Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Proper Restoration of Excitation-Contraction Coupling in the Dihydropyridine Receptor β1-null Zebrafish Relaxed Is an Exclusive Function of the β1a Subunit

2008; Elsevier BV; Volume: 284; Issue: 2 Linguagem: Inglês

10.1074/jbc.m807767200

ISSN

1083-351X

Autores

Johann Schredelseker, Anamika Dayal, Thorsten Schwerte, Clara Franzini‐Armstrong, Manfred Grabner,

Tópico(s)

Zebrafish Biomedical Research Applications

Resumo

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) β1a subunit. Lack of β1a results in (i) reduced membrane expression of the pore forming DHPR α1S subunit, (ii) elimination of α1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of β1a from rather general functions of β isoforms. Zebrafish and mammalian β1a subunits quantitatively restored α1S triad targeting and charge movement as well as intracellular Ca2+ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal β2a as the phylogenetically closest, and the ancestral housefly βM as the most distant isoform to β1a also completely recovered α1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca2+ transients and very limited tetrad formation compared with β1a. Consequently, larval motility was either only partially restored (β2a-injected larvae) or not restored at all (βM). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested β subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the β1a isoform. Consequently, we postulate a model that presents β1a as an allosteric modifier of α1S conformation enabling skeletal muscle-type EC coupling. The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) β1a subunit. Lack of β1a results in (i) reduced membrane expression of the pore forming DHPR α1S subunit, (ii) elimination of α1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of β1a from rather general functions of β isoforms. Zebrafish and mammalian β1a subunits quantitatively restored α1S triad targeting and charge movement as well as intracellular Ca2+ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal β2a as the phylogenetically closest, and the ancestral housefly βM as the most distant isoform to β1a also completely recovered α1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca2+ transients and very limited tetrad formation compared with β1a. Consequently, larval motility was either only partially restored (β2a-injected larvae) or not restored at all (βM). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested β subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the β1a isoform. Consequently, we postulate a model that presents β1a as an allosteric modifier of α1S conformation enabling skeletal muscle-type EC coupling. Excitation-contraction (EC) 3The abbreviations used are: EC, excitation-contraction; DHPR, 1,4-dihydropyridine receptor; RyR1, ryanodine receptor type-1; hpf, hours post fertilization; GFP, green fluorescent protein; nt, nucleotide(s); RE, restriction enzyme; WT, wild type; rb-β1a, rabbit β1a; zf-β1a, zebrafish β1a; MOPS, 4-morpholinepropanesulfonic acid. coupling in skeletal muscle is critically dependent on the close interaction of two distinct Ca2+ channels. Membrane depolarizations of the myotube are sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the sarcolemma, leading to a rearrangement of charged amino acids (charge movement) in the transmembrane segments S4 of the pore-forming DHPR α1S subunit (1Liman E.R. Hess P. Weaver F. Koren G. Nature. 1991; 353: 752-756Crossref PubMed Scopus (234) Google Scholar, 2Papazian D.M. Timpe L.C. Jan Y.N. Jan L.Y. Nature. 1991; 349: 305-310Crossref PubMed Scopus (431) Google Scholar). This conformational change induces via protein-protein interaction (3Schneider M.F. Chandler W.K. Nature. 1973; 242: 244-246Crossref PubMed Scopus (654) Google Scholar, 4Rios E. Brum G. Nature. 