164 on Ca_V1.2 channel inactivation with Na⁺ as charge carrier. Ca_Vβ_2b^T164D: n = 11; Ca_Vβ_2a^T164A: n = 8. F. Bar graph of mean r₁₀₀₀ values measured with Ca_Vβ_2a^T164A with Na⁺ as charge carrier were not significantly different.

Table 1.

	A1		t1 (msec)		A2		t2 (msec)		N
CaVb2b
WT	0.53	±0.03	612	±54	0.45	±0.05	155	±37	13
T164A	0.34	±0.05	688	±77	0.29	±0.03	132	±24	10
T164D	0.56	±0.06	626	±155	0.37	±0.02	146	±34	12
CaVb2a
T164A	0.27	±0.07	528	±331	0.27	±0.09	56	±15	4
T164D	0.21	±0.04	834	±89	0.33	±0.04	76	±5	4

Both voltage-dependent inactivation and Ca²⁺-dependent inactivation contribute substantially to the inactivation of Ca_V1.2 channels when Ca²⁺ is the charge carrier. In order to assess the effects of the Ca_Vβ_2b^T164 mutations on voltage-dependent inactivation of Ca_V1.2 channels specifically, we used Na⁺ as charge carrier [32]. We did not observe any significant difference in voltage-dependent inactivation between Ca_Vβ_2b^T164D and Ca_Vβ_2b^T164A (Fig. 8E and F). These results suggest that negative charge at the position of Ca_Vβ_2b^T164 enhances the Ca²⁺-dependent transition of Ca_V1.2 channels to the inactivated state with little effect on voltage-dependent inactivation. The results for WT Ca_Vβ_2b revealed slightly faster inactivation (Fig. 8E and F), as if the hydroxyl group of the native Ser at this position makes an interaction that slightly enhances the rate of voltage-dependent inactivation, and this interaction is prevented in both mutants.

4. Discussion

4.1. nctional effects of phosphorylation sites in the C-terminal of Ca_Vβ subunits

Previous studies have identified multiple sites of phosphorylation in the C-terminal domain of Ca_Vβ subunits by protein kinases in vitro [18, 27-29]. PKA phosphorylates three sites in the C-terminal domain of Ca_Vβ_2b subunits, and phosphorylation of these sites was reported to regulate the function of Ca_V1.2 channels expressed in nonmuscle cells [27]. Phosphatidylinositol-3 kinase phosphorylates a site in the C-terminal domain of the Ca_Vβ₂ subunit and regulates trafficking of the calcium channel complex to the plasma membrane [28]. Protein kinase G also phosphorylates a site in the C-terminal domain of the Ca_Vβ_2b subunit and inhibits the activity of Ca_V1.2 channels expressed in nonmuscle cells [10]. Although these studies suggest essential functional roles for the C-terminal domain of Ca_Vβ₂ subunits, deletion of the C-terminal domain including all three proposed sites of regulatory phosphorylation has no effect in mice [6]. The resulting C-terminal knockout mice are viable, fertile, and have no apparent physiological deficits [6]. Moreover, stimulation of the L-type calcium current in ventricular myocytes from these mice is normal [6]. Thus, these C-terminal phosphorylation sites on Ca_Vβ₂ subunits do not have essential functional roles that have been demonstrated in vivo. It is not known whether other Ca_Vβ subunits can effectively compensate for loss of regulation by phosphorylation at these sites in the Ca_Vβ₂ subunit, but Ca_Vβ subunits do compensate for each other when the complete genes are deleted [37]. Further work is required to determine whether the functional effects of phosphorylation of the C-terminal domain of Ca_Vβ₂ subunits observed in vitro in transfected nonmuscle cells are also important in vivo.

4.2. Phosphorylation sites in the Hook domain of Ca_Vβ_2b subunits

Unexpectedly, our mass spectrometry analysis did not detect phosphorylation of the C-terminal domain of the Ca_Vβ_1a subunit, but we found two previously unidentified sites of in-vivo phosphorylation in the Hook domain: Thr²⁰⁵ and Ser¹⁹³. These sites are located far from the site of interaction of Ca_Vβ subunits α₁ subunits (Fig. 9, AID) and far from the previously identified sites of phosphorylation in the C-terminal region. Thr²⁰⁵ was identified as a PKA phosphorylation site by in vitro phosphorylation with [γ³²P]ATP in early biochemical studies of purified Ca_V1.1 channels [18], and we confirmed those results with mass spectrometry in this work. Ser¹⁹³ was not previously identified as a site of protein phosphorylation of Ca_Vβ_1a in biochemical studies, and the kinase that is responsible for its phosphorylation in vivo remains unknown.

Figure 9. Structural model of the Ca_Vβ subunit.

The SH3 domain (yellow), HOOK domain (purple), Alpha Interaction Domain (AID, red), and Guanylate Kinase domain (GK, green) are illustrated in the indicated colors.

