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The Journal of Neuroscience, August 23, 2006, 26(34):8748-8757; doi:10.1523/JNEUROSCI.2764-06.2006
 Previous Article | Next Article 
Cellular/Molecular
The Calcium Channel  2 -2 Subunit Partitions with CaV2.1 into Lipid Rafts in Cerebellum: Implications for Localization and Function
Anthony Davies,1 *
Leon Douglas,1 *
Jan Hendrich,1 *
Jack Wratten,1
Alexandra Tran Van Minh,1
Isabelle Foucault,1
Dietlind Koch,1
Wendy S. Pratt,1
Helen R. Saibil,2 and
Annette C. Dolphin1
1Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom, and 2School of Crystallography, Birkbeck, University of London, London WC1E 7HX, United Kingdom
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Abstract
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The accessory  2 subunits of voltage-gated calcium channels are highly glycosylated transmembrane proteins that interact with calcium channel 1 subunits to enhance calcium currents. We compared the membrane localization and processing of native cerebellar 2-2 subunits with 2-2 stably expressed in tsA-201 cells. We identified that 2-2 is completely concentrated in cholesterol-rich microdomains (lipid rafts) in cerebellum, in which it substantially colocalizes with the calcium channel 1 subunit CaV2.1, although CaV2.1 is also present in the Triton X-100-soluble fraction. In tsA-201 cells, unlike cerebellum, 2-2 is not completely proteolytically processed into 2-2 and -2. However, this processing is more complete in the lipid raft fraction of tsA-201 cells, in which 2-2 also colocalizes with CaV2.1. Cholesterol depletion of intact cells disrupted their lipid rafts and enhanced CaV2.1/2-2/ 4 currents. Furthermore, 2-2 coimmunoprecipitates with lipid raft-associated proteins of the stomatin family. The apparent affinity of 2-2 for its ligand gabapentin is increased markedly in the cholesterol-rich microdomain fractions, in both cerebellum and the stable 2-2 cell line. In contrast, 2-2 containing a point mutation (R282A) has a much lower affinity for gabapentin, and this is not enhanced in the lipid raft fraction. This R282A mutant 2-2 shows reduced functionality in terms of enhancement of CaV2.1/4 calcium currents, suggesting that the integrity of the gabapentin binding site may be important for normal functioning of 2-2. Together, these results indicate that both 2-2 and CaV2.1 are normally associated with cholesterol-rich microdomains, and this influences their functionality.
Key words: calcium; channel; 2; gabapentin; lipid raft; cerebellum; cholesterol
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Introduction
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Voltage-gated Ca2+ (CaV) channels are composed of a pore-forming 1 subunit, associated, at least in the case of the CaV1 and 2 subfamilies, with an intracellular subunit responsible for trafficking (Walker and De Waard, 1998 ) and a transmembrane 2 subunit whose function is less well defined (Canti et al., 2003). The 1 subunit determines the main biophysical properties of the channel and is modulated by the other subunits (Walker and De Waard, 1998). Mammalian genes encoding 10 1, four , and four 2 subunits have been identified (Catterall, 2000; Ertel et al., 2000; Burgess et al., 2001).
The topology of the 2 protein appears to generalize for all four 2 subunits. They are all predicted to be type 1 transmembrane proteins, because all have a hydrophobic region in the C terminus (CT) that is likely to be a transmembrane domain. All have predicted N-terminal signal sequences, indicating that the N terminus is extracellular (for review, see Canti et al., 2003). From the early studies of 2-1 purified from skeletal and cardiac muscle, it was identified that the 2 subunit is disulfide bonded to a transmembrane subunit, and both subunits are the product of a single gene, encoding the 2 protein, which is posttranslationally cleaved into 2 and (Ellis et al., 1988; De Jongh et al., 1990; Gurnett et al., 1996). The 2-2 subunit is also clearly reduced in molecular mass by disulfide-bond reduction (Gong et al., 2001), and, when expressed heterologously, the signal sequence is cleaved and the 2 moiety is entirely extracellular (Brodbeck et al., 2002). However, there is poor sequence conservation between 2-1 and 2-2 around the identified site of cleavage between 2 and in 2-1 (Jay et al., 1991).
All 2 subunits enhance calcium currents through the high-voltage-activated (HVA) CaV1 and CaV2 channels (Gurnett et al., 1996; Klugbauer et al., 1999; Barclay et al., 2001). However, their mechanism of action remains unclear. Our recent work has shed light on this and suggests that the metal ion-dependent adhesion site (MIDAS) in the Von Willebrand factor-A (VWA) domain within 2-2 is of key importance in trafficking CaV1 subunits (Canti et al., 2005). This VWA domain is present in all 2 subunits and has a perfect MIDAS motif in 2-1 and 2-2 (Whittaker and Hynes, 2002). Both 2-1 and 2-2 bind the antiepileptic drugs gabapentin and pregabalin, which are also used in the treatment of neuropathic pain (Brown et al., 1998; Marais et al., 2001; Canti et al., 2003).