1987; 325: 717-720Crossref PubMed Scopus (654) Google Scholar) the opening of the sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca2+ influx through the DHPR (5Armstrong C.M. Bezanilla F.M. Horowicz P. Biochim. Biophys. Acta. 1972; 267: 605-608Crossref PubMed Scopus (301) Google Scholar). The release of Ca2+ from the sarcoplasmic reticulum via RyR1 consequently induces muscle contraction. The protein-protein interaction mechanism between DHPR and RyR1 requires correct ultrastructural targeting of both channels. In Ca2+ release units (triads and peripheral couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled to every other RyR1 and hence are geometrically arranged following the RyR-specific orthogonal arrays (6Block B.A. Imagawa T. Campbell K.P. Franzini-Armstrong C. J. Cell Biol. 1988; 107: 2587-2600Crossref PubMed Scopus (597) Google Scholar). The skeletal muscle DHPR is a heteromultimeric protein complex, composed of the voltage-sensing and pore-forming α1S subunit and auxiliary subunits β1a, α2δ-1, and γ1 (7Arikkath J. Campbell K.P. Curr. Opin. Neurobiol. 2003; 13: 298-307Crossref PubMed Scopus (423) Google Scholar). While gene knock-out of the DHPR γ1 subunit (8Freise 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, 9Ursu D. Sebille S. Dietze B. Freise D. Flockerzi V. Melzer W. J. Physiol. (Lond.). 2001; 533: 367-377Crossref Scopus (34) Google Scholar) and small interfering RNA knockdown of the DHPR α2δ-1 subunit (10Obermair G.J. Kugler G. Baumgartner S. Tuluc P. Grabner M. Flucher B.E. J. Biol. Chem. 2005; 280: 2229-2237Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 11Gach M.P. Cherednichenko G. Haarmann C. Lopez J.R. Beam K.G. Pessah I.N. Franzini-Armstrong C. Allen P.D. Biophys. J. 2008; 94: 3023-3034Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 12García K. Nabhani T. García J. J. Physiol. (Lond.). 2008; 586: 727-738Crossref Scopus (41) Google Scholar) have indicated that neither subunit is essential for coupling of the DHPR with RyR1, the lack of the α1S or of the intracellular β1a subunit is incompatible with EC coupling and accordingly null model mice die perinatally due to asphyxia (13Beam K.G. Knudson C.M. Powell J.A. Nature. 1986; 320: 168-170Crossref PubMed Scopus (185) Google Scholar, 14Gregg 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). β subunits of voltage-gated Ca2+ channels were repeatedly shown to be responsible for the facilitation of α1 membrane insertion and to be potent modulators of α1 current kinetics and voltage dependence (15Birnbaumer L. Qin N. Olcese R. Tareilus E. Platano D. Costantin J. Stefani E. J. Bioenerg. Biomembr. 1998; 30: 357-375Crossref PubMed Scopus (202) Google Scholar, 16Dolphin A.C. J. Bioenerg. Biomembr. 2003; 35: 599-620Crossref PubMed Scopus (314) Google Scholar). Whether the loss of EC coupling in β1-null mice was caused by decreased DHPR membrane expression or by the lack of a putative specific contribution of the β subunit to the skeletal muscle EC coupling apparatus (17Strube C. Beurg M. Powers P.A. Gregg R.G. Coronado R. Biophys. J. 1996; 71: 2531-2543Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 18Strube C. Beurg M. Sukhareva M. Ahern C.A. Powell J.A. Powers P.A. Gregg R.G. Coronado R. Biophys. J. 1998; 75: 207-217Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) was not clearly resolved. Recently, other β-functions were identified in skeletal muscle using the β1-null mutant zebrafish relaxed (19Schredelseker J. Di Biase V. Obermair G.J. Felder E.T. Flucher B.E. Franzini-Armstrong C. Grabner M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17219-17224Crossref PubMed Scopus (116) Google Scholar, 20Zhou W. Saint-Amant L. Hirata H. Cui W.W. Sprague S.M. Kuwada J.Y. Cell Calcium. 2006; 39: 227-236Crossref PubMed Scopus (36) Google Scholar). Like the β1-knock-out mouse (14Gregg 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) zebrafish relaxed is characterized by complete paralysis of skeletal muscle (21Granato M. van Eeden F.J. Schach U. Trowe T. Brand M. Furutani-Seiki M. Haffter P. Hammerschmidt M. Heisenberg C.P. Jiang Y.J. Kane D.A. Kelsh R.N. Mullins M.C. Odenthal J. Nusslein-Volhard C. Development. 1996; 123: 399-413Crossref PubMed Google Scholar, 22Haffter P. Granato M. Brand M. Mullins M.C. Hammerschmidt M. Kane D.A. Odenthal J. van Eeden F.J. Jiang Y.J. Heisenberg C.P. Kelsh R.N. Furutani-Seiki M. Vogelsang E. Beuchle D. Schach U. Fabian C. Nusslein-Volhard C. Development. 