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• W2016547597 abstract "Auxiliary channel subunits regulate membrane expression and modulate current properties of voltage-activated Ca2+ channels and thus are involved in numerous important cell functions, including muscle contraction. Whereas the importance of the α1S, β1a, and γ Ca2+ channel subunits in skeletal muscle has been determined by using null-mutant mice, the role of the α2δ-1 subunit in skeletal muscle is still elusive. We addressed this question by small interfering RNA silencing of α2δ-1 in reconstituted dysgenic (α1S-null) myotubes and in BC3H1 skeletal muscle cells. Immunofluorescence labeling of the α1S and α2δ-1 subunits and whole cell patch clamp recordings demonstrated that triad targeting and functional expression of the skeletal muscle Ca2+ channel were not compromised by the depletion of the α2δ-1 subunit. The amplitudes and voltage dependences of L-type Ca2+ currents and of the depolarization-induced Ca2+ transients were identical in control and in α2δ-1-depleted muscle cells. However, α2δ-1 depletion significantly accelerated the current kinetics, most likely by the conversion of slowly activating into fast activating Ca2+ channels. Reverse transcription-PCR analysis indicated that α2δ-1 is the exclusive isoform expressed in differentiated BC3H1 cells and that depletion of α2δ-1 was not compensated by the up-regulation of any other α2δ isoform. Thus, in skeletal muscle the Ca2+ channel α2δ-1 subunit functions as a major determinant of the characteristic slow L-type Ca2+ current kinetics. However, this subunit is not essential for targeting of Ca2+ channels or for their primary physiological role in activating skeletal muscle excitation-contraction coupling. Auxiliary channel subunits regulate membrane expression and modulate current properties of voltage-activated Ca2+ channels and thus are involved in numerous important cell functions, including muscle contraction. Whereas the importance of the α1S, β1a, and γ Ca2+ channel subunits in skeletal muscle has been determined by using null-mutant mice, the role of the α2δ-1 subunit in skeletal muscle is still elusive. We addressed this question by small interfering RNA silencing of α2δ-1 in reconstituted dysgenic (α1S-null) myotubes and in BC3H1 skeletal muscle cells. Immunofluorescence labeling of the α1S and α2δ-1 subunits and whole cell patch clamp recordings demonstrated that triad targeting and functional expression of the skeletal muscle Ca2+ channel were not compromised by the depletion of the α2δ-1 subunit. The amplitudes and voltage dependences of L-type Ca2+ currents and of the depolarization-induced Ca2+ transients were identical in control and in α2δ-1-depleted muscle cells. However, α2δ-1 depletion significantly accelerated the current kinetics, most likely by the conversion of slowly activating into fast activating Ca2+ channels. Reverse transcription-PCR analysis indicated that α2δ-1 is the exclusive isoform expressed in differentiated BC3H1 cells and that depletion of α2δ-1 was not compensated by the up-regulation of any other α2δ isoform. Thus, in skeletal muscle the Ca2+ channel α2δ-1 subunit functions as a major determinant of the characteristic slow L-type Ca2+ current kinetics. However, this subunit is not essential for targeting of Ca2+ channels or for their primary physiological role in activating skeletal muscle excitation-contraction coupling. Voltage-activated Ca2+ channels are important signaling proteins in many cellular processes including muscle contraction, secretion, synaptic function, and transcriptional regulation. Ca2+ channels are composed of a pore-forming α1 subunit and the auxiliary α2δ, β, and γ subunits (1Catterall W.A. Annu. Rev. Biochem. 