Proteolytic cleavage of the voltage-gated Ca2+ channel α₂δ subunit: structural and functional features

Abstract

By mediating depolarization-induced Ca²⁺ influx high voltage-activated (HVA) Ca²⁺ channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (α₁) and three auxiliary (α₂δ, β and γ) subunits. The α₂δ subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, α₂δ must undergo important post-translational modifications including a proteolytic cleavage that separates the extracellular α₂ from its transmembrane δ domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature α₂δ almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of α₂δ was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide reducing agent. Interestingly, these mutations significantly decreased whole-cell patch clamp currents without affecting the voltage-dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane.

Introduction

Ca²⁺ entry through voltage-activated Ca²⁺ (Ca_V) channels contributes to many physiological processes in the nervous system, including neurotransmitter release, neuronal excitability and plasticity, gene expression, neurite outgrowth, synaptogenesis and neuronal survival and differentiation. Ca_V channels have been subdivided according to their electrophysiological properties into: i) low voltage-activated (LVA or T-type) channels, and ii) high-voltage activated (HVA) channels, a class that includes the L-, N-, P/Q-, and R-types (Catterall et al., 2005; Lacinova, 2005). Molecular studies indicate that Ca_V channels of the HVA class are oligomeric complexes consisting of an ion-conducting α₁ subunit and auxiliary α₂δ, β and γ subunits (Felix, 2005; Lacinova, 2005). The auxiliary subunits modulate the trafficking and the biophysical properties of the α₁ subunit (Arikkath and Campbell, 2003; Lacinova, 2005).

The α₂δ subunit is a transmembrane protein complex that is encoded by a single gene. Since the identification of the first α₂δ subunit (Ellis et al., 1988), four genetically distinct α₂δ subunits have been described (Klugbauer et al., 1999; Qin et al., 2002). Each one of these proteins is differentially expressed in various tissues, including skeletal muscle, heart and brain. At the protein level, all four α₂δ subunits show conserved glycosylation sites, cysteine residues and predicted hydrophobicity profiles (Arikkath and Campbell, 2003; Klugbauer et al., 2003). It has been shown that heterologous expression of each of these α₂δ subunits induces an enhancement in the ionic current produced by the α₁ subunit expression, associated with an increase in the number of ligand binding sites for the channel (Felix, 1999; Arikkath and Campbell, 2003; Klugbauer et al., 2003). These data indicate that α₂δ produces an increase in cell surface expression of Ca²⁺ channels. Likewise, functional studies have revealed that the α₂δ subunits cause increases in current amplitude, faster activation and inactivation kinetics and hyperpolarizing shifts in the voltage dependence of the expressed currents (Singer et al., 1991; Welling et al., 1993; Shistik et al., 1995; Gurnett et al., 1996; Bangalore et al., 1996; Felix et al., 1997; Jones et al., 1998; Qin et al., 1998; Shirokov et al., 1998; Klugbauer et al., 1999; Hobom et al., 2000; Sandoval et al., 2004; Yasuda et al., 2004; Canti et al., 2005; Obermair et al., 2005).

Molecular studies have established that the α₂ domain of the protein is extensively glycosylated, a post-translational modification that is essential in maintaining the stability of the interaction with the α₁ subunit (Gurnett et al., 1996; 1997). It is also a major determinant in the ability of the protein to stimulate current amplitude (Gurnett et al., 1996; Sandoval et al., 2004). Likewise, it has been documented that the α₂δ subunit is translated as a precursor polypeptide that is post-translationally cleaved (De Jongh et al., 1990; Jay et al., 1991). The δ domain, which contains a single transmembrane segment, maintains the α₂ domain close to the plasma membrane. Despite proteolytic cleavage, the linkage of both domains is maintained by disulfide bridges formed between cysteine residues found in both proteins (Felix, 1999; Klugbauer et al., 2003; Lacinova, 2005). The mechanisms that underlie the proteolytic cleavage and the disulfide linkage still remain unclear.

