calciumcurrentsareenhancedby 2 -1lackingits · pdf file ·...

Calcium Currents Are Enhanced by �2�-1 Lacking ItsMembrane Anchor*□S

Received for publication, May 4, 2012, and in revised form, July 30, 2012 Published, JBC Papers in Press, August 6, 2012, DOI 10.1074/jbc.M112.378554

Ivan Kadurin1, Anita Alvarez-Laviada2, Shu Fun Josephine Ng, Ryan Walker-Gray, Marianna D’Arco,Michael G. Fadel, Wendy S. Pratt, and Annette C. Dolphin3

From the Department of Neuroscience, Physiology and Pharmacology, University College London, Gower St.,London WC1E 6BT, United Kingdom

Background:We examined the role of membrane anchoring of voltage-gated calcium channel �2� subunits.Results:We used a truncated �2�-1 construct (�2�-1�C-term), which still increases CaV2.1/�1b currents, despite beingmainlysecreted.Conclusion: The effect of �2�-1�C-term on calcium currents does not involve secretion and subsequent re-binding to theplasma membrane.Significance: C-terminal membrane anchoring of �2� is not essential for calcium current enhancement.

The accessory�2� subunits of voltage-gated calciumchannelsare membrane-anchored proteins, which are highly glycosy-lated, possess multiple disulfide bonds, and are post-transla-tionally cleaved into �2 and �. All �2� subunits have a C-termi-nal hydrophobic, potentially trans-membrane domain andweredescribed as type I transmembrane proteins, but we found evi-dence that they can be glycosylphosphatidylinositol-anchored.To probe further the function of membrane anchoring in �2�subunits, we have now examined the properties of �2�-1 con-structs truncated at their putative glycosylphosphatidylinositolanchor site, located before the C-terminal hydrophobic domain(�2�-1�C-term). We find that the majority of �2�-1�C-term issoluble and secreted into the medium, but unexpectedly, someof the protein remains associated with detergent-resistantmembranes, also termed lipid rafts, and is extrinsically bound tothe plasma membrane. Furthermore, heterologous co-expres-sion of �2�-1�C-term with CaV2.1/�1b results in a substantialenhancement of the calcium channel currents, albeit less thanthat produced by wild-type �2�-1. These results call into ques-tion the role of membrane anchoring of �2� subunits for cal-cium current enhancement.

Voltage-gatedCa2� (CaV)4 channels comprise an�1 subunit,which forms the pore and determines the main functional andpharmacological attributes of the channel (1). For the high volt-

age-activated channels, the �1 subunit is associated with anintracellular � subunit, which is required for the channel toreach the plasma membrane (2–4), and an �2� subunit, whosefunctions are less well understood but which also influencestrafficking of the channel (5–7). Genes encoding 10 �1, four �,and four �2� subunits have been identified (1, 8, 9).The topology of the �2� protein was initially determined for

skeletal muscle �2�-1 but is likely to generalize to all �2� sub-units (10, 11). The �2� subunits were predicted to be type Itransmembrane proteins, as they have an N-terminal signalpeptide sequence and a C-terminal hydrophobic and poten-tially transmembrane region (12). From the early studies of�2�-1 purified from skeletal and cardiac muscle, it was identi-fied that the�2 subunit is disulfide-bonded to a transmembrane� subunit (13). However, both subunits are the product of asingle gene, encoding the �2� protein, which is post-transla-tionally glycosylated and further processed with the formationof disulfide bond(s) and subsequent proteolytic cleavage into�2and � (12).In terms of function, the �2 moiety of �2�was found to play a

role in enhancement of calcium currents (11), and we showedthat the von Willebrand factor-A domain in �2 is essential forits trafficking function (6, 14). In contrast, the transmembrane �subunit was reported to function by modifying the voltage-de-pendent properties of the channels (10, 11).We have recently obtained evidence that �2� subunits can

form GPI-anchored proteins (15). In this study, we wished tofurther examine the role of membrane anchoring of �2�-1 bycreating an anchorless �2�-1, truncated at the putative GPI-anchor �-site, which removes the C-terminal hydrophobicdomain (Fig. 1, �2�-1�C-term, construct iii). A similarapproach has been taken with GPI-anchored prion protein,which was found to remain associated with lipid rafts despitethe loss of membrane anchoring (16). The interaction of atransmembrane formof prion proteinwith lipid raftswas foundto require interaction with glypicans, which are themselvesGPI-anchored (17).We have examined the role of membrane anchoring of �2�-1

on its biochemical properties, processing, subcellular localiza-

* This work was supported in part by Medical Research Council GrantsG0700368 and G0801756.Author’s Choice—Final version full access.

□S This article contains supplemental Figs. 1 and 2.1 To whom correspondence may be addressed. Tel.: 44-2076793276; Fax:

44-2076790042; E-mail: [email protected] Supported by a Biotechnology and Biological Sciences Research Council

studentship.3 To whom correspondence may be addressed. Tel.: 44-2076793276; Fax:

44-2076790042; E-mail: [email protected] The abbreviations used are: CaV, voltage-gated calcium; Ab, antibody; DRG,

dorsal root ganglion; DRM, detergent-resistant membrane; GPI, glycosyl-phosphatidylinositol; PNGase, peptide N-glycosidase; WCL, whole celllysate; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CFP, cyan fluorescent protein.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 40, pp. 33554 –33566, September 28, 2012Author’s Choice © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

33554 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 40 • SEPTEMBER 28, 2012

by guest on May 21, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/cgi/content/full/M112.378554/DC1

http://www.jbc.org/

tion, and function. We present the surprising evidence that�2�-1�C-term is still able to produce a significant enhance-ment of CaV2.1/�1b calciumchannel currents following its het-erologous expression, indicating that intrinsic membraneanchoring is not essential for this property. Furthermore, wehave found that although a large fraction (�75% after 3 days inculture) of �2�-1�C-term is soluble and secreted into themedium, some of this protein remains extrinsically associatedwith the external leaflet of the plasma membrane. Future stud-ies will be directed toward identifying the binding partner(s) of�2�-1�C-term mediating this extrinsic interaction.

EXPERIMENTAL PROCEDURES

Molecular Biology—�2�-1�C-term was constructed with aC-terminal HA tag, followed immediately by a STOP codoninserted directly after Cys-1059 (thus abolishing the Cys-1059/Gly-1060/Gly-1061-predicted GPI anchor �-site). A secondconstruct wasmade with anHA tag between Asn-549 and Asp-550, which was also truncated by a STOP codon immediatelyafter Cys-1059. All mutations were made by standard molecu-lar biological techniques and verified by DNA sequencing.Heterologous Expression of cDNAs—The calcium channel

cDNAsusedwere rabbit CaV2.1 (M64373), rat�2�-1 (M86621),and rat�1b (18). The cDNAswere cloned into the pMT2 vectorfor expression, unless otherwise stated. tsA-201 cells weretransfectedwith the cDNAcombinations stated. The cDNA forgreen fluorescent protein (mut3 GFP) (19) was also included toidentify transfected cells from which electrophysiologicalrecordings were made. Transfection was performed asdescribed previously (20). In control experiments where �2�was omitted, the ratio wasmade up as statedwith empty vector.Dorsal Root Ganglion (DRG) Neuron Culture and