1995; 64: 493-531Crossref PubMed Scopus (781) Google Scholar). Whereas the α1 subunits are responsible for voltage sensing and ion conduction, the auxiliary subunits have been implicated in functions of membrane targeting and modulation of channel properties (for review see Ref. 2Arikkath J. Campbell K.P. Curr. Opin. Neurobiol. 2003; 13: 298-307Crossref PubMed Scopus (423) Google Scholar). Much of our current knowledge about the specific properties of Ca2+ channel subunits has been obtained from heterologous expression in Xenopus oocytes and in mammalian expression systems. Moreover, null-mutant mice have provided important information about the roles of Ca2+ channel subunits in native tissues. In skeletal muscle the voltage-activated Ca2+ channel functions as a voltage sensor in excitation-contraction (EC) 1The abbreviations used are: EC, excitation-contraction; GFP, green fluorescent protein; eGFP, enhanced GFP; DHPR, dihydropyridine receptor; RyR1, ryanodine receptor type 1; siRNA, small interfering RNA; SR, sarcoplasmic reticulum; T-tubule, transverse tubule; RT, reverse transcription; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorter; nt, nucleotide. coupling. The role of the slowly activating L-type Ca2+ current, which is not necessary for the activation of skeletal muscle contraction, is not clear. A null-mutant of the skeletal muscle α1S subunit, the dysgenic mouse, lacks EC coupling and L-type Ca2+ currents and dies at birth from respiratory failure (3Tanabe T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (584) Google Scholar). A knock-out mouse of the skeletal β1a Ca2+ channel subunit results in a very similar lethal muscle phenotype (4Gregg R.G. Messing A. Strube C. Beurg M. Moss R. Behan M. Sukhareva M. Haynes S. Powell J.A. Coronado R. Powers P.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13961-13966Crossref PubMed Scopus (201) Google Scholar). In contrast, γ subunit knock-out mice are viable and show only mild effects on current properties (5Freise D. Held B. Wissenbach U. Pfeifer A. Trost C. Himmerkus N. Schweig U. Freichel M. Biel M. Hofmann F. Hoth M. Flockerzi V. J. Biol. Chem. 2000; 275: 14476-14481Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). α2δ-1 (6Ellis S.B. Williams M.E. Ways N.R. Brenner R. Sharp A.H. Leung A.T. Campbell K.P. McKenna E. Koch W.J. Hui A. Schwartz A. Harpold M.M. Science. 1988; 241: 1661-1664Crossref PubMed Scopus (442) Google Scholar) is the major α2δ subunit in skeletal muscle (2Arikkath J. Campbell K.P. Curr. Opin. Neurobiol. 2003; 13: 298-307Crossref PubMed Scopus (423) Google Scholar). Because homozygous α2δ-1 knock-out mice die during early embryonic development, 2J. Offord, personal communication. the role of this subunit in skeletal muscle or in any other native tissue is still elusive. A mutation of the α2δ-2 gene results in a severe reduction of P-type Ca2+ currents in cerebellar Purkinje cells, and homozygous α2δ-2 mutant mice develop ataxia and die within a few weeks after birth (7Barclay J. Balaguero N. Mione M. Ackerman S.L. Letts V.A. Brodbeck J. Canti C. Meir A. Page K.M. Kusumi K. Perez-Reyes E. Lander E.S. Frankel W.N. Gardiner R.M. Dolphin A.C. Rees M. J. Neurosci. 2001; 21: 6095-6104Crossref PubMed Google Scholar). The α2δ subunit is a product of a single gene that is post-translationally cleaved into α2 and δ peptides, which remain associated via disulfide bonds. The δ subunit is a single-pass membrane protein, and the α2 subunit is a highly glycosylated extracellular protein (8Gurnett C.A. De Waard M. Campbell K.P. Neuron. 1996; 16: 431-440Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). A total of four genes code for α2δ subunits (α2δ-1 to α2δ-4), which display distinct tissue distributions (2Arikkath J. Campbell K.P. Curr. Opin. Neurobiol. 