In the present report, we used site-directed mutagenesis and heterologous expression in HEK-293 cells to localize the putative site of proteolysis within α₂δ, and examine the impact of the proteolytic processing of this auxiliary subunit on the functional activity of neuronal recombinant Ca_V channels.

Materials and methods

Site-directed mutagenesis

Amino acids 941 and 946 were substituted by site directed mutagenesis of the corresponding cDNA to prevent proteolytic cleavage of the α₂δ-1b subunit. To this end, we used the recombinant bicistronic expression plasmid PIRES/α₂δ (Sandoval et al., 2004), which carried the entire protein-coding region for the rat brain α₂δ-1b Ca²⁺ channel auxiliary subunit (GenBank accession number M86621) and for the green fluorescent protein (GFP) coupled by an internal ribosomal entry site (IRES) sequence. The point mutations were introduced with ~40-mer synthetic oligonucleotides using the Quik-Change XL-mutagenesis kit (Stratagene, La Jolla, CA, USA). Initially, a single amino acid mutation changing a glutamic acid to a glutamine residue (E944Q) was created. Next, a quadruple mutation (P4) was done on the single cDNA mutant E944Q and lastly, a sextuple mutation (P6) was obtained on the P4 cDNA mutant by using suitable mutagenic primers (Fig. 1A). cDNAs of all three mutant channel subunits were sequenced on an automated sequencer (ABIPrism310, Perkin-Elmer Applied Biosystems, Foster City, CA, USA).

Figure 1. Expression of the α₂δ subunit proteolytic site mutants in HEK-293 cells.

A) Schematic representation of the Ca_Vα₂δ auxiliary subunit. The location of the two hydrophobic domains (H1 and H2), and the signal sequence (S) are given. The amino acid residues in the putative proteolytic site of the wild-type α₂δ-1 and its mutant versions are aligned and shown above the diagram. B) Western blot analysis of membranes from HEK-293 cells expressing the wild-type, and the mutant versions of the α₂δ subunit (E944Q, P4 and P6). Membranes were prepared as described under Material and Methods. Only the sextuple mutation (P6) is insensitive to proteolysis (do not change its electrophoretic pattern after dithiothreitol-DDT-treatment). C) Representative Ba²⁺ currents through recombinant Ca_V2.2/β₃ channels obtained in HEK-293 cells co-expressing the wild-type α₂δ-1 and its proteolysis mutants. Currents were elicited in response to 140 ms test pulses to +10 mV delivered from a holding potential of −80 mV.

Cell culture and recombinant Ca_V channel expression

Human embryonic kidney (HEK-293) cells were maintained in DMEM-high glucose supplemented with 10% equine serum, 1% L-glutamine, 110 mg/l sodium pyruvate and antibiotics, at 37°C in a 5% CO₂-95% air humidified atmosphere. Gene transfer was performed using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA, USA). Briefly, for a 35 mm Petri dish of HEK-293 cells, 2 μg of the plasmid cDNA encoding the rabbit brain N-type Ca²⁺ channel Ca_V2.2 pore-forming subunit (D14157; Fujita et al., 1993) in combination with 2 μg cDNA of the rat brain β₃ (M88751; Castellano et al., 1993) and 2 μg cDNA coding the wild-type rat brain α₂δ-1 or its mutants, were premixed with 6 μl of Lipofectamine in 100 μl serum-free medium according to the manufacturer’s instructions. The solution was then added to the dish and cells grown at 37°C for 24 h, when medium was changed. Cells were then harvested at 48 h.

SDS-PAGE and Western blotting

Microsomes from transfected HEK-293 cells were prepared as previously described (Felix et al., 1997; Gurnett et al., 1997). Samples were subjected to gel electrophoresis under denaturing conditions. All samples were heated at 95°C for 5 min, and 100 μg of protein/slot were loaded on 5% polyacrylamide gels. For Western blot analysis, proteins were transferred onto nitrocellulose membranes, and blots were developed as described (Felix et al., 1997; Gurnett et al., 1997) with specific Ca_Vα₂ polyclonal primary antibodies (Rabbit 136; Gurnett et al., 1996) used in a 1:4000 dilution. The secondary antibody was a goat anti-rabbit IgG horseradish peroxidase (Zymed Invitrogen, Carlsbad, CA, USA) used at a dilution of 1:6000. The specific protein bands were detected using the enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s instructions.