In this study, we identified that both native and heterologously expressed 2-2 subunits are concentrated together with CaV2.1 in Triton X-100-insoluble cholesterol-rich microdomain fractions (commonly known as lipid rafts), in which the affinity for binding [3H]gabapentin is strongly enhanced and in which they interact with proteins of the stomatin family.
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Materials and Methods
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Molecular biology.
Mouse 2-2 [GenBank accession number AF247139; common brain splice variant, lacking exon 23 and 6 bp of exon 38 (Barclay and Rees, 2000)] was engineered with an internal hemagglutinin (HA) tag between amino acids 652 (L) and 653 (Q), using standard molecular biology techniques to form 2-2(HA). The R282A mutation in mouse 2-2 was made by standard techniques. All constructs were sequenced before use. Other cDNAs used were rat CaV2.1 (GenBank accession number M64373), E1686R (Hans et al., 1999), and rat 4 (LO2315), cloned into the pMT2 vector for expression in mammalian cells.
Antibodies.
Ca channel antibodies (Abs) used were as follows: anti-mouse 2-2(16–29), 2-2(102–117) (Brodbeck et al., 2002), -2(1080–1094), and -2 CT (1133–1147) for 2-2 (all affinity-purified rabbit anti-peptide Abs) and anti-1b serum (Moss et al., 2002). Other primary antibodies and their sources were as follows: anti-flotillin-1 (BD Biosciences, Cowley, Oxford, UK), anti-prohibitin (Abcam, Cambridge, UK), anti-stomatin-like protein-2 (SLP-2) (BD Biosciences), anti-Akt (Cell Signaling Technology, Danvers, MA), and anti-CaV2.1 (Alomone Labs, Jerusalem, Israel).
Heterologous expression of cDNAs.
Cos-7 or tsA-201 cells were transfected with either 2 cDNA alone or with all calcium channel subunit cDNAs, using the ratios for 1, , 2, and green fluorescent protein (GFP) of 3:2:2:0.4. For electrophysiological recording, the cDNA for GFP (mut3 GFP) was included in the transfection to identify transfected cells from which recordings were made.
Creation of a tsA-201 stable cell line containing 2-2 with internal HA tag.
The internally HA-tagged 2-2 cDNA was cloned in-frame into the mammalian expression vector pcDNA3.1/Zeo(+) (Invitrogen, Carlsbad, CA) using KpnI and NotI, linearized with PvuI, and transfected into cultured tsA-201 cells using Fugene 6 (Roche, Mannheim, Germany). The vector conferred Zeocin (Invitrogen) resistance to cells that stably incorporated the plasmid. Clonal foci were isolated using trypsin-impregnated 5-mm-diameter sterile paper discs (Sigma, Poole, UK). Expression of the full-length 2-2 protein was confirmed by Western blotting and functionality of the protein by electrophysiology.
Cholesterol depletion of tsA-201 cells.
A 50 mM stock of methyl--cyclodextrin (Sigma) was prepared in serum-free medium, sterile filtered, and kept at room temperature for no longer than 10 h. For electrophysiological experiments, cells were replated onto collagen-coated dishes, before addition of methyl--cyclodextrin. Cells were treated with methyl--cyclodextrin (5 mM) in serum-free medium for 1 h at 37°C (Christian et al., 1997), immediately before use in electrophysiological or biochemical experiments. Control cells were incubated in serum-free medium for the same length of time.
Cell membrane preparation.
Membrane fractions from mouse cerebellum or cultured cells were prepared using the following general method. Mouse cerebella were homogenized in a buffer containing the following (in mM): 20 HEPES, pH 7.4, 50 NaCl, 300 sucrose, 2 EDTA, incomplete protease inhibitor cocktail (Roche), and 1 orthophenanthroline. Cultured cell pellets were resuspended in 25 vol of ice-cold 10 mM HEPES, pH 7.4, containing complete protease inhibitor cocktail (Roche). Cells were lysed by 10 passages through a 23 gauge needle, followed by three 10-s rounds of sonication. Cell debris was removed by centrifugation (1000 x g, for 15 min at 4°C), and the resultant supernatants were recentrifuged (100,000 x g, for 60 min at 4°C) to pellet membranes. Protein concentrations of the membrane preparations were determined by BCA assay (Perbio, Tatenhall, Cheshire, UK). Membranes were used immediately or stored in aliquots at –80°C.