Transfection—DRG neurons were isolated from P10 Sprague-Dawley rats and transfected by Amaxa nucleofection asdescribed in the manufacturer’s protocol (program G13,Lonza). Briefly, neurons were dissociated in dissection solutionas follows: Hanks’ basal salt solution buffer containing 5mg/mlDispase (Invitrogen), 2 mg/ml collagenase type 1A (Worthing-ton), and 0.1 mg/ml DNase, (Invitrogen), for 30 min at 37 °C,and then resuspended in 160 �l of nucleofection buffer (80 �lper sample). 2�g of total plasmidDNAwas used for each trans-fection condition. For expression,�2�-1mid-HAand�2�-1�C-term-HA were used in pcDNA3. Enhanced cyan fluorescentprotein (Clontech) was co-transfected with �2�-1 cDNA at aratio of 1:4. After transfection, DRGs were plated on poly-L-lysine-coated glass coverslips and cultured in DMEM/F-12medium (Invitrogen) supplemented with 10% FBS and 50ng/ml NGF. Culture medium was replaced 18 h aftertransfection.Primary Antibodies (Abs)—The following primary Abs were

used: anti-�2-1 (mouse monoclonal, Sigma); anti-HA (rabbitpolyclonal, Sigma, or rat monoclonal, Roche Applied Science);anti-flotillin-1 (mouse monoclonal, BD Biosciences); anti-Akt/PKB (rabbit polyclonal, Cell Signaling Technologies), and anti-GAPDH (mouse monoclonal, Ambion).Cell Lysis, Cell Surface Biotinylation, and Immunoblotting—

Theprocedures weremodified from those described previously(15, 20). 72 h after transfection, tsA-201 cells were rinsed with

phosphate-buffered saline (PBS, pH 7.4, Sigma) and then incu-bated with PBS containing 1 mg/ml EZ-link Sulfo-NHS-LC-Biotin (Thermo Scientific) for 30 min at room temperature.Cells were then rinsed twice with PBS containing 200 mM gly-cine to quench the reaction. The cells were scraped, resus-pended in cold PBS, and centrifuged at 1000 � g at 4 °C for 10min. The cell pellets were homogenized in PBS, pH 7.4, at 4 °Ccontaining 1% Igepal and protease inhibitors (complete, RocheApplied Science) by five passes through a 23-gauge needle, fol-lowed by sonication for 10 s, and were incubated on ice for 45min. The whole cell lysates (WCL) were then centrifuged at20,000 � g for 25 min at 4 °C, and the pellet was discarded.Aliquots of supernatant were assayed for total protein (Brad-ford assay, Bio-Rad). WCL corresponding to 20–40 �g of totalprotein was diluted with Laemmli sample buffer (15) supple-mented with 100 mM dithiothreitol, incubated at 60 °C for 10min, resolved by SDS-PAGE on 3–8% Tris-acetate or 4–12%Bis-Tris gels (Invitrogen), and transferred to polyvinylidene flu-oride (PVDF) membrane (Bio-Rad) byWestern blotting (semi-dry, Bio-Rad). 500 �g of the same lysate was used to precipitatebiotinylated protein by adding 50�l of prewashed streptavidin-agarose beads (Thermo Scientific) and overnight incubation at4 °C. The beads were washed five times with PBS containing0.1% Igepal and resuspended in an equal volumeof 2�Laemmlibuffer with 100 mM DTT, followed by 10 min of incubation at60 °C. The eluted protein was analyzed by immunoblotting, asdescribed above. The following secondary Abs were used forWestern blot: goat anti-rabbit coupled to horseradish peroxi-dase (HRP) and goat anti-mouse coupled to HRP (Bio-Rad).The signal was obtained by HRP reaction with fluorescentproduct (ECL Plus, GE Healthcare), and membranes werescanned on a Typhoon 9410 phosphorimager (GE Healthcare).Quantification of Western Blots—ImageJ software (rsb.info.

nih.gov)was used to draw a box around each band of interest, toquantify the mean gray intensity. The background was sub-tracted using an equally sized “background” box next to eachband. To quantify the cleavage of �2� to �2 and �, the �2�-1 and�2-1 bands were summed (total � cleaved � uncleaved), fromwhich the % cleavage was calculated.The proportion of �2�-1�C-term-HA secreted into the

medium 72 h after transfection of tsA-201 cells was quantifiedby measuring the mean intensity of �2�-1-associated bandsdetected by HA Ab in the media and in the WCL. Taking intoaccount the total volume of the media and the WCL for eachcondition, an estimate of the amount of �2�-1�C-term-HAprotein in each fraction was obtained and expressed as % of thetotal �2�-1�C-term-HA in all fractions.Deglycosylation with Peptide N-Glycosidase-F (PNGase-F)—

WCL were brought to 0.2–0.5 mg/ml protein in PNGase-Fbuffer (PBS, pH 7.4, supplementedwith 75mM �-mercaptoeth-anol, 0.5% Triton X-100, 0.1% SDS, and protease inhibitors). 1unit of PNGase-F (RocheApplied Science) was added per 10-�lvolume and incubated at 37 °C for 5–12 h.For the secreted proteins, equal amounts of concentrated

media were taken for each reaction. For PNGase-F deglycosy-lation, the concentrated media were diluted with 9 volumes ofPNGase-F buffer and incubated with PNGase-F as describedabove. Samples without enzyme were incubated in parallel in

Anchorless �2�-1 Enhances Calcium Currents

SEPTEMBER 28, 2012 • VOLUME 287 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 33555


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

both cases, and the whole reaction volume was analyzed byWestern blot.Collection ofMedium—tsA-201 cells were incubated for 72 h

post-transfection, and medium was collected and centrifuged(1000 � g) to remove any detached cells. The supernatant wasfiltered through a 0.22-�msyringe filter (Millipore). The result-ing cell-free medium was applied to 3-kDa cutoff filtration col-umn (Amicon) and centrifuged to concentrate the proteins(�150-fold). Aliquots of the concentrate were diluted in theappropriate amount of Laemmli sample buffer and analyzed byWestern blot.Preparation of Triton X-100-insoluble Membrane Fractions

(DRMs)—All steps were performed on ice. Confluent tsA-201cells from two 175-cm2 flasks were taken up in Mes-bufferedsaline (MBS, 25 mM Mes, pH 6.5, 150 mM NaCl, and completeprotease inhibitor mixture (Roche Applied Science)) contain-ing 1% (v/v) Triton X-100 (Thermo Scientific), resuspended by10 passages through a 1-mlGilson pipette tip, and left on ice for1 h. An equal volume of 90% (w/v) sucrose in MBS was thenadded. The sample was transferred to a 13-ml ultracentrifugetube and overlaid with 10ml 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 centrifugedat 33,000 rpm for 18 h at 4 °C (Beckman SW40 rotor). 1-mlfractions were subsequently harvested from the top to the bot-tom of the tube. When necessary, protein fractions from thegradient were washed free of sucrose by dilution into 25volumes of cold PBS and ultracentrifugation (150,000 � g,for 1 h at 4 °C) to pellet the cholesterol-enriched microdo-main material. Triton X-100-insoluble protein was resus-pended in deglycosylation buffer and treated with PNGase-Fas described.Basic pH Treatment of DRMs—Triton X-100-insoluble

membrane fractions were resuspended at 4 °C in 0.1 M K2CO3,pH 11.5, or 0.1 M Tris, pH 7.4, incubated on ice for 20 min, andthen centrifuged (150,000 � g for 1 h at 4 °C) (21). The super-natants and pellets from both washes were separated, and thesupernatants were concentrated by centrifugation through a10-kDa cutoff filtration column (Amicon). Equal volumes of eachfraction were analyzed by SDS-PAGE and immunoblotting.Immunocytochemistry on tsA-201Cells—Themethod used is

essentially as described previously (22). Cellswere fixedwith 4%paraformaldehyde in TBS for 5 min at room temperature andthen washed twice with TBS. Either no permeabilization stepwas used or cells were permeabilized for 15 min with 0.02%Triton X-100. The primary rat anti-HA (1:500) or mouse anti-�2-1 (1:100) Abs were incubated overnight at 4 °C, followed byTexas Red-conjugated anti-rabbit, Texas Red-conjugated anti-mouse, Alexa Fluor 488-conjugated anti-mouse (MolecularProbes) or biotinylated anti-rat Abs, and streptavidin-AlexaFluor 488 (Invitrogen). All secondary antibodies were used in1:500 dilution. 4�,6-Diamidino-2-phenylindole (DAPI) wasused to visualize the nuclei. Cells were mounted in Vectashield(Vector Laboratories, Burlingame, CA) to reduce photobleach-ing. Cells were examined on a confocal laser scanning micro-scope (Zeiss LSM), using a �40 (1.3 NA) or �63 (1.4 NA) oil-immersion objective. Confocal optical sections were 1 �m.Photomultiplier settings were kept constant in each experi-