Electrophysiological recording

Ionic currents from HEK-293 cells were recorded using the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981) 48 h after transfection. Ba²⁺ was used as the charge carrier. The extracellular solution contained (in mM): solution contained 10 BaCl₂, 125 TEA-Cl, 10 HEPES and 10 glucose (pH 7.3). The intracellular (in mM): 110 CsCl, 5 MgCl₂, 10 EGTA, 10 HEPES, 4 Na-ATP and 0.1 GTP (pH 7.3). Recordings were made with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) and acquired on-line using a Digidata 1320A interface with pClamp8 software (Molecular Devices). The offset potential between the pipette and bath solutions was zeroed prior to seal formation. Capacitive transients were canceled with the amplifier. Series resistance values were typically 2–10 MΩ and no records were used in which the voltage error (as defined by V_er = I_max × R_a) was greater than 5 mV. Currents were obtained from a holding potential (HP) of −80 mV applying test pulses every 20 s. Leak and residual capacitance currents were subtracted on-line by a P/4 protocol. Current signals were filtered at 2 kHz (internal 4 pole Bessel filter) and digitized at 5.71 kHz. Membrane capacitance (C_m) was determined as described previously (Avila et al., 2004) and used to normalize currents. Curve fitting and statistical analysis were carried out using the SigmaPlot 8.0 software package (SPSS Inc., Chicago, IL, USA). The significance of observed differences was evaluated by nonpaired Student’s t test. P<0.05 was considered to be significant.

Results

Before investigating the functional relevance of the proteolytic cleavage of the α₂δ auxiliary subunit on neuronal recombinant Ca_V channel activity, it was necessary to delimit the putative proteolytic site within the protein. Initial N-terminal sequencing studies on the rabbit skeletal muscle Ca_V channel δ subunit suggested that cleavage of the α₂δ-1 precursor occurs between residues Glu-934 and Ala-935 (De Jongh et al., 1990). Sequence alignment of this deduced proteolytic site with the rat brain α₂δ-1b retrieved from the GenBank corroborated the presence of these residues at positions 944 and 945, respectively. Given that many deduced sites of proteolysis are of 6 amino acid residues length, and the cleavage site occurs after the fourth amino acid we speculated that the proteolytic site of the rat brain α₂δ-1b used in this study might be positioned between Arg-941 and Val-946 (Fig. 1A).

To test whether this site is indeed post-translationally modified and whether such a modification entails any functional consequence, we produced one single and two multiple mutants of the rat brain α₂δ-1b (Fig. 1A; see Material and Methods for details). All cDNA constructs were sequenced to confirm the desired mutations. The wild-type α₂δ and its mutated versions were then expressed in HEK-293 cells and analyzed by immunoblotting. This analysis showed that under non-reducing conditions the wild-type protein and the α₂δ subunits harboring the single (E944Q) and the quadruple (P4) mutations migrate as high molecular mass proteins of about 150 kDa. Upon reduction, the majority of these proteins migrated as products of the predicted size for the δ₂ peptide (~130 kDa). This result suggested that the wild-type and the mutant proteins E944Q and P4 were proteolytically cleaved into separate α₂ and δ peptides (Fig. 1B). Expression of the protein that had all six amino acids of the putative proteolitic site mutated (P6) also resulted in the presence of a high molecular mass species (~150 kDa) under non-reducing conditions which was expected. However upon reduction, the size of this high molecular weight band remained unchanged, indicating that the P6 protein is not susceptible to proteolysis (Fig. 1B).