1996; 123: 1-36Crossref PubMed Google Scholar). While β1-knock-out mouse pups die immediately after birth due to respiratory paralysis (14Gregg 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), larvae of relaxed are able to survive for several days because of oxygen and metabolite diffusion via the skin (23Pelster B. Burggren W.W. Circ. Res. 1996; 79: 358-362Crossref PubMed Scopus (244) Google Scholar). Using highly differentiated myotubes that are easy to isolate from these larvae, the lack of EC coupling could be described by quantitative immunocytochemistry as a moderate ∼50% reduction of α1S membrane expression although α1S charge movement was nearly absent, and, most strikingly, as the complete lack of the arrangement of DHPRs in tetrads (19Schredelseker J. Di Biase V. Obermair G.J. Felder E.T. Flucher B.E. Franzini-Armstrong C. Grabner M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17219-17224Crossref PubMed Scopus (116) Google Scholar). Thus, in skeletal muscle the β subunit enables EC coupling by (i) enhancing α1S membrane targeting, (ii) facilitating α1S charge movement, and (iii) enabling the ultrastructural arrangement of DHPRs in tetrads. The question arises, which of these functions are specific for the skeletal muscle β1a and which ones are rather general properties of Ca2+ channel β subunits. Previous reconstitution studies made in the β1-null mouse system (24Beurg M. Sukhareva M. Strube C. Powers P.A. Gregg R.G. Coronado R. Biophys. J. 1997; 73: 807-818Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 25Beurg M. Sukhareva M. Ahern C.A. Conklin M.W. Perez-Reyes E. Powers P.A. Gregg R.G. Coronado R. Biophys. J. 1999; 76: 1744-1756Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) using different β subunit constructs (26Coronado R. Ahern C.A. Sheridan D.C. Cheng W. Carbonneau L. Bhattacharya D. Biol. Res. 2004; 37: 565-575Crossref PubMed Google Scholar) did not allow differentiation between β-induced enhancement of non-functional α1S membrane expression and the facilitation of α1S charge movement, due to the lack of information on α1S triad expression levels. Furthermore, the β-induced arrangement of DHPRs in tetrads was not detected as no ultrastructural information was obtained. In the present study, we established zebrafish mutant relaxed as an expression system to test different β subunits for their ability to restore skeletal muscle EC coupling. Using isolated myotubes for in vitro experiments (19Schredelseker J. Di Biase V. Obermair G.J. Felder E.T. Flucher B.E. Franzini-Armstrong C. Grabner M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17219-17224Crossref PubMed Scopus (116) Google Scholar, 27Ono F. Higashijima S. Shcherbatko A. Fetcho J.R. Brehm P. J. Neurosci. 2001; 21: 5439-5448Crossref PubMed Google Scholar) and complete larvae for in vivo expression studies (28Westerfield M. The Zebrafish Book. University of Oregon Press, Eugene2000Google Scholar, 29Nüsslein-Volhard C. Dahm R. Nüsslein-Volhard C. Dahm R. Zebrafish. Oxford University Press, Oxford2002Google Scholar, 30Hirata H. Saint-Amant L. Waterbury J. Cui W. Zhou W. Li Q. Goldman D. Granato M. Kuwada J.Y. Development. 2004; 131: 5457-5468Crossref PubMed Scopus (63) Google Scholar, 31Hirata H. Watanabe T. Hatakeyama J. Sprague S.M. Saint-Amant L. Nagashima A. Cui W.W. Zhou W. Kuwada J.Y. Development. 2007; 134: 2771-2781Crossref PubMed Scopus (93) Google Scholar) and freeze-fracture electron microscopy, a clear differentiation between the major functional roles of β subunits was feasible in the zebrafish system. The cloned zebrafish β1a and a mammalian (rabbit) β1a were shown to completely restore all parameters of EC coupling when expressed in relaxed myotubes and larvae. However, the phylogenetically closest β subunit to β1a, the cardiac/neuronal isoform β2a from rat, as well as the ancestral βM isoform from the housefly (Musca domestica), could recover functional α1S membrane insertion, but led to very restricted tetrad formation when compared with β1a, and thus to impaired DHPR-RyR1 coupling. This impairment caused drastic changes in skeletal muscle function. The present study shows that the