Functional studies of Ca_V1.1 channels are made difficult by poor cell-surface expression in nonmuscle cells. Therefore, we analyzed the potential functional effects of phosphorylation of the homologous Ser¹⁵² and Thr¹⁶⁴ residues through studies of mutants of the Ca_Vβ_2b subunit co-expressed with Ca_V1.2 channels in human embryonic kidney tsA-201 cells. The functional effects of phosphomimetic and phosphoinhibitory mutations at these sites suggest complementary regulatory roles. Addition of a negative charge at Ser¹⁵² decreases peak Ca_V1.2 channel currents and shifts both activation and inactivation to more positive membrane potentials. Together, these effects would significantly decrease channel activity. Because co-expression of Ca_Vβ_2b subunits causes a negative shift in the voltage dependence of channel activation [38], addition of negative charge at this position by protein phosphorylation would oppose this functional effect of the Ca_Vβ_2b subunit. In contrast, addition of negative charge at the position of Thr¹⁶⁴ in Ca_Vβ_2b subunits has no effect on the voltage dependence of activation or inactivation, but it significantly increases the rate of calcium-dependent inactivation. These results suggest possible interactions between the Hook domain in the Ca_Vβ_2b subunit and the proximal C-terminal domain of the Ca_Vα₁ subunit where calcium-dependent inactivation is mediated by interaction of calcium/calmodulin with an IQ motif [15].

4.3. Possible physiological significance of phosphorylation of the Hook domain

Our results revealing functional effects of phosphomimetic mutations in the Hook domain are in agreement with previous work showing that the Hook domain of Ca_Vβ can regulate Ca_V channel inactivation [39]. Deletion of the Hook domain of Ca_Vβ_2a enhanced Ca_V2.2 channel inactivation suggesting that the Hook domain normally interacts with Ca_V2.2 α-subunit to impede Ca_V2.2 channel inactivation [39]. The Ca_Vβ Hook domain was also shown to regulate the binding affinity of Gβγ to Ca_Vβ subunits to regulate Ca_V2.2 channel activity [40]. It is possible that phosphorylation sites in the Hook domain could regulate the binding affinity of Gβγ for Ca_Vβ subunits and thereby modulate their effects on Ca_V2.2 channel activity because the interaction sites for Gβγ and Ca_Vβ-subunits are located close to each other in the intracellular linker connecting domains I and II of Ca_Vα₁ subunits [41-45].

Determining the significance of phosphorylation of the Hook domain of Ca_Vβ-subunits for regulation of basal and PKA-stimulated activity of Ca_V1.2 channels is an important aim for future experiments, but will require a detailed analysis of the complex signaling network that controls the activity of this channel. The basal activity of Ca_V1.2 channels expressed as an autoinhibitory signaling complex with bound AKAP and PKA in nonmuscle cells is much reduced by mutations that prevent phosphorylation of Ser¹⁷⁰⁰ and Thr¹⁷⁰⁴, which are located at the interface between the distal and proximal C-terminal domains of the Ca_Vα₁ subunit [23, 24]. Ser¹⁷⁰⁰ is a substrate for phosphorylation by PKA and CaMKII, whereas Thr¹⁷⁰⁴ is a substrate for casein kinase II [23, 24]. If phosphorylation of Ser¹⁵² in Ca_Vβ_2b inhibits Ca_V1.2 channel activity, as implied by our results, this effect would oppose up-regulation of basal channel activity by phosphorylation at Ser¹⁷⁰⁰ and Thr¹⁷⁰⁴ in the Ca_Vα₁ subunit.

β-adrenergic stimulation of the heart in the fight-or-flight response leads to both increased contractility, which is caused by increased peak L-type calcium currents in atrial and ventricular myocytes, and to increased beating rate, which is caused jointly by activation of hyperpolarization- and cyclic nucleotide-gated channels and Ca_V1.3 channels in the sino-atrial node [46, 47]. PKA stimulation of Ca_V1.2 channels expressed as an autoinhibitory signaling complex in nonmuscle cells is blocked by mutations that prevent phosphorylation of Ser¹⁷⁰⁰ in the Ca_Vα₁ subunit [23], and mutation of Ser¹⁷⁰⁰ and Thr¹⁷⁰⁴ in mice greatly reduces the response to β-adrenergic stimulation in ventricular myocytes [25, 26]. If phosphorylation of Thr¹⁶⁴ in the Ca_Vβ_2b subunit has no effect on peak current, as implied by our results, this site of phosphorylation would not contribute directly to the up-regulation of Ca_V1.2 channel activity by PKA. However, increased beating rate in response to β-adrenergic stimulation of the heart requires shortening of the ventricular action potential. Enhanced activation of K_V channels by PKA phosphorylation is a well-documented mechanism contributing to shortening of the ventricular action potential during β-adrenergic stimulation of the heart [48]. However, phosphorylation of Thr¹⁶⁴ in the Ca_Vβ_2b subunit would increase the rate of calcium-dependent inactivation, shorten the L-type calcium current, and thereby contribute to shortening the plateau of the ventricular action potential. Knock-in mutations in mice will be require to rigorously test these potential physiological effects of phosphorylation of the Hook domain of Ca_Vβ_2b subunits.

Highlights.

• Ca²⁺ channel β subunits are phosphorylated on two sites in the Hook domain in vivo

• Ser¹⁵² in Ca_Vβ_2b has a casein kinase II consensus sequence; Thr 164 is a PKA site

• Phosphomimetic mutation S152E in Ca_Vβ_2b decreased peak current and shifted activation

• Phosphomimetic mutation T164D in Ca_Vβ_2b increased Ca²⁺-dependent inactivation

• Phosphorylation of sites in the Hook domain may regulate Ca_V1.2 channels in vivo