2003; 13: 298-307Crossref PubMed Scopus (423) Google Scholar). The α2δ-1 subunit is expressed at high levels in skeletal and cardiac muscle, in vascular smooth muscle, and in brain (9Angelotti T. Hofmann F. FEBS Lett. 1996; 397: 331-337Crossref PubMed Scopus (66) Google Scholar) and may associate with several different α1 subunit isoforms. Coexpression of α2δ subunits with various α1 subunits in heterologous expression systems demonstrated that α2δ enhances the membrane expression of functional Ca2+ channels, increases ligand-binding sites, and alters the voltage dependence and kinetics of the Ca2+ currents (2Arikkath J. Campbell K.P. Curr. Opin. Neurobiol. 2003; 13: 298-307Crossref PubMed Scopus (423) Google Scholar). Interestingly, the extracellular α2 specifically interacts with the α1 subunit and conveys the effects of increased membrane expression. However, the δ peptide is sufficient for the modulation of current properties (10Felix R. Gurnett C.A. De Waard M. Campbell K.P. J. Neurosci. 1997; 17: 6884-6891Crossref PubMed Google Scholar). The manifestation and the magnitude of these multiple effects vary with the combination of α1 and α2δ isoforms expressed in heterologous systems. Finally, α2δ-1 is the target for the anticonvulsant drugs gabapentin and pregabalin, two highly effective agents to medicate neuropathic pain (11Luo Z.D. Calcutt N.A. Higuera E.S. Valder C.R. Song Y.H. Svensson C.I. Myers R.R. J. Pharmacol. Exp. Ther. 2002; 303: 1199-1205Crossref PubMed Scopus (351) Google Scholar, 12Dworkin R.H. Corbin A.E. Young Jr., J.P. Sharma U. LaMoreaux L. Bockbrader H. Garofalo E.A. Poole R.M. Neurology. 2003; 60: 1274-1283Crossref PubMed Scopus (703) Google Scholar). Undoubtedly, the α2δ-1 subunit is an important component of Ca2+ channel complexes in many tissues and of great interest as a drug target. Nevertheless, its exact role in specific cell functions is still unknown. This is particularly critical in the case of skeletal muscle where the Ca2+ channel plays a vital role in the activation of muscle contraction. Therefore, we studied the role of the α2δ-1 subunit in skeletal muscle using siRNA knock-down of α2δ-1. This first loss-of-function analysis of α2δ-1 in a differentiated cell type indicated that in skeletal muscle cells α2δ-1 is required neither for the targeting of the channel into the triads nor for normal EC coupling. However, α2δ-1 is a critical determinant of the characteristic slow kinetics of skeletal muscle L-type Ca2+ currents. Design of siRNA Plasmids—siRNA target sequences corresponding to the α2δ-1 coding region (Cacna2d1, GenBank™ accession number NM_009784) were selected as recommended (13Elbashir S.M. Harborth J. Weber K. Tuschl T. Methods. 2002; 26: 199-213Crossref PubMed Scopus (1031) Google Scholar), and siRNAs targeting all known α2δ-1 splice variants (9Angelotti T. Hofmann F. FEBS Lett. 1996; 397: 331-337Crossref PubMed Scopus (66) Google Scholar) were expressed as hairpins from the pSilencer1.0-U6 siRNA expression vector (Ambion Ltd., Huntington, Cambridgeshire, UK). Specificity was confirmed by BLAST searches (www.ncbi.nlm.nih.gov/BLAST). Hairpin siRNA templates were designed as two complementary DNA strands of 70 bp consisting of a 5′ 4-nt overhang followed by an ApaI restriction site, a 19-nt siRNA sense sequence, a hairpin loop sequence TTCAAGAGA, a 19-nt siRNA antisense sequence, (T)6 and a DraI restriction site with a 3′ 4-nt overhang. The two complementary DNA strands were annealed in 30 mm HEPES, 100 mm potassium acetate, and 2 mm magnesium acetate, pH 7.4, by incubation for 2 min at 95 °C and subsequently slow cooling to room temperature. Double strands