ment, and all images were scanned sequentially. Image process-ing was performed using ImageJ. Data illustrated are represent-ative of more than 10 cells each.Live Labeling of DRG Neuron Cultures—DRG cultures were

incubated with monoclonal rat anti-HA Ab (1:250, RocheApplied Science) for 1 h at 37 °C inmedium containing (inmM)the following: 145 NaCl; 5 KCl; 2 CaCl2; 1MgSO4; 10 Hepes; 10glucose, pH 7.4. Neurons were washed with PBS, fixed with 4%paraformaldehyde in PBS for 5min and then blocked for 30–60min with PBS supplemented with 10% goat serum. SecondaryAb (anti-rat Alexa Fluor 555, 1:500, Invitrogen) was applied for2 h at room temperature. Samples were mounted and scannedas above.Electrophysiology—Calcium channel currents were recorded

in tsA-201 cells by whole cell patch-clamp recording, essen-tially as described previously (23). The internal (pipette) andexternal solutions and recording techniques were similar tothose previously described (24). The patch pipette solutioncontained in mM: cesium aspartate, 140; EGTA, 5; MgCl2, 2;CaCl2, 0.1; K2ATP, 2;Hepes, 10; pH7.2, 310mosMwith sucrose.The external solution for recording Ba2� currents contained inmM: tetraethylammonium bromide, 150; KCl, 3; NaHCO3, 1.0;MgCl2, 1.0; Hepes, 10; glucose, 4; BaCl2, 1 or 5 as indicated, pH7.4, 320mosMwith sucrose. Pipette of resistance 2–4megohmswere used. An Axopatch 1D amplifier (Axon Instruments, Bur-lingame, CA) was used, and data were filtered at 1–2 kHz anddigitized at 5–10 kHz. Current records were subjected to leakand residual capacitance current subtraction (P/8 protocol).Analysis was performed using PCLAMP9 (Molecular Devices)and Origin 7 (Microcal Origin, Northampton, MA).Current-voltage (I-V) plots were fit with a modified Boltz-

mann equation as described previously (25), for determinationof the voltage for 50% activation (V50, act).Where data are givenas mean � S.E., statistical comparisons were performed usingeither Student’s t test or analysis of variance with post hoc test,as appropriate.

RESULTS

Expression of�2�-1�C-term—Ananchorless�2�-1 construct(�2�-1�C-term-HA) was made with a C-terminal HA tag (Fig.1A, construct iii), to monitor expression. It was expressed intsA-201 cells, and expression was compared with WT �2�-1(Fig. 1A, construct i) and �2�-1mid-HA (Fig. 1A, construct ii) intheWCL (Fig. 1B, lanes 2–4). The presence of a mid-HA tag inthis position within �2-1 does not affect the function of thefull-length �2�-1 (see Fig. 4A). Similar expression levels and asimilar level of N-linked glycosylation, as shown by treatmentwith PNGase-F, were observed (Fig. 1B, top two panels, �2-1Ab).As found previously for heterologous expression of WT �2�

subunits (15, 26), both �2�-1 mid-HA and �2�-1�C-term-HAwere only partially cleaved into �2 and � (Fig. 1B, lanes 6–8).Partial cleavage is the reason that the �2-1 Ab recognizes twobands in reduced samples. These can best be distinguished fol-lowing deglycosylation and have molecular masses of �130kDa (�2�-1 “uncleaved form”) and �105 kDa (�2-1 “cleavedform”) (Fig. 1B, top right panel). As expected from the locationof the HA epitope, in reduced deglycosylated samples, the HA




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Ab revealed bands associated with uncleaved �2�-1�C-term-HA (�130 kDa) and �-1�C-term-HA peptide (�19 kDa)(Fig. 1B, lane 8). Note that the �2�-1�C-term-HA showedincreased HA immunoreactivity, compared with full-length�2�-1 mid-HA (Fig. 1B, middle panels). This is likely due tobetter accessibility of the C-terminally located HA epitope tag,rather than to increased expression levels of �2�-1�C-term-HA, as the corresponding bands revealed by the�2-1Abwere ofsimilar intensities (Fig. 1B, top panel). To examine whether the

C-terminal HA tag was affecting the properties of the protein,we also made a truncated �2�-1�C-term construct with aninternal HA tag (�2�-1 mid-HA �C-term) (supplemental Fig.1A). This construct was similarly expressed (supplemental Fig.1B) and glycosylated (data not shown), compared with �2�-1mid-HA, and it was recognized by both �2-1 and HA Abs.Truncated �2�-1�C-term Is Secreted into the Medium—In

medium conditioned by tsA-201 cells expressing �2�-1�C-term-HA, we observed a band of �165 kDa, which was immu-noreactive to both�2-1 andHAAbs (Fig. 2A, lane 4), indicatingthat the anchorless construct was secreted. This band wasabsent from medium conditioned by untransfected cells orthose expressing full-length WT �2�-1 or �2�-1 mid-HA (Fig.2A, lanes 1–3).The secreted anchorless protein was glycosylated, because

PNGase-F treatment shifted the predominant band from�165to�130 kDa, equivalent to the observed size of the unprocessedform of �2�-1�C-term-HA after deglycosylation (Fig. 2B, lanes1 and 2; see also Fig. 1B). Surprisingly, we barely detected anyproteolytically processed �2�-1�C-term in the medium, asdemonstrated by the absence of �-1�C-term-HA peptide inFig. 2A (lane 4).Transfection with increasing amounts of �2�-1�C-term-HA

cDNA (3, 6, or 10 �g) resulted in increased expression andsecretion of �2�-1�C-term-HA at 72 h, as demonstrated byWestern blot analysis (Fig. 2, C and D). We quantified the per-centage of secreted �2�-1�C-term-HA, relative to the totalamount expressed, by measuring the mean intensity of bandsand taking into account the total volume of each fraction, asdescribed under “Experimental Procedures.” For this calcula-tion, we have summed the cleaved and uncleaved �2�-1 bandsdetectable inWCL and in the media to obtain the total expres-sion. The proportion of �2�-1�C-term-HA secreted into themedium was 75.6 � 2.8% of the total amount of �2�-1�C-term-HA expressed (n � 3; Fig. 2E). Truncation within thehydrophobic domain resulting in a longer �2�-1 construct waspreviously shown to result in a construct that was partiallysecreted into themedium (27). The truncated constructwith aninternal HA tag (�2�-1 mid-HA �C-term) also showed secre-tion into the medium as the uncleaved protein (supplementalFig. 1C).Despite Removal of the Membrane Anchor, �2�-1�C-term

Remains in Part Associated with the Plasma Membrane—Wecompared the distribution of full-length �2�-1 mid-HA to thatof anchorless �2�-1�C-term-HA, following heterologousexpression in tsA-201 cells. Unexpectedly, we found that �2�-1�C-term-HA was also associated with the plasma membranein nonpermeabilized cells (Fig. 3A, panel ii), to a similar extentto the robust cell surface staining observed for�2�-1mid-HA innonpermeabilized cells (Fig. 3A, panel i) and to WT �2�-1, asdemonstrated by anti-�2-1Ab staining (supplemental Fig. 2). Inpermeabilized cells, immunostaining was also observed intra-cellularly in both conditions (Fig. 3A, panels iii and iv). Associ-ation of�2�-1�C-term-HAwith the plasmamembranewas notaffected by co-transfection with an �1 and � subunit (CaV2.2and �1b; Fig. 3B; supplemental Fig. 2, compare A and B), indi-cating that �2�-1�C-term-HA does not require associationwith other calcium channel subunits for its membrane localiza-