enhancement of functional α1S membrane expression is a common function of all the tested β subunits, from β1a to even the most distant βM, whereas the effective formation of tetrads and thus proper skeletal muscle EC coupling is an exclusive function of the skeletal muscle β1a subunit. In context with previous studies, our results suggest a model according to which β1a acts as an allosteric modifier of α1S conformation. Only in the presence of β1a, the α1S subunit is properly folded to allow RyR1 anchoring and thus skeletal muscle-type EC coupling. Zebrafish Embryos—Adult zebrafish, heterozygous for the β1-null redts25 (relaxed) mutation were maintained and bred under standard aquarium conditions (28Westerfield M. The Zebrafish Book. University of Oregon Press, Eugene2000Google Scholar, 29Nüsslein-Volhard C. Dahm R. Nüsslein-Volhard C. Dahm R. Zebrafish. Oxford University Press, Oxford2002Google Scholar). Freshly spawned eggs were directly used for zygote RNA microinjection (see below) and/or raised until 25-32 h post-fertilization (hpf) at 28 °C to be used for experiments. Expression Plasmids—All β subunit cDNAs were N-terminally fused in-frame to GFP cDNA and cloned into expression vector pCI-neo (Promega) that allows both, in vitro RNA synthesis for zygote injection as well as transient expression in cultured relaxed myotubes. Constructs were designed as follows, with nucleotide numbers (nt) given in parentheses and asterisks indicating restriction enzyme (RE) sites introduced by the PCR technique using proofreading Pfu Turbo DNA polymerase (Stratagene). The integrity of cDNA sequences generated by PCR was confirmed by sequence analysis (Eurofins MWG Operon, Martinsried, Germany). zf-β1a—Total RNA from adult wild type (WT) zebrafish muscle was isolated using the RNeasy Mini kit (Qiagen) and reverse transcribed using the Ready-To-Go T-primed first-strand kit (Amersham Biosciences). From the first-strand cDNA, the zf-β1a open reading frame (GenBank™ AY952462) was PCR-generated in three fragments: HindIII*-XhoI (nt -5-502), XhoI-HindIII (nt 502-1352), and HindIII-BamHI* (nt 1352-1577). A subclone was created by co-ligating fragments XhoI-HindIII (nt 502-1352) and HindIII-BamHI* (nt 1352-1577) into the XhoI/BamHI polylinker RE sites of pBlue-script SK+ (pBS) (Stratagene). For N-terminal GFP tagging, fragment HindIII*-XhoI (nt -5-502) was in-frame ligated together with the excised fragment XhoI-BamHI* (nt 502-1577) into the HindIII/BamHI polylinker RE sites of the proprietary expression plasmid pGFP37 (32Grabner M. Dirksen R.T. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1903-1908Crossref PubMed Scopus (136) Google Scholar). From this subclone GFP-zf-β1a cDNA was excised with PstI-XhoI (nt -734-502) and XhoI-BamHI* (nt 502-1577) and ligated into the PstI/BamHI cut pBS. For the final construct zf-β1a, the SalI-BamHI* (nt -771-1577) insert was co-ligated with the 226-bp poly(A) tail excised with BamHI-NotI from the proprietary transcription plasmid pNKS2 (a gift of O. Pongs) into the XhoI/NotI cut polylinker of pCI-neo. rb-β1a—The open reading frame of rabbit β1a cDNA (Gen-Bank NM_001082279) was isolated from plasmid pcDNA3 (33Neuhuber B. Gerster U. Mitterdorfer J. Glossmann H. Flucher B.E. J. Biol. Chem. 1998; 273: 9110-9118Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) as the HindIII-BstXI fragment (nt -20-834) and as the BstXI-BamHI (nt 834-1575) PCR fragment, reintroducing its original stop codon at nt 1572. For N-terminal GFP tagging, both fragments were co-ligated into the HindIII/BamHI polylinker RE sites of pGFP37. From this subclone GFP-rb-β1a cDNA was excised with PstI-KpnI (nt -734 to -10) and KpnI-BamHI* (nt -10-1575) and ligated into the PstI/BamHI cut pBS. To gain the final construct for rb-β1a, the SalI-BamHI* (nt -771-1575) insert was co-ligated with the BamHI-NotI-excised poly(A) tail (see above) into the XhoI/NotI cut polylinker of pCI-neo. β2a—The open reading frame of rat β2a cDNA (GenBank M80545) was isolated from plasmid p91023(B) (34Perez-Reyes E. Castellano A. Kim H.S. Bertrand P. Baggstrom E. Lacerda A.E. Wei X.Y. Birnbaumer L. J. Biol. Chem. 1992; 267: 1792-1797Abstract