In order to evaluate the functional role of the proteolytical processing of the α₂δ subunit, the patch clamp technique was used to study macroscopic Ba²⁺ currents (I_Ba) through recombinant Ca_V channels (Ca_V2.2/β₃) in HEK-293 cells transiently expressing the wild-type α₂δ and its mutants. As can be seen in Fig. 1C, the amplitude of the Ba²⁺ currents (I_Ba) through Ca_V2.2/β₃ channels, was enhanced by co-expression of the wild-type α₂δ-1 subunit, as previously reported (Sandoval et al., 2004; Yasuda et al., 2004). Interestingly, recordings performed in cells transfected with the single (E944Q) and the quadruple (P4) mutations, indicated that the stimulatory effect of the auxiliary subunit was preserved. In contrast, replacement of all six residues in the proteolytic site of the protein significantly decreased whole-cell patch clamp currents (Fig. 1C).

To facilitate comparisons from cells of different size, peak I_Ba amplitude was normalized to membrane capacitance (C_m) and expressed as I_Ba density (in picoamperes per picofarad). The effects of the wild-type α₂δ and its mutant variants E944Q, P4 and P6 on peak I_Ba density (determined for a test pulse to +10 mV) are summarized in Figure 2A. When compared with the wild-type, E944Q or P4 expressing cells, I_Ba density in HEK-293 cells transfected with the P6 mutant construct was significantly reduced (>52%).

Figure 2. Functional effects of heterologous expression of the wild-type α₂δ subunit and its proteolytic site mutants.

A) Average ± SEM I_Ba density in HEK-293 cells expressing the wild-type α₂δ-1 and its mutant variants (filled bars). I_Ba density was calculated from I_Ba amplitude at a test pulse of +10 mV normalized to cell membrane capacitance. The open bar shows the average current amplitude in the absence of the α₂δ subunit. The number of recorded cells is indicated in parentheses, and the asterisks denote significant differences (P<0.05). B) Average ± SEM I–V relationships for I_Ba recorded from HEK-293 cells expressing the wild-type α₂δ-1 and its mutant variants (E944Q, P4 and P6). I_Ba density was calculated at a series of test pulses applied from a holding potential of −80 mV in 10 mV steps between −50 and +80 mV (n = 6–8). Open circles represent the average current density in the absence of the α₂δ subunit.

Figure 2B compares mean ± SEM current density-voltage (I–V) relationships from HEK-293 cells expressing the wild-type α₂δ protein and the E944Q, P4 and P6 mutants. As expected, the E944Q and P4 constructs were not different than wild-type α₂δ-1 for effects on I_Ba density. In contrast, mean peak I_Ba density in cells expressing the α₂δ-1 P6 mutant was severely depressed across the entire voltage range investigated. In line with this, the macroscopic conductance (G_max) for the P6 α₂δ-1 mutant subunit, determined from the I–V relationships, was significantly decreased by ~70% compared with the Ca_V2.2/β₃/α₂δ-1 combination. In contrast, a small but no significant inhibition on G_max was observed by co-expression of the E944Q or P4 mutant subunits (Fig. 3A; Table 1). The G_max value for the P6 mutant containing channels was comparable to the G_max value obtained in cells transfected with Ca_V2.2/β₃ in the absence of the α₂δ-1 subunit (Table 1), suggesting a role for the proteolytic cleavage of α₂δ-1 in surface expression of HVA Ca²⁺ channels.

Figure 3. Activation properties of Ca_V2.2/β₃ channels co-expressed with the wild-type α₂δ-1or its proteolysis mutations.

A) Left, voltage dependence of activation of Ca_V2.2/β₃ channels co-expressed with wild-type α₂δ-1 (filled circles), its proteolysis mutants (E944Q, P4 and P6) or without any α₂δ subunit (open circles). Maximum conductance (G_max) plotted data were derived from Fig. 2B. The mean data were fitted with Boltzmann functions, the V_½ values of which are given in Table 1. Right, comparison of the mean half-activation voltage (V_1/2) for Ca_V2.2/β₃ channels co-expressed with the P6 proteolysis mutation or without the α₂δ subunit (n = 6–8). B) Mean time to peak and time constants of activation (τ_act) obtained by fitting the rising phase of I_Ba at +10 mV with a single exponential, for Ca_V2.2/β₃ channels co-expressed with wild-type α₂δ-1, its proteolysis mutants, or without any α₂δ subunit (n = 11–25).