FIGURE 1. Biochemical properties of �2�-1�Cterm-HA expressed in tsA-201 cells. A, scheme of �2�-1 constructs used. The site of truncation and theposition of the HA epitope (light gray box) are marked as follows: construct i,wild-type (WT) �2�-1; construct ii, full-length �2�-1 with an internal HAepitope (�2�-1 mid-HA); construct iii, truncated �2�-1 with C-terminal HA(�2�-1�C-term-HA). The amino acid sequence of the C terminus of rat �2�-1,with the site of truncation at the GPI anchor attachment site predicted in ourprevious study (15) (�-site; C in CGG underlined), and the C-terminal hydro-phobic sequence (underlined) are shown at top. B, WCL from untransfectedtsA-201 cells (lanes 1 and 5) or cells transfected with WT �2�-1 (lanes 2 and 6),�2�-1 mid-HA (lanes 3 and 7), or �2�-1�C-term-HA (lanes 4 and 8), eitheruntreated (left panel) or treated with PNGase-F (right panel). Bands were visu-alized with the indicated Abs, either against �2-1 (top panel) or against the HAepitope (middle two panels). The arrows on the right indicate bands corre-sponding to the deglycosylated proteins shown in the scheme in A, eitheruncleaved �2�-1 (upper band), cleaved �2-1 (lower band), or free �-1. Thelower part of the same membrane was blotted with anti-GAPDH Ab as aloading control (bottom panel). WB, Western blot.




http://ww

w.jbc.org/

Dow

nloaded from









http://www.jbc.org/

tion. Furthermore, the fact that the HA epitope is exposed innonpermeabilized �2�-1�C-term-HA-transfected cells indi-cates that it has not utilized another hydrophobic region in theprotein as a transmembrane domain, in which case the C-ter-minal HA tag would be intracellular. We also observed that,when expressed by nucleofection in DRG neurons, �2�-1�C-term-HA was associated with the cell surface, both in the cellbodies and in neurites, to a similar extent to�2�-1mid-HA (Fig.3, C and D).

To rule out that the C-terminal HA tag was in some wayartifactually mediating noncovalent membrane association, wealso used the�2�-1mid-HA�C-term construct.We found that

this construct behaved similarly to �2�-1�C-term-HA, beingassociated with the plasma membrane in nonpermeabilizedcells (supplemental Fig. 1D), as well as being secreted (supple-mental Fig. 1C).

�2�-1�C-term-HA Retains Partial Functionality, in Terms ofEnhancing Calcium Currents—First, we showed that the full-length �2�-1 mid-HA construct used in this study retained fullfunctionality in comparison with untagged full-length �2�-1(WT �2�-1), in terms of increasing calcium currents relative toCaV2.1/�1b alone (Fig. 4A). We then examined the ability of�2�-1�C-term-HA to enhance CaV2.1/�1b calcium currents,and surprisingly, we found that it retained substantial ability to

FIGURE 2. Truncated �2�-1�C-term-HA, but not full-length WT �2�-1 or �2�-1 mid-HA, is secreted into the medium as a glycosylated protein. A,conditioned medium from untransfected tsA-201 cells (lane 1) or cells transfected with the full-length WT �2�-1 (lane 2), �2�-1 mid-HA (lane 3), or �2�-1�C-term-HA (lane 4) was concentrated and analyzed as described under “Experimental Procedures.” The Western blots (WB) were revealed with �2-1 Ab (upperpanel) and HA Ab (lower panel). The arrows indicate a single band of �165 kDa corresponding to the glycosylated uncleaved �2�-1�C-term-HA, which was themain form found secreted in the medium. No secreted material was seen using WT �2�-1 or �2�-1 mid-HA-transfected cells (lanes 2 and 3) or untransfected cells(lane 1). B, conditioned medium, from cells transfected with �2�-1�C-term-HA, was either untreated (lane 1) or treated with PNGase-F (lane 2). Bands werevisualized with HA Ab. C, WCL from cells transfected with 3, 6, or 10 �g of �2�-1�C-term-HA cDNA was analyzed on blots visualized with anti-HA Ab. 20 �g oftotal deglycosylated protein was loaded for each transfection condition. D, conditioned medium from the same cells was also blotted against HA Ab. An equalamount of medium was loaded for each condition. E, % of the total �2�-1�C-term-HA that was either secreted (black bar) or present in the WCL (gray bar) wasdetermined from three independent experiments (�S.E.), taking into account both the total volumes of the samples and the intensity of Western blot bands(see “Experimental Procedures”). Examples of gels used for this quantification are shown in C and D. Both the cleaved and uncleaved forms of �2�-1-�C-term-HA were included in this analysis.




http://ww

w.jbc.org/

Dow

nloaded from




http://www.jbc.org/

cause an increase in these currents, relative to no �2� co-ex-pression (Fig. 4, B and C), although the enhancement wassmaller than that observed with WT �2�-1 (Fig. 4, B and C). Inthe absence of �2�-1, peak IBa at � 5 mV (from Fig. 4B) was9.0 � 2.7% of that in the presence of WT �2�-1, whereas for�2�-1�C-term-HA it was 38.5 � 7.6%, representing a 3.8-foldincrease over no �2� (p � 0.001 compared with no �2�-1, anal-ysis of variance, and Dunnett’s post hoc test).As observed previously (28), WT �2�-1 increased the inacti-

vation rate of the peak CaV2.1/�1b IBa (Fig. 4, D and E), �inactbeing reduced from 177 ms in the absence of �2�-1 to 120 mswhen WT �2�-1 was co-expressed. The truncated �2�-1�C-term-HA had a less marked effect on inactivation, with �inactbeing 145 ms (Fig. 4, D and E).Does Intercellular Transfer Occur from Secreted Anchorless

�2�-1?—To examine whether transcellular transfer of secreted�2�-1�C-term-HAmight occur via themedium to neighboringcells and contribute to plasma membrane association or cal-

cium current enhancement, tsA-201 cells expressing either�2�-1 mid-HA or �2�-1�C-term-HA were co-cultured withcells expressing CaV2.1/�1b/GFP. The cells were transfectedseparately and then washed and mixed after 5 h in culture.After a further 48 h in culture, cell surface �2�-1 was exam-ined by immunocytochemistry in fixed nonpermeabilizedcells, for both �2�-1�C-term-HA and �2�-1 mid-HA (Fig.5A, panels i and ii, red staining, white arrows), whereas GFP-positive cells were rarely found to be associated with any redstaining. We found a very small amount of evidence of pos-sible transfer of �2�-1�C-term-HA to areas of the plasmamembrane of CaV2.1/�1b/GFP-transfected cells (indicatedby a yellow arrow in Fig. 5A, panel i). No evidence of transferof full-length �2�-1 mid-HA was observed (Fig. 5A, panel ii).These results suggest that attachment of �2�-1�C-term-HAto cell surface components occurs mainly during the secre-tory process, rather than via secretion into the medium andreattachment.

FIGURE 3. A proportion of �2�-1�C-term-HA is associated with the plasma membrane, independently of other CaV channel subunits. A, confocalimages of tsA-201 cells expressing either �2�-1 mid-HA (left, panels i and iii) �2�-1�C-term-HA (right, panels ii and iv). Immunostaining was performed usinganti-HA Ab. Upper row, nonpermeabilized cells showing surface expression of both constructs. Lower row, cells were permeabilized with 0.02% Triton X-100 toshow total expression. DAPI was used to visualize the nucleus (blue). B, confocal images of tsA-201 cells expressing �2�-1 mid-HA (left, panels i and iii) or�2�-1�C-term-HA (right, panels ii and iv) both co-transfected with CaV2.2 and �1b, stained with anti-HA Ab as in A. C, three-dimensional projection images ofDRG neurons transfected with CFP/�2�-1 mid-HA or CFP/�2�-1�C-term-HA. Cell surface detection of exogenous �2�-1 subunits was obtained by incubation oflive neuronal cultures with anti-HA Ab (right) in CFP-positive neurons (left). Images are representative of nine CFP-positive neurons examined in each condition.D, higher magnification images of a neuronal process expressing CFP and labeled for surface �2�-1-mid-HA (upper panels) or �2�-1�C-term-HA (lower panels).Scale bars, 20 �m in A and B and 10 �m in C and D.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Furthermore, co-culture of cells expressing CaV2.1/�1b for24–36 h directly with cells transfected with �2�-1�C-term-HA, with �2�-1 mid-HA, or with empty vector as a control didnot cause any increase in IBa recorded from these cells (Fig. 5B).Similarly, culture of tsA-201 cells expressingCaV2.1/�1b for 5 hwith medium conditioned by cells expressing �2�-1�C-term-HA did not cause any increase in IBa compared with cellsincubated with unconditioned medium (data not shown).