Preparation of Triton X-100-insoluble membrane fraction.
All steps were performed on ice. Confluent cells from four 175 cm2 flasks, or pelleted whole homogenate derived from two mouse cerebella, were taken up in 1.5 ml of MES-buffered saline (MBS) (25 mM MES, pH 6.5, 150 mM NaCl, and complete protease inhibitor cocktail) containing 1% (v/v) Triton X-100 (Perbio), resuspended by five passages through a 23 gauge needle and three 7-s rounds of sonication, and then left on ice for 1 h. An equal volume of 90% (w/v) sucrose in MBS was then added. The 3 ml sample was transferred to a 13 ml ultracentrifuge tube and overlaid with 10 ml of discontinuous sucrose gradient, consisting of 35% (w/v) sucrose in MBS (5 ml) and 5% (w/v) sucrose in MBS (5 ml). The sucrose gradients were centrifuged at 140,000 x g for 18 h at 4°C in a Beckman Instruments (Fullerton, CA) SW40 rotor; 1 ml fractions were subsequently harvested from the top to the bottom of the tube. When necessary, protein fractions from the gradient were washed free of sucrose by dilution into 25 vol of MBS and centrifugation (100,000 x g, for 60 min at 4°C) to pellet the cholesterol-enriched microdomain material.
Immunoprecipitation.
2-2(HA) was immunoprecipitated from stably transfected tsA-201 cells as follows. Triton X-100-insoluble membranes prepared from these cells were resuspended in immunoprecipitation (IP) solubilization buffer (1% Igepal, 50 mM Tris, 75 mM NaCl, and protease inhibitor cocktail, pH 7.4) for 1 h at 4°C. Insoluble material was removed by centrifugation at 12,000 x g at 4°C for 15 min. The clarified material was incubated for 2 h at 4°C with  2 µg of the appropriate Ab. Protein G-agarose (30 µl of 50% slurry; Sigma) was added and incubated for 1 h at 4°C. Beads were then washed three times in low detergent buffer (0.15% Igepal, 50 mM Tris, 75 mM NaCl, and protease inhibitor cocktail, pH 7.4), and bound protein was removed from the beads by the addition of SDS sample buffer containing 100 mM DTT, with heating at 70°C for 10 min. For protein sequencing, samples were separated on Nu-Page 4–12% Bis-Tris gels (Invitrogen) and stained with Coomassie blue (Simply Blue safe stain; Invitrogen). Bands of interest were excised from the gel, and protein identification was performed at Imperial College London Proteomics Facility by tryptic mass fingerprinting, confirmed by quadrupole time of flight (Q-ToF) mass spectrometry. The probability of correct identification from peptide mass fingerprinting is based on the molecular weight search (MOWSE) score using the Matrix Science Mascot search engine, which is –10*Log(P), where P is the probability that the observed match is a random event. Individual peptide ion scores >52 indicate identity or extensive homology (p < 0.05).
Immunoblotting.
Immunoblot analysis was performed essentially as described previously (Page et al., 2004). SDS-PAGE-resolved samples were transferred to polyvinylidene difluoride membranes and probed with relevant primary Abs and the appropriate horseradish peroxidase-conjugated secondary Abs, followed by enhanced chemiluminescence detection. Ca channel and prohibitin primary Abs were used at 1–5 µg/µl, and anti-flotillin-1 and anti-SLP-2 were used at 1:500 and 1:250, respectively.
Electrophysiology.
Calcium channel expression in tsA-201 cells was investigated by whole-cell patch-clamp recording. The internal (pipette) and external solutions and recording techniques were similar to those described previously (Campbell et al., 1995). The patch pipette solution contained the following (in mM): 140 Cs-aspartate, 5 EGTA, 2 MgCl2, 0.1 CaCl2, 2 K2ATP, and 10 HEPES, pH 7.2 (310 mOsm with sucrose). The external solution for recording Ba2+ currents contained the following (in mM): 150 tetraethylammonium Br, 3 KCl, 1.0 NaHCO3, 1.0 MgCl2, 10 HEPES, 4 glucose, and 10 BaCl2, pH 7.4 (320 mOsm with sucrose). Pipettes of resistance 2–4 M were used. An Axopatch 1D amplifier (Molecular Devices, Palo Alto, CA) was used, and data were filtered at 1–2 kHz and digitized at 5–10 kHz. Analysis was performed using pClamp7 (Molecular Devices) and Origin 7 (Microcal, Northampton, MA). Current records are shown after leak and residual capacitance current subtraction (P/4 protocol). For determination of the voltage for 50% current activation (V50,act), the current density–voltage (I–V) relationships were fitted between –30 and +50 mV with a modified Boltzmann equation as follows: I = Gmax x (V – Vrev)/(1 + exp(–(V – V50,act)/k)), where I is the current density (in picoamperes per picofarad), Gmax is the maximum conductance (in nanosiemans per picofarad), Vrev is the reversal potential, and k is a slope factor. Steady-state inactivation data were fitted with a single Boltzmann equation of the following form: I/Imax = ((A1 – A2)/[1 + exp((V – V50,inact)/k)] + A2, where Imax is the maximal current, V50,inact is the half-maximal voltage for current inactivation, and A1 and A2 represent the total and non-inactivating current, respectively.