Full Text PDF PubMed Google Scholar) as the HindIII-XhoI fragment (nt -11-1064) and the XhoI-BamHI* (nt 1064-1816) PCR fragment. For GFP tagging, fragments were co-ligated into the HindIII/BamHI polylinker RE sites of pGFP37. From this subclone GFP-β2a cDNA was excised with PstI-BamHI* (nt -740-1816) and ligated into the PstI/BamHI opened pBS. As a final step, the SalI-BamHI* (nt -777-1816) insert was co-ligated with the BamHI-NotI cut poly(A) tail into the XhoI/NotI opened polylinker of pCI-neo. βM—Musca β (βM) cDNA (GenBank X78561) was isolated from plasmid βM-pNKS2 (35Grabner M. Wang Z. Mitterdorfer J. Rosenthal F. Charnet P. Savchenko A. Hering S. Ren D. Hall L.M. Glossmann H. J. Biol. Chem. 1994; 269: 23668-23674Abstract Full Text PDF PubMed Google Scholar) as HindIII-DraIII (nt 8-2369) and DraIII-XbaI (nt 2369-3506) fragments and was co-ligated in-frame with GFP cDNA into the HindIII/XbaI polylinker of pGFP37. GFP-βM cDNA was isolated as PstI-DraIII (nt -721-2369) and DraIII-XbaI (nt 2369-3506) fragments and ligated into the PstI/XbaI opened pBS. To generate the final βM construct, fragments SalI-DraIII (nt -758-2369) and DraIII-XbaI (nt 2369-3506) were ligated into the XhoI/XbaI cut polylinker of pCI-neo. GFP—For standardizing experimental conditions, GFP alone was cloned into expression vector pCI-neo in the following way: the GFP cDNA was excised EcoRI-HindIII (nt -24-716) from subclone GFP-zf-β1a in pBS (see above; GFP-open reading frame numbering) and was co-ligated with the HindIII-NotI cut poly(A) tail into the EcoRI/NotI-cleaved polylinker of pCI-neo. Primary Culture of Zebrafish Myotubes—For the isolation of myoblasts, 25-28 hpf chorionated embryos derived from heterozygous relaxed parental fish were surface-sterilized using 0.5% sodium hypochlorite for 2 min and then enzymatically dechorionated using 2 mg/ml Pronase (Protease, Type XIV, Sigma) (28Westerfield M. The Zebrafish Book. University of Oregon Press, Eugene2000Google Scholar) for 20 min at 28 °C and collected in 0.5× Hanks' buffered salt saline (Sigma). Homozygous relaxed larvae were identified by their inability to move despite tactile stimulation. Motile "normal" siblings (i.e. heterozygous and WT) were used for control experiments. 100-150 larvae were anesthetized with 0.02% tricaine (MS-222; Sigma), decapitated, and the tails digested for 1 h in 200 units/ml collagenase type I in Hanks' buffered salt saline (Sigma) at 28 °C in a thermomixer with continuous trituration. Collagenase digestion was stopped by adding 7 ml of zebrafish culture medium containing 60% L-15 medium (Sigma) with 3% fetal calf serum, 3% horse serum (both Invitrogen), and 4 mm l-glutamine (Sigma). After centrifugation for 5 min at 200 × g cells were resuspended and transfected with 2 μg of expression plasmid cDNA using the AMAXA™ rat neonatal cardiomyocyte nucleofector kit (AMAXA Biosystems, Köln, Germany) according to the manufacturer's manual. Myocytes were resuspended in 200 μl of zebrafish medium supplemented with 4 units/ml penicillin/streptomycin (Invitrogen) ("full zebrafish medium") and plated on carbon, gelatin, and collagen-coated glass coverslips (for immunocytochemical experiments) or as droplets in the center of collagen-coated plastic dishes (for electrophysiological experiments). After 20 min, 1.5 ml of full zebrafish medium was added and cells were cultured at 28 °C for 4 to 6 days. Immunocytochemistry—Myotubes cultured on glass coverslips were washed in phosphate-buffered saline supplemented with 100 μm N-benzyl-p-toluene sulfonamide. Cells were fixed with 4% paraformaldehyde in 0.1 m sodium phosphate buffer for 20 min, permeabilized, and blocked by incubating with 5% normal goat serum in phosphate-buffered saline supplemented with 0.2% bovine serum albumin and 0.2% Triton X-100 (PBT) for 30 min, followed by incubation with primary antibodies in PBT overnight at 4 °C. Primary antibodies used were monoclonal antibody 1A against α1S (Affinity Bioreagents) at 1:2,000 (36Morton M.E. Froehner S.C. J. Biol. Chem. 1987; 262: 11904-11907Abstract Full Text PDF PubMed Google Scholar, 37Kugler G. Grabner M. Platzer J. Striessnig J. Flucher B.E. Arch. Biochem. Biophys. 