These results indicate that the enhancement of calcium cur-rents only occurs after co-expression of �2�-1�C-term-HAwith the calcium channel �1 and � subunits, and it is likely toinvolve an interaction of this construct with CaV2.1/�1b chan-nel complexes intracellularly, rather than once the �1/� com-plex has reached the plasma membrane.Anchorless �2�-1�C-term-HA at the Plasma Membrane Is

Proteolytically Processed to �2 and � to a Greater Extent than

FIGURE 4. �2�-1�C-term-HA produces a partial enhancement of calcium channel currents. A, I-V relationships for CaV2.1 plus �1b, either alone (blacksquares, n � 6) or co-expressing either WT �2�-1 (black circles, n � 6) or �2�-1 mid-HA (white triangles, n � 7). Data are mean � S.E. Data between 30 and �50 mV were fit with a modified Boltzmann function. The mean V50, act values obtained were 4.2, 7.3, and 5.3 mV, respectively. B, I-V relationships for CaV2.1plus �1b, either alone (white squares, n � 8) or co-expressing either WT �2�-1 (black circles, n � 8) or �2�-1�C-term-HA (gray triangles, n � 12). Data are mean �S.E. Data between 30 and � 45 mV were fit with a modified Boltzmann function. The V50, act values obtained were 4.1, 4.7, and 5.5 mV, respectively. C,representative current traces for the three transfection conditions in response to depolarizing steps from 30 to �10 mV from VH of 90 mV as shown;calibration bars refer to all traces. D, representative IBa (normalized to the peak current), in response to 900 ms of depolarization to �5 mV, to show differencesin inactivation kinetics between CaV2.1/�1b plus WT �2�-1 (rapidly inactivating black trace), without �2� (slowly inactivating black trace) and with �2�-1�C-term-HA (gray trace). E, mean �inact for IBa at �5 mV for WT �2�-1 (black bar, n � 7), in comparison with no �2� (white bar, n � 5) and �2�-1�C-term-HA (gray bar,n � 6). IBa was recorded using 1 mM Ba2�. Statistical differences were determined using one-way analysis of variance and Dunnett’s post hoc test, where **, p �0.01, NS � nonsignificant.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

the Full-length �2�-1 mid-HA—We next examined the proper-ties of the �2�-1�C-term-HA on the cell surface by cell surfacebiotinylation (Fig. 6A). Interestingly, we observed that �2�-1�C-term-HA in the cell surface biotinylated fractions wasproteolytically cleaved to form �2-1 to a greater extent, com-pared with the WCL (Fig. 6A, compare lanes 3 and 6). Theproteolytic cleavage, quantified from three independent exper-iments, revealed an almost 2-fold increase of processing for�2�-1�C-term-HA from the cell surface biotinylated fraction(80 � 4.7% cleavage) compared with the corresponding WCL(43 � 6%) (Fig. 6B). In contrast, there was a smaller increasefrom 38 � 4.5 to 50 � 3.3%, respectively, for full-length �2�-1mid-HA (Fig. 6). Therefore, the anchorless �2�-1�C-term-HAthat remained attached to the cell surface by an as yet unknown

mechanismwasmainly processed to�2-1 and �. In contrast, thesecreted formof�2�-1�C-term-HAwas predominantly unpro-cessed, as demonstrated by the absence of �-HA in themedium(Fig. 2A).Some �2�-1�C-term Is Associated with Lipid Rafts—Because

some�2�-1�C-term-HAwas associated with the plasmamem-brane, we also examined whether it was still associated withDRMs, also termed lipid rafts, as demonstrated for WT �2�-1and other �2� subunits (15, 26). We isolated DRMs from cellsexpressingWT�2�-1,�2�-1mid-HA, or�2�-1�C-term-HAbydiscontinuous sucrose gradient centrifugation as describedpreviously (26). Untransfected tsA-201 cells express a smallamount of endogenous �2�-1 (Fig. 7A), which localizes inDRMs. In transiently transfected tsA-201 cells, 66 � 5.7% of

FIGURE 5. Examination of whether intercellular transfer occurs for �2�-1�C-term-HA. A, images of cells expressing CaV2.1 plus �1b and GFP, mixed andco-cultured with cells expressing either �2�-1�C-term-HA (left) or �2�-1 mid-HA (right). Cells were not permeabilized, and the HA tag is seen as red immuno-staining, on cells transfected with �2�-1�C-term-HA or WT �2�-1 mid-HA (white arrows). Cells expressing GFP were rarely found to have some small regions ofred surface stain in the �2�-1�C-term-HA condition (solid yellow arrow) but not in the �2�-1 mid-HA condition. DAPI was used to visualize cell nuclei (blue). Scalebars, 10 �m. B, left, I-V relationship for cells expressing CaV2.1 plus �1b and GFP, co-cultured with cells expressing empty vector (white squares, n � 7) or either�2�-1 mid-HA (black circles, n � 5) or �2�-1�C-term-HA (gray triangles, n � 7). Data are mean � S.E. Right, representative current traces for each condition inresponse to depolarizing steps from 30 to �10 mV from VH of 90 mV as shown; calibration bars refer to all traces. IBa was recorded using 5 mM Ba2� to recordthe small currents accurately.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

WT �2�-1 (Fig. 7B, fractions 5–7; n � 3) and 59.5 � 5.7% of�2�-1 mid-HA (Fig. 7C, n � 3) were found in DRM fractions.TheDRM localization of the endogenousmarker flotillin-1 wasquantified to be 85.4 � 3.9%, whereas the transferrin receptor,which was used a marker for the soluble fractions, was essen-

tially absent from DRMs (n � 3; Fig. 7, A--D). In contrast, weobserved a large proportion of �2�-1�C-termHA (Fig. 7D) and�2�-1 mid-HA �C-term (supplemental Fig. 1) to be in the sol-uble fractions (11–13), as judged by both �2-1 and HA immu-noreactivity. This distribution would be expected for a solubleprotein in the process of being secreted. However, a significantfraction of both anchorless constructs (22.9 � 4.7% of �2�-1mid-HA �C-term and 29.1 � 2.1% of �2�-1�C-term-HA, n �3) remained associatedwith theDRMs (Fig. 7D and supplemen-tal Fig. 1E). This result suggests that the GPI anchor is not theonly means by which the protein is retained in DRMs.As observed previously, proteolytic cleavage of �2�-1 to �2

and �wasmore pronounced in isolatedDRMs than inWCL (15,26). However, that increase was greater for �2�-1�C-term-HA(Fig. 7E) than for full-length �2�-1 mid-HA (Fig. 7F, quantifi-cation included in Fig. 6B). Therefore, similarly to the cell sur-face biotinylated �2�-1�C-term-HA, isolated DRM fractionsalso contained more processed �2�-1�C-term-HA (Fig. 6B).How Is�2�-1�C-termAssociatedwith the PlasmaMembrane—

We examined a number of possibilities that could be responsi-ble for the extrinsic interaction of �2�-1�C-term with the cellsurface. The fact that theHA epitope at theC terminus is acces-sible in nonpermeabilized cells strongly suggested that thetruncated construct does not adopt a transmembrane configu-ration. To rule out the possibility that �2�-1�C-term-HAformed an integral membrane protein, we treated DRMs iso-lated from cells expressing �2�-1�C-term-HA or �2�-1mid-HA with neutral (pH 7.4) or basic carbonate (pH 11.5)buffers. This method has been used previously to examinewhether proteins are extrinsically associated with the mem-brane (16, 21, 29). We found that a high pH wash could releasemore �2�-1�C-term-HA from DRMs into the supernatant,which was not the case for �2�-1 mid-HA. Less �2�-1�C-term-HAwas released by neutral pH washes (Fig. 8). This indi-cates that �2�-1�C-term-HA is not an integral membrane pro-tein, rather the interaction involves electrostatic association.To test whether, similar to prion protein, the interaction of

�2�-1�C-term-HA with DRMs and cell surface involved inter-action with glypicans, we treated cells with heparin (100�g/ml), which should interfere with any interaction with hepa-ran sulfate proteoglycans. Incubation of either isolated DRMsor transfected cells with heparin had no effect on the associa-tion of �2�-1�C-term-HA with DRMs or the plasma mem-brane (data not shown). We also found that mutation of themetal ion-dependent adhesionmotif in the vonWillebrand fac-tor-A domain (6) of �2�-1�C-term-HA did not prevent theprotein from interacting with the plasma membrane (data notshown), indicating that the interaction does not require thissite.