Calcium current recording from isolated Purkinje cells was performed as described previously (Barclay et al., 2001), except for the following points: cerebellar slices (300 µm) were prepared from postnatal days 7–9 mice using a vibrating tissue slicer and kept in 95%O2/5% CO2-saturated Krebs' solution for 30 min at 36°C before being cooled to room temperature. Cells were isolated immediately before use by digestion of slices with papain (20 U/ml) for 5–15 min, washed and triturated in Ringer's solution, and plated onto poly-L-lysine-coated coverslips.
Gabapentin binding assay.
Binding of [3H]gabapentin was performed as described previously (Canti et al., 2005), in a final volume of 250 µl at room temperature for 2 h. Membranes (50 µg/tube) or cholesterol-rich microdomains (3 µg/tube) were incubated with various concentrations of [3H]gabapentin (ARC, St. Louis, MO) in 10 mM HEPES/KOH, pH 7.4, and then rapidly filtered through GF/B filters, presoaked with 0.3% polyethyleneimine. Filters were washed three times with 3 ml of ice-cold 50 mM Tris/HCl, pH 7.4, and the amount of bound [3H]gabapentin was determined by scintillation counting. Concentrations of [3H]gabapentin >50 nM were achieved by adding nonradioactive gabapentin and correcting the specific binding by the dilution factor, as described previously (Marais et al., 2001). Nonspecific binding was determined in the presence of 1000-fold excess of nonradioactive gabapentin (or pregabalin, with identical results). Replicate (three to five) independent experiments were performed, each in triplicate, and data were analyzed by fitting an equation for a rectangular hyperbola to the specific binding data from each experiment, to obtain the dissociation constant (KD) and maximum number of binding sites (Bmax).
Data are presented as mean ± SEM, and statistical significance of differences between data were determined using Student's unpaired, two-tailed t test.
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Results
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Processing of native 2-2 in cerebellar tissue
In parallel with the posttranslational processing that has been determined for native muscle 2-1 (Jay et al., 1991), the theoretical processing of 2-2 is shown in Figure 1A. The mature form of 2-2 is thought to be proteolytically cleaved into 2-2 and -2, which remain disulfide bonded, until subjected to disulfide-bond reduction in vitro (Hobom et al., 2000; Brodbeck et al., 2002). The cerebellum is rich in native 2-2, and we confirmed that the protein from cerebellum that is recognized by the 2-2(102–117) Ab used in these studies is indeed derived from 2-2, because it is absent from du2J/du2J cerebellum (Fig. 1B). This mouse strain has a 2 bp mutation in the Cacna2d2 gene (calcium channel, voltage-dependent, alpha2delta subunit-2), which is predicted to result in a complete loss of 2-2 protein (Barclay et al., 2001).
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Figure 1. Processing of 2-2 from cerebellum. A, Schematic showing the proposed products of posttranslational and in vitro modifications of the 2-2 subunit. SS, Signal sequence; S-S, disulfide bond; TM, transmembrane domain. B, Presence of 2-2(102–117)-immunoreactive band in wild-type but absence from du2J/du2J cerebellar membranes, separated under reducing conditions, in the presence of DTT (100 mM). Akt immunoreactivity was used as a loading control. C, Western blot of cerebellar membranes separated under nonreducing conditions (–) or reducing conditions (+100 mM DTT) and probed with Ab 2-2(102–117). Immunolabeled bands corresponding to mature full-length 2-2 (180 kDa) and glycosylated 2-2 (145 kDa) are arrowed. D, Western blot of cerebellar membranes prepared under reducing conditions, treated without (–) or with (+) endo-F and then probed with Ab 2-2(102–117). Bands corresponding to glycosylated 2-2 and deglycosylated 2-2 (107 kDa) are arrowed.