were digested with ApaI and DraI and ligated into the corresponding polylinker sites of the pSilencer1.0-U6 vector. The following gene-specific sequences were selected and tested (nucleotide numbers in the α2δ-1 coding sequence): 264–282, 945–963, 1099–1117, 1326–1344, 2332–2350, and a random control sequence 5′-GCCTAGGCGAACTGATACA-3′. Cell Cultures and Transfections—Myotubes of the homozygous dysgenic (mdg/mdg) mouse cell line GLT were cultured as described previously (14Powell J.A. Petherbridge L. Flucher B.E. J. Cell Biol. 1996; 134: 375-387Crossref PubMed Scopus (82) Google Scholar). At the onset of myoblast fusion (2 days after addition of differentiation medium) GLT cultures were transfected by using Fu-GENE according to the manufacturer's instructions (Roche Diagnostics). A total of 2 μg of plasmid DNA was used per 35-mm culture dish; siRNA plasmids were used at a 12–24-fold molar excess in combination with GFP-α1S. Three to 5 days later transfected cultures were fixed for immunofluorescence labeling or were analyzed in combined patch clamp and Ca2+ release measurements. BC3H1 nonfusing skeletal muscle cells were cultured as described previously (15Protasi F. Franzini-Armstrong C. Flucher B.E. J. Cell Biol. 1997; 137: 859-870Crossref PubMed Scopus (71) Google Scholar). Before plating the cells were transfected by electroporation with the AMAXA nucleofection system (AMAXA Biosystems, Köln, Germany). A total of 5 μg of plasmid DNA was used for 106 cells; the siRNA plasmids were transfected at a 12–24-fold molar excess with a plasmid coding for eGFP as a marker for transfected cells. Differentiated myoblasts were analyzed 6 days after addition of differentiation medium. Expression plasmids used were GFP-α1S (16Grabner M. Dirksen R.T. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1903-1908Crossref PubMed Scopus (136) Google Scholar) and pβA-eGFP (17Obermair G.J. Szabo Z. Bourinet E. Flucher B.E. Eur. J. Neurosci. 2004; 19: 2109-2122Crossref PubMed Scopus (126) Google Scholar). Immunofluorescence Analysis—Differentiated cultures were simultaneously immunostained as described by Flucher et al. (18Flucher B.E. Andrews S.B. Daniels M.P. Mol. Biol. Cell. 1994; 5: 1105-1118Crossref PubMed Scopus (75) Google Scholar) with the monoclonal α2δ-1 antibody mAb 20A (19Morton M.E. Froehner S.C. J. Biol. Chem. 1987; 262: 11904-11907Abstract Full Text PDF PubMed Google Scholar) at a dilution of 1:1,000 or the monoclonal α1S antibody mAb 1A (20Morton M.E. Caffrey J.M. Brown A.M. Froehner S.C. J. Biol. Chem. 1988; 263: 613-616Abstract Full Text PDF PubMed Google Scholar) at a dilution of 1:2,000, together with a rabbit affinity-purified anti-GFP antibody (Molecular Probes, Eugene, OR) at a dilution of 1:5,000 or the affinity-purified antibody162 against the RyR1 (21Giannini G. Conti A. Mammarella S. Scrobogna M. Sorrentino V. J. Cell Biol. 1995; 128: 893-904Crossref PubMed Scopus (491) Google Scholar) at a dilution of 1:5,000. An Alexa-488-conjugated goat anti-rabbit antibody (Molecular Probes, Eugene, OR) was used with anti-GFP so that the antibody label and the intrinsic GFP signal were both recorded in the green channel. Alexa-594-conjugated goat anti-mouse (Molecular Probes, Eugene, OR) was used to detect anti-α2δ-1 or anti-α1S. Controls, for example the omission of primary antibodies and incubation with inappropriate antibodies, were routinely performed. Images were recorded on a Zeiss Axiophot microscope equipped with a cooled CCD camera and METAVIEW image-processing software (Universal Imaging, West Chester, PA). Quantitative analysis of α2δ-1 expression and clustering was performed by systematically screening of cover glasses for well differentiated transfected (GFP-positive) myotubes (GLT) or myoblasts (BC3H1) and classifying their clustered α2δ-1 expression (in the red channel) as strong (+++), medium (++), weak (+), or absent (-), whereby strong labeling was defined as the staining intensity and density of clusters observed in the majority of the control myotubes. Representative examples of these labeling patterns are shown in Fig. 2. For the analysis of GFP-α1S expression and clustering, well differentiated GLT myotubes were identified based on the RyR1 expression (red channel), and GFP-α1S expression was classified as normal (clustered in triads), not targeted (ER/SR distribution; see Ref. 22Flucher B.E. Kasielke N. Grabner M. J. Cell Biol. 2000; 151: 467-478Crossref PubMed Scopus (62) Google Scholar) or absent (nontransfected myotubes). In BC3H1 cells, α1S expression was analyzed in eGFP-positive myocytes. All counts were performed in duplicate cover glasses from at least four independent experiments. Data are given as mean ± S.E. Statistical significance was determined by unpaired Student's t test. Patch Clamp Analysis and Ca2+ Release Measurements—Ca2+ currents were recorded with the whole cell patch clamp technique. Patch pipettes had resistances of 1.5–3 megohms when filled with (mm): 145 cesium aspartate, 2 MgCl2, 10 HEPES, 0.1 Cs-EGTA, 2 Mg-ATP (pH 7.4 with CsOH). Fluorescent signals of intracellular Ca2+ transients were collected microphotometrically (Photon Technology International, S. Brunswick, NJ) during whole cell recordings by including 0.2 mm pentapotassium Fluo-4 (Molecular Probes, Eugene, OR) in the pipette solution. The bath solution contained (mm): 10 CaCl2, 145 tetraethyl-ammonium chloride, 10 HEPES (pH 7.4 with tetraethylammonium hydroxide). Currents were recorded with an Axopatch 200B amplifier (Axon Instruments Inc., Foster City, CA). Data acquisition and command potentials were controlled by pClamp software (version 7.0, Axon Instruments). Test pulses were preceded by a 1-s prepulse to -50 mV to inactivate endogenous T-type Ca2+ channels (23Adams B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: 569-572Crossref PubMed Scopus (222) Google Scholar). Currents were determined with 200-ms depolarizing steps from a holding potential of -80 mV to test potentials between -50 and +80 mV in 10-mV increments. Leak currents were digitally subtracted by a P/3 prepulse protocol. Recordings were low-pass Bessel filtered at 2 kHz and sampled at 5 kHz. Recordings were performed at room temperature (≈23 °C). Only currents with a voltage error ≤5 mV attributable to series resistance were further analyzed with Clampfit® 8.0 (Axon Instruments, Foster City, CA) and SigmaPlot® 8.0 (SPSS Science, Chicago) software. For the selection of recordings for the kinetic analysis additional constraints (noise and stability) applied. The activation phase of currents (0 to current level to 98% of peak) was fitted with both a single and a double-exponential function using Clampfit® 8.0 software. The more accurate fit for each current was determined based on the χ2 values (see also Supplement 2). Data are given as mean ± S.E. Statistical significance was determined by unpaired Student's t test. Fluorescent Cell Sorting and Protein Biochemistry—Cells were harvested by trypsin treatment, pelleted, and resuspended at a density of 1–2 million cells/ml in serum-free medium. EGFP-positive cells were sorted using a FACSVantage SE flow cytometry system (BD Biosciences). Collected cells were pelleted and lysed for 15 min in ice-cold lysis buffer (10 mm Tris-HCl, 0.2 mm phenylmethylsulfonyl fluoride, 0.5 mm benzamidine, 1 μm pepstatin A, 1 μm leupeptin, 0.3 μm aprotinin, 2 mm iodoacetamide, 0.1 mg/ml trypsin inhibitor, 10 mm EDTA, pH 7.4) and homogenized by 15–20 strokes