Table 1.

Differential effects of the wild-type α₂δ subunit and its proteolysis mutants on the biophysical properties of the Ca_V2.2/δ₃ currents

	Wild-type	E944Q	P4	P6	α2δ-1 (−)
Potential for half-activation (mV)	−4.5.3±1.8	−4.0±0.9	−4.7±1.8	−3.0±2.4	−9.6±2.4
Maximal conductance at +10 mV (nS)	19.3±4.3	15.9±3.3	14.9±2.8	6.1±2.3	5.7±1.3
Potential for half steady−state inactivation (mV)	−60.2±3.6	−59.7±4.9	−56.8±3.0	−52.3±2.2	−44.6±2.1
Rate of inactivation at +10 mV (μs−1)	20.4±1.2	20.1±1.0	18.9±1.1	19.6±1.4	12.3±1.1

It is well established, that alterations in the maximal conductance in the absence of kinetic changes of the macroscopic currents may depend on variations in the number of functional channels. Therefore, we next investigated whether the effects of the P6 mutation involved alterations in the kinetic properties of the currents recorded. Normalized currents obtained from cells expressing the wild-type α₂δ and its mutants showed that the temporal course of the current traces was similar, suggesting that the activation and inactivation rate of the channels were not altered. The channel activation rate was quantitatively compared by fitting current traces to an exponential equation (Sandoval et al., 2004). Accordingly, neither the time to peak nor the time constant for the activation of the current (τ_act) were significantly modified (Fig. 3B). Likewise, we generated complete voltage-dependent inactivation curves for the recombinant Ca_V channels expressed. The parameters describing the inactivation curves were similar to the corresponding values obtained from cells expressing the wild-type α₂δ-1 (Fig. 4A; Table 1). Lastly, the time constant for the inactivation of the current (τ_inact), obtained by fitting the decaying phase of the currents with an exponential function, and the percentage of current remaining after 140 ms activating pulses were practically indistinguishable between cells expressing the wild-type α₂δ and cells expressing the mutant subunits (Fig. 4B; Table 1). Although a decrease in channel open probability cannot be ruled out, our data suggest that the proteolytic cleavage site may be required for cell surface expression.

Figure 4. Inactivation properties of Ca_V2.2/β₃ channels co-expressed with the wild-type α₂δ-1 or its proteolysis mutations.

A) Left, voltage dependence of steady-state inactivation of Ca_V2.2/β₃ channels co-expressed with wild-type Ca_Vα₂δ-1 (filled circles), its proteolysis mutants E944Q, P4 and P6 or without any α₂δ subunit (open circles). Currents were recorded after conditioning pulses of 1 s duration, applied from a holding potential of −80 mV in 10 mV steps between −110 and +40 mV, followed by a 140 ms test pulse to +10 mV. The normalized data are plotted against the conditioning potentials (n = 5–11). The mean data were fitted with a Boltzmann function, the V_½ values of which are given in Table 1. Right, comparison of the mean half-inactivation voltage (V_1/2) for Ca_V2.2/β₃ channels co-expressed with the P6 mutation or without the α₂δ subunit. B) Average time constants of inactivation (τ_inact) and percentage of current remaining 140 ms into the depolarizing pulse for Ca_V2.2/β₃ channels co-expressed with wild-type α₂δ-1, its proteolysis mutants, or without any α₂δ subunit (n = 11–25).