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Additional examination of the native species shows it to have the expected molecular mass of 180 kDa before in vitro reduction with DTT and 145 kDa for the reduced form (Fig. 1A,C). Subsequent in vitro deglycosylation with endoglycosidase F (endo-F) resulted in the expected reduction in mass for the reduced 2-2(102–117)-immunoreactive species from 145 kDa before deglycosylation to 107 kDa after deglycosylation (Fig. 1D). This is in accordance with the predicted processing shown in the schematic diagram in Figure 1A. The specificity of the 2-2(102–117) Ab was further confirmed by showing that it recognized heterologously expressed 2-2 but not 2-1 or 2-3 transfected into tsA-201 cells (data not shown).
Native 2-2 from cerebellum is colocalized in cholesterol-rich microdomains with CaV2.1
The punctate cell-surface distribution of heterologously expressed 2-2 that we observed previously after heterologous expression of 2-2 (Canti et al., 2005) suggested to us that it might be concentrated in cholesterol-rich microdomains. Cell lysates were prepared from mouse cerebellum and separated on a sucrose gradient. These showed native 2-2 to be entirely concentrated in cholesterol-rich lipid raft microdomains (Fig. 2A, fractions 5, 6), in which it colocalizes with the lipid raft marker flotillin-1 (Fig. 2A). Within the lipid raft fraction, 2-2 is entirely in its mature disulfide-bond linked form, as shown by the fact that, when subjected to SDS-PAGE under reducing conditions, it is separated into 2-2 (145 kDa) (Fig. 2A,B) and -2 (32 kDa) (Fig. 2B) and, as expected, has a cleaved signal sequence (Fig. 2B). No 2-2 was present in the Triton X-100-soluble fraction (Fig. 2A, fractions 10–13). We also examined the distribution of native CaV2.1 in the cerebellar sucrose gradient fractions and found that it substantially colocalized with 2-2 in the cholesterol-rich microdomain fractions, although some was also present in the Triton X-100-soluble fractions (fractions 10–13), which contain a large percentage of the total protein (Fig. 2C) (for quantification, see Fig. 6A). Two main immunoreactive bands were observed for CaV2.1, of mass 250 and 190 kDa, with the lower band being the predominant species. Multiple mass species of CaV2.1 have been attributed previously to the existence of different C-terminal splice variants and to proteolytic cleavage of the C terminus (Sakurai et al., 1995). Furthermore, there is evidence that these species may have differing locations (Sakurai et al., 1996).
Gabapentin binding to cerebellar 2-2 is enhanced in cholesterol-rich microdomains
Gabapentin is a ligand for 2-2 as well as 2-1 (Canti et al., 2003, 2005; Klugbauer et al., 2003). We therefore examined the [3H]gabapentin binding affinity in cholesterol-rich microdomains compared with membrane preparations from cerebellum. The affinity for [3H]gabapentin was increased 4.8-fold in the lipid raft fraction, with the KD being reduced from 385 ± 56 nM in membranes (n = 3) to 79.7 ± 19.8 nM (n = 3) in lipid rafts (Fig. 2D). The Bmax was 5.23 ± 0.73 pmol/mg protein in cerebellar membranes and 1.67 ± 0.45 pmol/mg protein for cerebellar rafts.
Gabapentin has no acute effect on Purkinje cell calcium currents
It is assumed in much of the literature that gabapentin has its effect via binding to 2 subunits and consequently inhibiting calcium currents (for review, see Sutton and Snutch, 2001) and reducing transmitter release (van Hooft et al., 2002). However, the effect of gabapentin on calcium currents is controversial because, although it has been found to produce a small inhibition of native calcium currents in sensory neurons (Sutton et al., 2002), it does not produce acute inhibition of calcium currents in heterologous expression systems (Vega-Hernandez and Felix, 2002; Canti et al., 2003). Because 2-2 is very strongly expressed in cerebellar Purkinje cells (Barclay et al., 2001) and has also been found to be associated with many other GABAergic neurons (Cole et al., 2005), we therefore examined the effect of gabapentin on Purkinje cell calcium currents. An inhibition of calcium currents in GABAergic neurons and consequent inhibition of GABA release would not be compatible with the well established antiepileptic action of gabapentin. We found no significant effect of 1–10 µM gabapentin on Purkinje cell calcium currents from wild-type mice (Fig. 3A,B).
Colocalization of heterologously expressed CaV1 subunits with 2-2 in cholesterol-rich microdomains
We wanted to examine whether heterologously expressed 2-2 was also localized in lipid rafts in a similar manner to native 2-2. For these experiments, we used either transient transfection of wild-type 2-2 or a cell line stably expressing either wild-type 2-2 or 2-2 with an internal HA tag [2-2(HA)].