DISCUSSION

The recent discovery that �2� subunits can be anchored tothe membrane via a GPI moiety rather than a transmembraneprotein domain has provided a novel point of view concerningsome of their previously investigated properties as key modu-lators of CaV currents (10, 11). However, it has also opened newquestions related to the role of membrane anchoring for thephysiological function of �2� proteins. The initial aim of this

FIGURE 6. �2�-1 �C-term-HA associated with the plasma membrane ishighly processed to �2-1 and �-1. A, samples of deglycosylated WCL (leftpanel) and precipitated cell surface-biotinylated proteins (right panel) fromuntransfected cells (U/T, lanes 1 and 4) and cells transfected with �2�-1mid-HA (lanes 2 and 5) or �2�-1�C-term-HA (lanes 3 and 6) were resolved on a3– 8% Tris acetate gel. Western blots (WB) were revealed with �2-1 Ab. Lowerpanel, Western blotting with anti-Akt Ab (cytoplasmic protein) was used as abiotinylation control. Note the difference in relative proportions between thebands corresponding to �2�-1 and �2-1 in WCL (lanes 2 and 3) and cell surfacebiotinylated fractions (lanes 5 and 6). B, proteolytic cleavage of �2�-1 to �2-1was calculated for different subcellular fractions for �2�-1 mid-HA (white bars)and �2�-1�C-term-HA (black bars) using blots revealed with �2-1 Ab (n � 3independent experiments � S.E.); an example of one of the blots used forquantification of cleavage in the WCL and on the cell surface is shown in A,and for the DRM fraction is shown in Fig. 7, E and F. *, p � 0.05 compared with�2�-1 mid HA, Student’s t test.




http://ww

w.jbc.org/

Dow

nloaded from




http://www.jbc.org/

FIGURE 7. �2�-1�C-term-HA is partially associated with DRMs. A–D, WCL from untransfected tsA-201 cells (A) or those expressing WT �2�-1 (B), �2�-1mid-HA (C), or �2�-1�C-term-HA (D) were subjected to sucrose gradient fractionation to isolate DRMs (fractions 5–7). Fractions were examined using �2-1 (toppanels) and HA (middle panels) Abs. In each case, the distributions of the endogenous DRM marker, flotillin-1, and non-raft marker, transferrin receptor (TfR)were also examined (bottom two panels). Data are representative of at least three experiments. Quantification of % of material present in DRMs is given in the“Results.” E and F, isolated DRM fractions of �2�-1�C-term-HA (E) and �2�-1 mid-HA (F)- transfected cells were deglycosylated with PNGase-F (lane 2 comparedwith lane 1) to show the presence of �2-1 (E and F) and �-1-HA (E). The % cleavage of �2�-1 into �2-1 was quantified for three independent experiments andincluded in the graph shown in Fig. 6B. Note that proteolytic cleavage of the protein into �2-1 and �-1 in DRMs is increased relative to WCL, as also shown inquantification on Fig. 6B.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

study was to address some of those issues with respect tomem-brane anchoring of �2�-1.

Previous in vitro studies have shown that �2�-1, �2�-2, and�2�-3 subunits all increase themaximumconductance ofwholecell calcium channel currents arising from �1/� subunit com-binations for the CaV1 and CaV2 classes, in several differentexpression systems (30–34). For�2�-1, it was previously shownthat expression of the �2-1 or �-1 alone did not enhance cal-cium currents through CaV2.1 channels (10). Furthermore,these authors also found that expression of �2�-1 with thetransmembrane segment from another protein (adhalin) didnot enhance calcium currents, which is nowunsurprising in thelight of our recent finding that the �2� subunits can form GPI-anchored proteins (15). Replacing the transmembrane segmentwith an unrelated sequencemight interfere with the cleavage ofGPI-anchoring signal sequence and the subsequent attachmentof the GPI moiety. It has been found that uncleaved GPI pre-cursor proteins show aggregation in the endoplasmic reticulum(35).It was initially suggested that the transmembrane segment of

� was required for calcium current stimulation, and the entireextracellular portion of �2�-1 was implicated in subunit inter-action with CaV2.1 (10). Moreover, co-expression of CaV2.1channels with �-1 alone affected the biophysical properties ofthe currents but did not enhance their amplitudes (26). Wehave now revisited this issue with respect to our recent findingsthat �2� subunits can form GPI-anchored proteins.We have created an anchorless �2�-1 (�2�-1�C-term) by

adding a stop codon immediately prior to the predicted site ofattachment of the GPI moiety (15, 36). This approach has pre-viously been employed successfully to study the role of GPI

anchoring on the behavior of the prion protein (16). By thismeans, we obtained a soluble protein deprived of hydrophobicmembrane anchoring but containing �2-1 and all the extracel-lular parts of �-1, both of which were both previously shown tobe of major importance for channel regulation and interaction(11, 26). In our study we found the surprising result that theC-terminalmembrane anchoring is not the only determinant ofthe ability of �2�-1 to enhance calcium channel currents,because �2�-1�C-term still produced a substantial increase ofcalcium currents when co-expressed with CaV2.1 and �1b.Interestingly, we did not observe such effects upon externalapplication of secreted �2�-1�C-term to cells previously trans-fected with CaV2.1 and �1b, suggesting that an intracellularinteraction with other subunits is required for the functionalityof �2�-1 in the calcium channel complex. Moreover, this resultimplies that other factors than membrane anchoring are likelyto be involved in the current-potentiating effects of �2�-1.

As expected, a large proportion of �2�-1�C-term is secretedinto the medium, when it is expressed in tsA-201 cells. How-ever, we also found, using both immunocytochemistry and cellsurface biotinylation, that �2�-1�C-term constructs, despitethe lack of a C-terminal membrane anchor, remain partiallyassociated with the plasma membrane. However, �2�-1�C-term-HA does not utilize another hydrophobic region as atransmembrane anchor, because both the�2-1 andHAAbs canaccess their epitopes in nonpermeabilized cells.The interaction of �2�-1�C-term with membranes occurs

via a noncovalent linkage, because alkaline carbonate treatmentdisrupted theDRMassociation. Furthermore, the interaction isnot affected by the presence or absence of other calcium chan-nel subunits. Our finding that secreted �2�-1�C-term does notre-attach to the plasma membrane following secretion furthersuggests that its association with the plasma membrane occursduring the maturation and trafficking of the protein.There are 16 predictedN-linked glycosylation sites in the rat