in a Dounce homogenizer. The homogenate was centrifuged at 300 × g (20 min; 4 °C); the supernatant was collected, and homogenization and centrifugation steps were repeated with the pellet. The combined supernatants were centrifuged at 200,000 × g (60 min; 4°), and pellets were resuspended in lysis buffer at a concentration equivalent to 2,500 cells/μl, quickly frozen in liquid N2, and stored at -80 °C. The equivalent to 50,000 cells per lane was separated by SDS-gel electrophoresis and analyzed by Western blot using the monoclonal α2δ-1 antibody mAb 20A (19Morton M.E. Froehner S.C. J. Biol. Chem. 1987; 262: 11904-11907Abstract Full Text PDF PubMed Google Scholar) at a dilution of 1:2,000. Loading and transfer was routinely examined with Coomassie Blue staining of the blots. RT-PCR and Quantitative TaqMan PCR—Total RNA was isolated from FACS-sorted transfected BC3H1 cells, from nontransfected differentiated BC3H1 cells, and from embryonic mouse brain (E16.5) using the TRIzol method (Invitrogen). RNA samples (2–5 μg) were reverse-transcribed by using the Ready-To-Go T-primed first-strand kit (Amersham Biosciences). The expression of α2δ isoforms 1–4 in differentiated BC3H1 myoblasts was investigated by conventional RT-PCR (40 cycles; 55 °C annealing) and TaqMan quantitative PCR (50 cycles). mRNA from embryonic mouse brain was used as a positive control for the α2δ isoforms 1–4. The relative abundance of α2δ-1 mRNA was assessed by TaqMan quantitative PCR using the standard curve method (User Bulletin 2: ABI PRISM 7700 Sequence Detection System. P/N 4303859B, Applied Biosystems, Foster City, CA) and the β-actin transcript as reference. Between 5 and 37.5 ng of total RNA equivalents, 200 nm of each primer, and 120 nm TaqMan probe were used for each 25-μl reaction in Brilliant QPCR Master Mix (Stratagene, La Jolla, CA). Analysis was performed using the Mx4000® Multiplex Quantitative PCR System (Stratagene, La Jolla, CA). Each TaqMan probe was modified with a 5′ reporter dye (6-carboxylfluorescein) and a 3′ quencher dye (6-carboxytetramethylrhodamine). Sets of primers were designed in a way that either the probe itself or the product was spanning an exon-exon boundary. Primers and probes specifically designed for β-actin (Actb, GenBank™ NM_007393), α2δ-1 (Cacna2d1, GenBank™ NM_009784), α2δ-2 (Cacna2d2, GenBank™ NM_020263), α2δ-3 (Cacna2d3, GenBank™ NM_009785), and the putative α2δ-4 (Cacna2d4, GenBank™ BK005394) were purchased from Metabion (Metabion, Planegg-Martinsried, Germany). Primer sequences are listed in Supplement 4. Exon-intron structures of the genes Actb (β-actin) and Cacna2d1–3 (α2δ-1–3) were derived from the Ensembl Genome Browser (www.ensembl.org). BLAST searches (www.ensemble.org; www.ncbi.nlm.nih.gov/BLAST) using the human α2δ-4 cDNA sequence (GenBank™ AF516695) as screening template revealed the putative cDNA sequence of a mouse α2δ-4 subunit. The translated protein sequence shares 84% identity (MULTALIN; see Ref. 24Combet C. Blanchet C. Geourjon C. Deleage G. Trends Biochem. Sci. 2000; 25: 147-150Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar) with the human α2δ-4 subunit (Supplement 3). The nucleotide sequence data of the putative mouse α2δ-4 subunit were submitted to GenBank™ and are available in the Third Party Annotation Section of the DDBJ/EMBL/GenBank™ data bases under the accession number TPA BK005394. α2δ-1 Requires the α1 Subunit for Normal Targeting into Skeletal Muscle Triads—Previously, we observed that in skeletal myotubes lacking the dihydropyridine receptor (DHPR) α1S subunit, the α2δ-1 subunit is mistargeted (25Flucher B.E. Phillips J.L. Powell J.A. J. Cell Biol. 1991; 115: 1345-1356Crossref PubMed Scopus (45) Google Scholar). Fig. 1b shows the