It was first important to determine whether 2-2 with an internal HA tag was fully functional. Coexpression of 2-2 enhanced calcium currents through all HVA calcium channels in all expression systems we tested (Canti et al., 2005). In the example shown in Figure 4, A and B, 2-2 increased currents through CaV2.1/4 channels in tsA-201 cells by 3.8 fold (n = 16). This is a physiologically relevant subunit combination, likely to be the main functional heteromeric calcium channel in cerebellar Purkinje cells. Transient transfection of the 2-2(HA) construct enhanced CaV2.1/4 currents to the same extent as wild-type 2-2, and these currents were very similar to those obtained after transient transfection of CaV2.1/4 into the stable 2-2(HA) cell line (Fig. 4B). Furthermore, both 2-2 and 2-2(HA) produced a similar small hyperpolarization of the steady-state inactivation compared with CaV2.1/4 alone (Fig. 4C).
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Figure 4. Comparison of the effect of expression of 2-2 and 2-2(HA) on currents through Cav2.1/4 channels in tsA-201 cells. Cav2.1/4 were expressed alone or with 2-2 or 2-2 with internal HA tag [2-2(HA)] in tsA-201 cells, and currents were recorded using Ba2+ as charge carrier. A, Representative current traces elicited between –30 and +15 mV in 5 mV steps from a holding potential of –90 mV for Cav2.1/4 (left), Cav2.1/4/2-2 (middle left), Cav2.1/4/2-2(HA) (middle right), or Cav2.1/4 in stable 2-2(HA) cell line (right). B, Current density–voltage relationships (mean ± SEM) for the four experimental conditions.  , Cav2.1/4 (n = 13);  , with 2-2 (n = 16);  , with 2-2(HA) (n = 7);  , Cav2.1/4 in 2-2(HA) cell line (n = 21). C, Steady-state inactivation (mean ± SEM) for the four experimental conditions: Cav2.1/4 (; n = 7; V50 = –23.7 ± 1.2 mV); with 2-2 (; n = 2; V50 = –27.3 mV); with 2-2(HA) (; n = 3; V50 = –30.6 ± 1.9 mV; p < 0.01 compared with no 2-2), and Cav2.1/4 in 2-2(HA) cell line (; n = 8; V50 = –29.5 ± 1.9 mV; p < 0.03 compared with no 2-2).
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The processing of stably expressed 2-2(HA) was examined in either the additional presence of transiently transfected CaV2.1 and 4 (Fig. 5A) or their absence (Fig. 5B). Cells were lysed, and the Triton X-100-soluble and -insoluble fractions were separated on a discontinuous sucrose gradient. Fractions from the sucrose gradient were analyzed by SDS-PAGE under reducing conditions in the presence of DTT. As was the case for cerebellar 2-2, the heterologously expressed 2-2 was clearly present in the flotillin-1-positive lipid raft fractions (Fig. 5A,B, fractions 5, 6), although it was also present in the Triton X-100 soluble fractions (fractions 10–13). This was true in both the presence (Fig. 5A) and absence (Fig. 5B) of coexpressed CaV2.1.
Whereas a single 2-2(102–117)-immunoreactive band of 180 kDa was predominant in the Triton X-100-soluble fractions (fractions 10–13), two bands of 180 and 145 kDa were present in the lipid raft fractions 5 and 6 (Fig. 5A,B), which are likely to represent intact noncleaved 2-2 and fully mature 2-2 derived from disulfide-bonded 2-2, respectively. In agreement with this, a -2(1080–1094)-immunoreactive band at 32 kDa was only observed in the lipid raft fractions (Fig. 5B). Similar results were obtained using transient transfection of wild-type 2-2, indicating that this partial processing of heterologously expressed 2-2 is not a result of introduction of the HA tag (data not shown).
Additional investigation of the peak lipid raft fraction was undertaken to confirm the nature of the two protein bands that were immunoreactive against 2-2(102–117), by subjecting this fraction to in vitro deglycosylation before separation under reducing conditions on SDS-PAGE (Fig. 5C). Deglycosylation tended to increase the immunoreactivity with the anti-peptide Abs, probably by increasing the exposure of the peptide epitopes. This analysis showed that the 180 and 145 kDa bands were reduced in mass to 125 and 107 kDa after in vitro deglycosylation (Fig. 5C, left panel), consistent with their identification as full-length 2-2 and free 2-2. Additional confirmation of this conclusion is that only the upper band is immunoreactive against the -2(1080–1094) Ab (Fig. 5C, middle panel). Free -2 is also present (Fig. 5C, right panel).