�2�-1 sequence. We have found that secreted �2�-1�C-termwas heavily glycosylated, because PNGase-F removed allN-gly-cosylation (�35 kDa) producing a shift to an apparent mass of�130 kDa, corresponding to unprocessed deglycosylated �2�-1�C-term (Fig. 2B).We have shown in our previous studies that �2� subunits are

strongly localized in DRMs, both in native tissue and followingheterologous expression (15, 26). The GPI-anchoring of �2�subunits, as for other proteins, is likely to be an importantdeterminant of their localization in these domains (15) but notthe sole factor (16, 37). This is reinforced by the finding that�2�-1�C-term remains, in part, associated with DRMs. Muchof theDRMfraction is derived from the cholesterol-rich plasmamembrane, as determined by combined cell surface biotinyla-tion and DRM studies (17). Thus, the partial association of the�2�-1�C-term constructs with DRMs is in agreement with ourevidence that some �2�-1�C-term is associated with theplasma membrane.Removal of the C-terminal GPI anchor signal sequence from

prion protein did not completely prevent its lipid raft or mem-brane association (16), although the anchorless prion proteinwas mainly secreted. Furthermore, prion protein was found tointeract with GPI-anchored heparan sulfate proteoglycans

FIGURE 8. �2�-1�C-term-HA, but not �2�-1 mid-HA, is released frommembranes by alkaline carbonate treatment. Isolated DRM fractions fromtsA-201 cells transfected with �2�-1 mid-HA (lanes 1, 2, 5, and 6) or �2�-1�C-term-HA (lanes 3, 4, 7, and 8) were treated with buffer composed of either 0.1M Tris, pH 7.4, or 0.1 M K2CO3, pH 11.5, as indicated, to dissociate extrinsicallyassociated proteins. After centrifugation, the pellets (left panel) and superna-tants (right panel) were deglycosylated with PNGase-F followed by Westernblotting (top panel �2-1 Ab; bottom panel anti-HA Ab to reveal the �-1-HApeptide).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

(glypicans), which play a role in retaining it in DRM fractions(17). In contrast, in this study heparin, which should disruptsuch an interaction, did not prevent cell surface or DRM asso-ciation of �2�-1�C-term.We have found previously that heterologously expressed �2�

proteins are only partially proteolytically processed into �2 and� in many expression systems (15, 26, 38). This behavior ofexpressed �2� subunits contrasts with the complete processingof native �2� proteins, where no full-length �2� is observed (15,38). In this study, we found that �2�-1�C-term is also incom-pletely processed, and in particular the secreted form showsvery little proteolytic cleavage. In contrast, �2�-1�C-term inboth the DRM fraction and the cell surface-biotinylated frac-tion exhibits a much greater proportion of cleaved �2-1 and�-1�C-term than the WCL or the secreted fraction. Theseresults indicate that the protease in question is likely to beabsent from the constitutive secretory pathway, but it is presentin the biogenesis pathway for membrane components. Theincreased proteolytic cleavage of plasmamembrane and DRM-associated �2�-1�C-term, compared with full-length �2�-1,may result from its greater flexibility and availability as a sub-strate. Furthermore, a number of proteases have also beenlocalized to lipid rafts (39, 40), whichmay relate to the increasedprocessing of �2� in DRM fractions.

Because it has been found that the proteolytic cleavage of�2�-1 is important for its function to enhance calcium channelcurrents (41), it is likely that the proteolytically cleaved �2�-1�C-term associated with the plasmamembrane is responsiblefor its function, but this remains to be conclusively demon-strated by using a protease-deficient mutant of �2�-1�C-term.The main physiological relevance of this study is that the

truncation of �2�-1 at its predicted GPI anchor site does notprevent the ability of this construct to affect calcium channelfunction. This indicates that intrinsic association of �2�-1 tothe plasmamembrane is not essential for its function. Our find-ing that anchorless �2�-1 is still in part extrinsically associatedwith the plasma membrane and DRMs suggests that �2�-1�C-term may be processed by two alternative routes, a secretorypathway and a membrane biogenesis pathway, and in the latterpathway it becomes associated with one or more binding part-ners that determine its association with membranes. This nowgives us an important means of identifying the physiologicalbinding partner(s) of �2� proteins involved in controlling theirtrafficking and cell surface localization.Our future researchwill therefore be aimed at identifying the

binding partner(s) with which �2�-1�C-term is interactingduring the trafficking process, and which may also serve totether it to the plasmamembrane. It will also be of great interestto determine whether this interaction is related to the surpris-ing ability of �2�-1�C-term to produce a partial enhancementof calcium channel currents.

REFERENCES1. Catterall, W. A. (2000) Structure and regulation of voltage-gated Ca2�

channels. Annu. Rev. Cell Dev. Biol. 16, 521–5552. Dolphin, A. C. (2003)� subunits of voltage-gated calcium channels. J. Bio-

energ. Biomembr. 35, 599–6203. Leroy, J., Richards,M.W., Richards,M. S., Butcher, A. J., Nieto-Rostro,M.,

Pratt, W. S., Davies, A., and Dolphin, A. C. (2005) Interaction via a key

tryptophan in the I-II linker ofN-type calcium channels is required for �1but not for palmitoylated �2, implicating an additional binding site in theregulation of channel voltage-dependent properties. J. Neurosci. 25,6984–6996

4. Waithe, D., Ferron, L., Page, K. M., Chaggar, K., and Dolphin, A. C. (2011)�-Subunits promote the expression of CaV2.2 channels by reducing theirproteasomal degradation. J. Biol. Chem. 286, 9598–9611

5. Cantí, C., Davies, A., and Dolphin, A. C. (2003) Calcium channelalpha2delta subunits: structure, functions and target site for drugs. Curr.Neuropharmacol. 1, 209–217

6. Cantí, C., Nieto-Rostro,M., Foucault, I., Heblich, F.,Wratten, J., Richards,M.W.,Hendrich, J., Douglas, L., Page, K.M., Davies, A., andDolphin, A. C.(2005) The metal ion-dependent adhesion site in the vonWillebrand fac-tor-A domain of �2� subunits is key to trafficking voltage-gated Ca2�

channels. Proc. Natl. Acad. Sci. U.S.A. 102, 11230–112357. Davies, A., Hendrich, J., Van Minh, A. T., Wratten, J., Douglas, L., and

Dolphin, A. C. (2007) Functional biology of the �2� subunits of voltage-gated calcium channels. Trends Pharmacol. Sci. 28, 220–228

8. Ertel, E. A., Campbell, K. P., Harpold,M.M., Hofmann, F.,Mori, Y., Perez-Reyes, E., Schwartz, A., Snutch, T. P., Tanabe, T., Birnbaumer, L., Tsien,R.W., and Catterall,W. A. (2000) Nomenclature of voltage-gated calciumchannels. Neuron 25, 533–535

9. Burgess, D. L., Gefrides, L. A., Foreman, P. J., and Noebels, J. L. (2001) Acluster of three novel Ca2� channel � subunit genes on chromosome19q13.4. Evolution and expression profile of the � subunit gene family.Genomics 71, 339–350

10. Gurnett, C. A., DeWaard,M., and Campbell, K. P. (1996) Dual function ofthe voltage-dependent Ca2� channel �2� subunit in current stimulationand subunit interaction. Neuron 16, 431–440

11. Gurnett, C. A., Felix, R., and Campbell, K. P. (1997) Extracellular interac-tion of the voltage-dependent Ca2� channel �2� and �1 subunits. J. Biol.Chem. 272, 18508–18512

12. Ellis, S. B., Williams, M. E., Ways, N. R., Brenner, R., Sharp, A. H., Leung,A. T., Campbell, K. P., McKenna, E., Koch, W. J., and Hui, A. (1988)Sequence and expression of mRNAs encoding the �1 and �2 subunits of aDHP-sensitive calcium channel. Science 241, 1661–1664

13. Jay, S. D., Sharp, A. H., Kahl, S. D., Vedvick, T. S., Harpold, M. M., andCampbell, K. P. (1991) Structural characterization of the dihydropyridine-sensitive calcium channel�2-subunit and the associated � peptides. J. Biol.Chem. 266, 3287–3293

14. Hoppa,M. B., Lana, B., Margas,W., Dolphin, A. C., and Ryan, T. A. (2012)�2� expression sets presynaptic calcium channel abundance and releaseprobability. Nature 486, 122–125