typical expression pattern of α2δ-1 in myotubes from the dysgenic cell line GLT. The α2δ-1 subunit is expressed diffusely throughout the plasma membrane and is partially retained in cytoplasmic membrane compartments. When dysgenic myotubes were reconstituted by transfection with GFP-α1S, the distribution pattern of α2δ-1 changed dramatically. Both the α1S subunit and the α2δ-1 subunit were coexpressed in clusters (Fig. 1, c and d), and these DHPR clusters were also colocalized with the ryanodine receptor type 1 (RyR1) (Fig. 1, e and f). This distribution pattern is characteristic for triad proteins in skeletal myotubes, and the colocalization of the DHPR subunits with the RyR1 is indicative of the location of these proteins in junctions of the plasma membrane or the T-tubules with the SR (18Flucher B.E. Andrews S.B. Daniels M.P. Mol. Biol. Cell. 1994; 5: 1105-1118Crossref PubMed Scopus (75) Google Scholar), which will collectively be referred to as triads. Thus, the restoration of normal distribution of α2δ-1 in dysgenic myotubes transfected with GFP-α1S confirms that the α2δ-1 needs the α1S subunit for its own incorporation into the EC coupling apparatus located in the triad. But is the reverse also the case and does the α1S subunit need α2δ-1 for its targeting into skeletal muscle triads? Depletion of α2δ-1 with siRNA Does Not Affect Expression and Targeting of the α1S Subunit—To address this question, we used siRNA depletion of α2δ-1 in dysgenic myotubes. This system allows us to simultaneously express the GFP-α1S fusion protein, which facilitates the analysis of targeting properties and serves as marker for transfected myotubes, and plasmids coding for siRNAs. Expression of siRNAs from plasmids was chosen to ensure a continuous synthesis of siRNA for the duration of myotube differentiation. Five specific siRNA probes for unique mouse α2δ-1 sequences were designed, and their efficacy to reduce α2δ-1 expression was determined by immunofluorescence labeling. The probe resulting in the strongest reduction of α2δ-1 immunoreactivity (nucleotides 1099–1117) was used for all subsequent experiments, and its effects were compared with those of a control siRNA probe consisting of a nonspecific nucleotide sequence. Fig. 2 shows the effects of α2δ-1 siRNA on the expression and distribution of the α2δ-1 and GFP-α1S subunits in dysgenic myotubes. In the majority of myotubes transfected with GFP-α1S and α2δ-1 siRNA, the α2δ-1 immunoreactivity was reduced to below detectability (Fig. 2A, b), and others showed strongly reduced α2δ-1 expression compared with untransfected myotubes (Fig. 2A, d). The overall percentage of transfected myotubes showing any α2δ-1 immunoreactivity at all was reduced from 88 ± 5% (mean ± S.E., n = 181) in control siRNA-treated cultures to 44 ± 6% (n = 171) in α2δ-1 siRNA-treated cultures (Fig. 2C). However, only 7 ± 3% of the α2δ-1 siRNA-transfected myotubes showed α2δ-1 expression levels similar to the +++ levels in 57 ± 4% of the controls (Fig. 2, A, f, and C). The fact that our cultures, obviously, also contain many nontransfected muscle cells, which express normal levels of α2δ-1 (see Fig. 2A, b and d), precluded a meaningful Western blot analysis of α2δ-1 depletion by siRNA. Nevertheless, the immunofluorescence analysis clearly demonstrated the reduction of α2δ-1 by the specific α2δ-1 siRNA qualitatively and quantitatively and further allowed us to study the effects of α2δ-1 depletion on α1S targeting in exactly those myotubes where α2δ-1 reduction was most robust. Fig. 2A, a and b, demonstrate that in myotubes in which α2δ-1 was no longer detectable, the GFP-α1S subunit was expressed at normal levels and in the characteristic subcellular distribution pattern. Quantitative