We also observed that a substantial proportion of coexpressed CaV2.1 was present in the Triton X-100-insoluble lipid raft fractions in tsA-201 cells (Fig. 5A). In this study, the long C-terminal isoform of CaV2.1 was expressed, and the mass of 250 kDa is in agreement with its predicted mass. To compare the localization in lipid rafts of CaV2.1 and 2-2 in cerebellum and tsA-201 cells, we quantified the 2-2(102–117) and CaV2.1 immunoreactivity in the sucrose gradient fractions per milligram of total protein (Fig. 6). This shows that 2-2 is strongly concentrated in the lipid raft fraction in both cerebellum (Fig. 6A) and tsA-201 cells (Fig. 6B), whereas CaV2.1 is more strongly concentrated in lipid raft fractions in tsA-201 cells than in cerebellum. This suggests that, in cerebellum, some CaV2.1 is either associated with an 2 that is not concentrated in lipid rafts or that not all native CaV2.1 is associated with 2. In this regard, we observed that 2-1 is also associated with lipid rafts (data not shown). It is therefore possible that some native CaV2.1 is not associated with these 2 subunits.
Effect of cholesterol depletion of tsA-201 cells
The 2-2(HA) tsA-201 cell line was incubated with methyl--cyclodextrin (5 mM) for 1 h to deplete membrane cholesterol (Christian et al., 1997), and calcium channel currents were recorded in cells that had also been transiently transfected with CaV2.1 and 4 (Fig. 7). Lipid rafts became disrupted by the methyl--cyclodextrin treatment, as shown previously (Fagan et al., 2000). We confirmed there was a reduced concentration of flotillin-1 in the Triton X-100-insoluble fraction (data not shown). Calcium channel currents were significantly elevated after cholesterol depletion (Fig. 7). The increase at +15 mV was 44.1 ± 16.5% (n = 17; p < 0.05). There was no effect on the V50,act, which was +2.8 ± 1.2 mV after treatment with cyclodextrin compared with +3.7 ± 0.8 mV for controls performed in parallel (n = 17 for both).
Coimmunoprecipitation of 2-2 with other lipid raft constituent proteins
We examined whether 2-2 is associated with other protein constituents of lipid rafts and found that IP of 2-2(HA) from the lipid raft fraction, by means of its HA tag, consistently resulted in coimmunoprecipitation with three related proteins of the SPFH family (stomatin/prohibitin/flotillin/HflK), which have been identified to be lipid raft constituent proteins (for review, see Langhorst et al., 2005). They were identified by peptide mass fingerprinting and Q-ToF mass spectrometry. The proteins identified were SLP-2 (Owczarek et al., 2001), which was present in the excised 60 kDa band, presumably complexed to another protein or as a dimer, because its monomer protein mass is 39 kDa. Three peptides were identified for this protein giving an MOWSE score of 85 (p < 0.05). Stomatin (for review, see Rivera-Milla et al., 2006) was identified to be present in the excised 40 kDa band. Four peptides were identified for stomatin, giving an MOWSE score of 108 (p < 0.05). Prohibitin was also identified in the excised 40 kDa band, with an MOWSE score of 285 (p < 0.05), and, in addition, the same protein was identified in the excised 32 kDa band, which is similar to its protein mass of 30 kDa (Fig. 8A). We found that SLP-2 is strongly, although not exclusively, expressed in the peak Triton X-100-insoluble lipid rafts from tsA-201 cells (Fig. 8B), as is prohibitin (data not shown). Immunoprecipitation of 2-2, followed by immunoblotting confirmed the presence of coimmunoprecipitating SLP-2 and prohibitin (Fig. 8C). No commercial Ab was available for stomatin, and thus its interaction with 2-2 has not yet been verified by immunoblotting.
Gabapentin binding to heterologously expressed 2-2 is enhanced in cholesterol-rich microdomains
For comparison with cerebellum, we examined the concentration of [3H]gabapentin binding sites and their affinity for [3H]gabapentin in cholesterol-rich microdomains compared with membrane preparations, both derived from the 2-2(HA) cell line (Fig. 9A).