15. Davies, A., Kadurin, I., Alvarez-Laviada, A., Douglas, L., Nieto-Rostro, M.,Bauer, C. S., Pratt, W. S., and Dolphin, A. C. (2010) The �2� subunits ofvoltage-gated calcium channels formGPI-anchored proteins, a posttrans-lational modification essential for function. Proc. Natl. Acad. Sci. U.S.A.107, 1654–1659

16. Campana, V., Caputo, A., Sarnataro, D., Paladino, S., Tivodar, S., andZurzolo, C. (2007) Characterization of the properties and trafficking of ananchorless form of the prion protein. J. Biol. Chem. 282, 22747–22756

17. Taylor, D. R., Whitehouse, I. J., and Hooper, N. M. (2009) Glypican-1mediates both prion protein lipid raft association and disease isoformformation. PLoS. Pathog. 5, e1000666

18. Tomlinson, W. J., Stea, A., Bourinet, E., Charnet, P., Nargeot, J., andSnutch, T. P. (1993) Functional properties of a neuronal class C L-typecalcium channel. Neuropharmacology 32, 1117–1126

19. Cormack, B. P., Valdivia, R. H., and Falkow, S. (1996) FACS-optimizedmutants of the green fluorescent protein (GFP). Gene 173, 33–38

20. Page, K. M., Heblich, F., Davies, A., Butcher, A. J., Leroy, J., Bertaso, F.,Pratt, W. S., and Dolphin, A. C. (2004) Dominant-negative calcium chan-nel suppression by truncated constructs involves a kinase implicated inthe unfolded protein response. J. Neurosci. 24, 5400–5409

21. Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P. B. (1982) Isolation ofintracellularmembranes bymeans of sodium carbonate treatment. Appli-cation to endoplasmic reticulum. J. Cell Biol. 93, 97–102

22. Brice, N. L., Berrow, N. S., Campbell, V., Page, K. M., Brickley, K., Tedder,I., and Dolphin, A. C. (1997) Importance of the different � subunits in the




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

membrane expression of the �1A and �2 calcium channel subunits. Stud-ies using a depolarization-sensitive �1A antibody. Eur. J. Neurosci. 9,749–759

23. Berrow, N. S., Brice, N. L., Tedder, I., Page, K. M., and Dolphin, A. C.(1997) Properties of cloned rat �1A calcium channels transiently ex-pressed in the COS-7 cell line. Eur. J. Neurosci. 9, 739–748

24. Campbell, V., Berrow, N. S., Fitzgerald, E. M., Brickley, K., and Dolphin,A. C. (1995) Inhibition of the interaction of G protein Go with calciumchannels by the calcium channel�-subunit in rat neurones. J. Physiol. 485,365–372

25. Cantí, C., Davies, A., Berrow,N. S., Butcher, A. J., Page, K.M., andDolphin,A. C. (2001) Evidence for two concentration-dependent processes for�-subunit effects on �1B calcium channels. Biophys. J. 81, 1439–1451

26. Davies, A., Douglas, L., Hendrich, J., Wratten, J., Tran Van Minh, A.,Foucault, I., Koch, D., Pratt, W. S., Saibil, H. R., and Dolphin, A. C. (2006)The calcium channel �2� 2 subunit partitions with CaV2.1 into lipid raftsin cerebellum. Implications for localization and function. J. Neurosci. 26,8748–8757

27. Brown, J. P., andGee, N. S. (1998) Cloning and deletionmutagenesis of the�2� calcium channel subunit from porcine cerebral cortex. Expression ofa soluble form of the protein that retains [3H]gabapentin binding activity.J. Biol. Chem. 273, 25458–25465

28. Felix, R., Gurnett, C. A., De Waard, M., and Campbell, K. P. (1997) Dis-section of functional domains of the voltage-dependent Ca2� channel�2�subunit. J. Neurosci. 17, 6884–6891

29. Brown, D. A., and Rose, J. K. (1992) Sorting of GPI-anchored proteins toglycolipid-enrichedmembrane subdomains during transport to the apicalcell surface. Cell 68, 533–544

30. Walker, D., and DeWaard, M. (1998) Subunit interaction sites in voltage-dependent Ca2� channels. Role in channel function. Trends Neurosci. 21,148–154

31. Mori, Y., Friedrich, T., Kim,M. S.,Mikami, A., Nakai, J., Ruth, P., Bosse, E.,Hofmann, F., Flockerzi, V., and Furuichi, T. (1991) Primary structure andfunctional expression from complementaryDNAof a brain calcium chan-nel. Nature 350, 398–402

32. Klugbauer, N., Lacinová, L., Marais, E., Hobom, M., and Hofmann, F.(1999)Molecular diversity of the calcium channel�2� subunit. J. Neurosci.19, 684–691

33. Dolphin, A. C.,Wyatt, C. N., Richards, J., Beattie, R. E., Craig, P., Lee, J. H.,Cribbs, L. L., Volsen, S.G., and Perez-Reyes, E. (1999)The effect of�2� andother accessory subunits on expression and properties of the calciumchannel �1G. J. Physiol. 519, 35–45

34. Hobom, M., Dai, S., Marais, E., Lacinova, L., Hofmann, F., and Klugbauer,N. (2000) Neuronal distribution and functional characterization of thecalcium channel �2�-2 subunit. Eur. J. Neurosci. 12, 1217–1226

35. Delahunty, M. D., Stafford, F. J., Yuan, L. C., Shaz, D., and Bonifacino, J. S.(1993) Uncleaved signals for glycosylphosphatidylinositol anchoringcause retention of precursor proteins in the endoplasmic reticulum. J. Biol.Chem. 268, 12017–12027

36. Pierleoni, A., Martelli, P. L., and Casadio, R. (2008) PredGPI. A GPI-an-chor predictor. BMC Bioinformatics 9, 392

37. Robinson, P., Etheridge, S., Song, L., Shah, R., Fitzgerald, E. M., and Jones,O. T. (2011) Targeting of voltage-gated calcium channel �2�-1 subunit tolipid rafts is independent from a GPI-anchoring motif. PLoS ONE 6,e19802

38. Douglas, L., Davies, A., Wratten, J., and Dolphin, A. C. (2006) Do voltage-gated calcium channel�2� subunits require proteolytic processing into�2and � to be functional? Biochem. Soc. Trans. 34, 894–898

39. Cordy, J. M., Hussain, I., Dingwall, C., Hooper, N. M., and Turner, A. J.(2003) Exclusively targeting �-secretase to lipid rafts by GPI anchor addi-tion up-regulates�-site processing of the amyloid precursor protein. Proc.Natl. Acad. Sci. U.S.A. 100, 11735–11740

40. Vetrivel, K. S., Cheng, H., Lin, W., Sakurai, T., Li, T., Nukina, N., Wong,P. C., Xu, H., and Thinakaran, G. (2004) Association of �-secretase withlipid rafts in post-Golgi and endosome membranes. J. Biol. Chem. 279,44945–44954

41. Andrade, A., Sandoval, A., Oviedo, N., De Waard, M., Elias, D., and Felix,R. (2007) Proteolytic cleavage of the voltage-gated Ca2� channel �2� sub-unit. Structural and functional features. Eur. J. Neurosci. 25, 1705–1710




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Marianna D'Arco, Michael G. Fadel, Wendy S. Pratt and Annette C. DolphinIvan Kadurin, Anita Alvarez-Laviada, Shu Fun Josephine Ng, Ryan Walker-Gray,

-1 Lacking Its Membrane Anchorδ2αCalcium Currents Are Enhanced by

doi: 10.1074/jbc.M112.378554 originally published online August 6, 20122012, 287:33554-33566.J. Biol. Chem.

10.1074/jbc.M112.378554Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2012/08/06/M112.378554.DC1

http://www.jbc.org/content/287/40/33554.full.html#ref-list-1

This article cites 41 references, 17 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M112.378554

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;287/40/33554&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/287/40/33554

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=287/40/33554&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/287/40/33554

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2012/08/06/M112.378554.DC1

http://www.jbc.org/content/287/40/33554.full.html#ref-list-1

http://www.jbc.org/

calciumcurrentsareenhancedby 2 -1lackingits · pdf file ·...

Documents