An interesting finding was that the average current amplitude in the presence of the P6 mutant was comparable to the magnitude of the currents in the absence of the α₂δ-1 subunit (Fig. 2). However, the waveforms and the voltage dependence of the currents in the two conditions were different (Figs. 3 and 4). Currents in the absence of any α₂δ subunits activate and inactivate significantly slower (Figs. 3B and 4B). Likewise, the half-maximal voltage for current activation and inactivation was shifted >5 mV to the hyperpolarizing direction (Figs. 3A and 4A, right panels; Table 1). Taken together, these data suggest that all mutant proteins, including P6 interact with the channel. However, only the P6 mutant is inefficient in trafficking to the cell membrane or is less stable once it reaches the membrane. In keeping with this view, the functional properties of the channels were practically unaltered (Figs. 3 and 4), and only major alterations were observed in the maximal conductance which depends on the number of functional channels in the plasma membrane. Thus conceivably, alterations in the number of Ca_V channels at the membrane might be accounting for the effects of the P6 α₂δ-1 mutant, which might be particularly prone to structural alterations.

Discussion

In the present report we found that the proteolytic site of the rat brain Ca_Vα₂δ-1 subunit is localized between amino acid residues Arg-941 and Val-946. Likewise, we found also that a single amino acid substitution in the putative proteolytic site is not sufficient to prevent cleavage, and that replacement of the six residues (Arg-Leu-Leu-Glu-Ala-Val) is required to make the Ca_Vα₂δ-1 protein insensitive to proteolysis. Furthermore, mutation of the entire hexapeptide prevents the whole-cell current stimulation (normally observed after co-expression of the α₂δ-1 without affecting its kinetic properties, suggesting either changes in channel open probability or a regulation on the number of functional channels at the plasma membrane.

One observation of particular interest in our studies is that hampering normal proteolysis has no biophysical consequences on the Ca_V channels regulation by α₂δ-1b. Interestingly, recent studies have shown that though an important fraction of the α₂δ-2 subunit expressed heterologously may not be proteolytically processed into α₂-2 and δ-2, it might show full functionality and being expressed on the cell membrane (Douglas et al., 2006). Consequently, it has been suggested that cleavage into α₂-2 and δ-2 subunits may not be essential for functional enhancement of macroscopic Ca²⁺ currents. However, it has been also found that native α₂-2 is fully cleaved in the mouse cerebellum (Douglas et al., 2006), and the possibility exists that the fraction of α₂δ-2 that is proteolytically cleaved into α₂-2 and δ-2 in the heterologous system is the active form of the protein. Therefore, it remains an open question for future research whether the α₂δ auxiliary subunits are functional in their proteolytically cleaved and non-cleaved forms.

Likewise, previous studies have shown that the Ca_Vα₂δ subunits increase the whole-cell conductance in different expression systems (Felix, 1999; Arikkath and Campbell, 2003; Klugbauer et al., 2003). Relevant to this, it has been shown that α₂δ-2have only a small influence on the properties of the channels at the single level (Barclay et al., 2001; Brodbeck et al., 2002). Taken together, these data imply that the α₂δ subunit is possibly acting on the lifetime of the channel complex at the cell surface, either by enhancing trafficking to the plasma membrane or by reducing turnover of channels (Douglas et al., 2006). In agreement with this view, it has been reported that the α₂δ subunits increase the amount of Ca_V1 and Ca_V2 proteins in the cell membrane (Felix, 1999; Arikkath and Campbell, 2003; Klugbauer et al., 2003).

Although the precise mechanism by which the α₂δ subunit controls Ca²⁺ channel turnover is presently unclear, Bernstein and Jones (2006) have recently suggested a model where α₂δ serves to maintain Ca_V2.2 channel levels at the cell surface by limiting their entry into a degradative pathway. According to this model, any free (possibly less physiologically desirable) Ca_V2.2/β complexes at the cell surface, are more rapidly removed and diverted to a non-degradable pool, perhaps for further attempts at complexation with α₂δ subunits (Bernstein and Jones, 2006). Therefore, investigating to what extent proteolytic processing alterations of α₂δ interfere with Ca_V channel subunit proper folding and trafficking is an interesting topic for future studies.