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Figure 9. Gabapentin binding to 2-2(HA) cell line is enhanced in cholesterol-enriched microdomains and prevented by R282A mutation. A, Examples of experiments to determine the concentration of [3H]gabapentin binding sites and affinity for [3H]gabapentin of a membrane fraction (; KD of 468 nM; representative example from n = 3) and cholesterol-rich microdomains (; KD of 42.6 nM; representative example from n = 4) derived from the 2-2(HA) cell line. Also included on this graph are examples of the lack of binding of [3H]gabapentin to membranes (; representative example from n = 5) or lipid rafts ( ; representative example from n = 2) prepared from Cos-7 cells transiently expressing the R282A 2-2 mutant. B, Western blot analysis of the distribution of proteins found in fractions from a typical discontinuous sucrose gradient, derived from transient expression of R282A 2-2. Fractions 1–13 from the sucrose gradient (run under disulfide-bond-reducing conditions) were probed for 2-2-derived products with Ab 2-2(102–117) (top). The bottom shows the location of the cholesterol-rich microdomain marker protein flotillin-1. C, Comparison of the effect of expression of 2-2 and R282A 2-2 on currents through Cav2.1/4 channels in tsA-201 cells, using Ba2+ as charge carrier. Current density–voltage relationships (mean ± SEM) are shown for the three experimental conditions: , Cav2.1/4 (n = 14); , with 2-2 (n = 14); and , with R282A 2-2 (n = 18). D, Comparison of the effect of expression of 2-2 and R282A 2-2 on the steady-state inactivation of currents through Cav2.1/4 channels in tsA-201 cells. Conditioning prepulses of 5 s to the potentials shown were given before a constant test pulse to +10 mV. The normalized data (mean ± SEM) are shown for the three experimental conditions: , Cav2.1/4 (n = 11); , with 2-2 (n = 11); and , with R282A 2-2 (n = 9). The V50 values are –30.5 ± 0.4, –36.2 ± 0.5, and –36.5 ± 0.4 mV, and the k values are 8.4 ± 0.4, 8.0 ± 0.4, and 8.1 ± 0.4 mV, respectively.
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Initial studies showed that the presence of the HA tag had no effect on the gabapentin binding affinity. For wild-type 2-2, stably expressed in tsA-201 cells, the apparent KD for [3H]gabapentin in membranes was 450.6 ± 36.9 nM (n = 4), very similar to the apparent KD for gabapentin binding to membranes prepared from the 2-2(HA) cell line, which was 474.4 ± 32.9 nM (n = 3). Just as we observed in the cerebellum, the affinity for [3H]gabapentin was enhanced in the lipid raft fraction compared with the membrane fraction. The affinity of 2-2(HA) for [3H]gabapentin was approximately ninefold greater in the cholesterol-rich microdomain fraction compared with the membrane fraction, the apparent KD decreasing to 53.3 ± 8.4 nM (n = 4). Unlike in the cerebellum, the Bmax also increased from 5.1 ± 0.4 pmol/mg protein (n = 3) in the membrane fraction to 71.1 ± 29.8 pmol/mg protein (n = 4) in the lipid raft fraction, indicating a 14-fold purification of [3H]gabapentin binding sites in this fraction. No specific [3H]gabapentin binding was observed in the absence of transfected 2-2 (data not shown). One explanation for the increase in Bmax is that it may relate to the lower relative concentration of total protein in the lipid raft fraction of the tsA-201 cell line.
The effect of a mutation R282A in 2-2 that prevents [3H]gabapentin binding
The binding of [3H]gabapentin was almost completely abrogated by a mutation R282A in 2-2, which is situated just before the VWA domain. The affinity for [3H]gabapentin was reduced very substantially by this mutation to an unmeasurably high KD of >2 µM (n = 5) (Fig. 9A). Although the R282A mutant 2-2 did partition into cholesterol-rich microdomains (Fig. 9B), its affinity for gabapentin was not enhanced in this fraction (Fig. 9A). An equivalent R217A mutation in 2-1 has been described previously to prevent [3H]gabapentin binding to 2-1 (Wang et al., 1999), and a knock-in mouse carrying this mutation has been reported to be nonresponsive to gabapentin (Taylor, 2004). We confirmed that the R282A mutant 2-2 was still functional, although it enhanced CaV2.1/4 currents to a significantly smaller extent than wild-type 2-2 in tsA-201 cells. The peak IBa at +15 mV was enhanced only 2.6-fold by R282A 2-2 compared with 3.8-fold for wild-type 2-2 (p < 0.05) (Fig. 9C). However, both the wild-type and R282A 2-2 hyperpolarized the steady-state inactivation to similar extents, indicating that, despite a reduction in the peak currents, the R282A mutant 2-2 is still able to influence the properties of the expressed channels (Fig. 9D). For 2-1, hyperpolarization of the steady-state inactivation was found to be a property of the subunit (Felix et al., 1997).
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Discussion
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Previous in vitro studies have shown that 2-1, 2-2, and 2-3 subunits all increase the maximum conductance of a number of expressed calcium channel 1/ subunit combinations at the whole-cell level in several different expression systems (Mori et al., |
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Abstract
Introduction