analysis of GFP-α1S expression revealed no significant difference (p = 0.39) in the degree of GFP-α1S clustering in myotubes cotransfected with either control or α2δ-1 siRNA (96 ± 2%, n = 83, versus 93 ± 4%, n = 76; Fig. 2B). Thus, the depletion of α2δ-1 expression did not show an effect on the expression and distribution of GFP-α1S, suggesting that normal targeting and incorporation of the α1S subunit into the triad complex does not depend on the presence of substantial amounts of the α2δ-1 subunit. siRNA Depletion of α2δ-1 Does Not Affect Amplitudes and Voltage Dependence of EC Coupling and L-type Ca2+ Currents—Because immunocytochemistry revealed that despite the lack of the α2δ-1 subunit GFP-α1S was still incorporated into triads, we could address the question as to how the depletion of α2δ-1 affects the normal function of the DHPR as Ca2+ channel and as the voltage sensor in EC coupling. To this end, combined whole cell patch clamp and intracellular fluorescence Ca2+ measurements were performed in transfected myotubes loaded with the fluorescent Ca2+ indicator Fluo-4 (Fig. 3 and Table I). Fig. 3A shows representative examples of recordings from GLT myotubes transfected with GFP-α1S in combination with either control (left) or α2δ-1 siRNA (right). Most importantly, the α2δ-1-depleted myotubes responded to depolarization with intracellular Ca2+ transients and Ca2+ currents (Fig. 3A). The averaged amplitudes and voltage dependence of the transients and currents are displayed in Fig. 3B and show no difference between control and α2δ-1 siRNA transfected myotubes. Peak current densities were similar also to recordings from myotubes transfected with GFP-α1S but without any siRNA (not shown). Thus, the physiological role of the DHPR in skeletal muscle EC coupling is not compromised by the depletion of the α2δ-1 subunit.Table IProperties of intracellular Ca2+ transients and Ca2+ currents Properties of depolarization-induced Ca2+ transients and Ca2+ currents from control and α2δ-1 siRNA-transfected dysgenic myotubes and BC3H1 muscle cells were determined from combined whole cell patch clamp and intracellular Fluos-4 Ca2+ recordings as described under “Experimental Procedures” and the Supplemental Material.ParametersGLT GFP-α1S + siRNABC3H1 eGFP + siRNAControl siRNAα2δ siRNAControl siRNAα2δ siRNACa2+ transients(ΔF/F)max1.46 ± 0.14aAll data are presented as mean ± S.E1.44 ± 0.28VF1/2 (mV)8.6 ± 1.65.2 ± 1.8NDbND indicates not determinedNDkF (mV)10.5 ± 0.79.4 ± 0.7n2214Ca2+ currentsIpeak (pA/pF)–2.7 ± 0.3–2.9 ± 0.3–6.0 ± 0.4–5.7 ± 0.4Gmax (nS/nF)100 ± 7101 ± 9161 ± 7151 ± 10VG1/2 (mV)35.2 ± 1.033.7 ± 1.123.3 ± 1.221.6 ± 1.0kG (mV)8.1 ± 0.37.7 ± 0.36.7 ± 1.26.7 ± 0.2Vrev (mV)78.9 ± 1.480.6 ± 1.479.4 ± 1.378.0 ± 0.9n24171919ActivationTime to peak (ms)98 ± 9cStatistical significance is p < 0.00137 ± 5cStatistical significance is p < 0.001115 ± 7cStatistical significance is p < 0.00157 ± 11cStatistical significance is p < 0.001n24171919τmono (ms)8.5 ± 1.16.0 ± 03Amono (pA/pF)2.9 ± 0.36.2 ± 06τfast (ms)5.0 ± 0.51.8 ± 02τslow (ms)30.0 ± 4.532.4 ± 2.3Afast (pA/pF)1.1 ± 0.20.9 ± 0.1Aslow (pA/pF)1.8 ± 0.25.3 ± 0.3Relative Afast/Aslow38/6215/85(%)n17141710InactivationR2002.0 ± 1.1cStatistical significance is p < 0.00113.9 ± 1.7cStatistical significance is p < 0.0017.6 ± 1.4dStatistical significance is p < 0.0117.8 ± 3.3dStatistical significance is p < 0.01Incidence (%)22938994n23141816a All data are presented as mean ± S.Eb ND indicates not determinedc Statistical significance is p < 0.001d Statistical significance is p < 0.01 Open table in a new tab siRNA Depletion of α2δ-1 Accelerates Activation and Inactivation Kinetics of Skel" @default.
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