2004; 427: 91-100Crossref PubMed Scopus (16) Google Scholar) and rb-anti-GFP (Invitrogen) at 1:5,000 dilutions. After several washes with PBT, secondary antibodies, goat anti-mouse Alexa Fluor 594, and goat anti-rabbit Alexa Fluor 488 (Invitrogen) at a concentration of 1:4,000 in PBT were applied for 1 h at room temperature. Specimens were mounted in 90% glycerol, 0.1 m Tris with 5 mg/ml p-phenylendiamine to retard photobleaching (38Flucher B.E. Andrews S.B. Daniels M.P. Mol. Biol. Cell. 1994; 5: 1105-1118Crossref PubMed Scopus (75) Google Scholar). Images were taken with a cooled CCD camera (Diagnostic Instruments) mounted on a Zeiss Axiophot microscope equipped with a ×63, 1.4 NA objective lens, using MetaVue image-processing software (Universal Imaging, West Chester, PA). For quantification of α1S triad expression, images were acquired with identical exposure times, followed by background subtraction and shading correction. Transfected cells were identified by positive anti-GFP staining. Quantification of α1S triad expression was determined by measuring the average fluorescence intensity of Alexa Fluor 595 along a line across a row of α1S clusters (triadic junctions; see Fig. 1A) in 5 measurements on each myotube, which were obtained from at least 2 different cultures. Myotubes that barely expressed GFP-β (and as a consequence also α1S) and were only visible because of signal amplification by anti-GFP/Alexa Fluor 488 staining were excluded from the α1S quantification to allow a quantitative link to our patch-clamp data. To this aim we determined the percentage of expressing myotubes, either identified by direct GFP fluorescence (patch-clamp approach) or by GFP-antibody enhancement (immunocytochemical approach) from the total number of myotubes. Calculations were done from 2 different preparations for both approaches. Fractions of expressing cells were 7 ± 1%, n = 460; and 21 ± 6%, n = 325, for the patch-clamp and immunocytochemical approach, respectively. Thus, to enable a link between both approaches, only the values of the highest1/3 of expressing myotubes were considered for α1S fluorescence quantification. Whole-cell Patch Clamp Analysis—Immobilization-resistant intramembrane charge movement, as a measure of functional α1S expression (39Adams B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: 569-572Crossref PubMed Scopus (222) Google Scholar), as well as intracellular Ca2+ transients were recorded from myotubes cultured for 4-6 days after transfection. GFP fluorescing myotubes were patch clamp analyzed on an Olympus IX70 inverted fluorescence microscope equipped with Hoffmann modulation contrast. Patch pipettes were pulled from borosilicate glass (Harvard Instruments), fire-polished (Microforge MF-830, Narishige), and had resistances of 3.5-5 MΩ after back-filling with pipette solution containing 100 mm Cs-aspartate, 10 mm HEPES, 0.5 mm CsEGTA, 3 mm MgATP, and 0.2 mm Fluo-4 (pH 7.4 with CsOH). The bath solution consisted of 10 mm Ca(OH)2, 100 mm l-aspartate, and 10 mm HEPES (pH 7.4 with tetraethylammonium hydroxide). Contractions of myotubes were blocked by adding 100 μm of the myosin-II blocker N-benzyl-p-toluene sulfonamide (Sigma) to the bath solution (40Cheung A. Dantzig J.A. Hollingworth S. Baylor S.M. Goldman Y.E. Mitchison T.J. Straight A.F. Nat. Cell Biol. 2002; 4: 83-88Crossref PubMed Scopus (222) Google Scholar). Recordings were performed with an Axopatch 200B amplifier controlled by pClamp software (version 7.0; Axon Instruments Inc., Foster City, CA) and leak currents were subtracted by a P/4 prepulse protocol. To inactivate endogenous T-type currents all test pulses were preceded by a 1-s prepulse to -30 mV (39Adams B.A. Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 346: 569-572Crossref PubMed Scopus (222) Google Scholar). Recordings were low-pass Bessel-filtered at 1 kHz and sampled at 5 kHz. DHPR charge movement was measured in 20-ms depolarizing test pulses starting from a test potential of +70 down to -60 mV in 10-mV increments. Total charge movement was calculated by integrating the ON-component of gating currents. 0.2 mm Fluo-4 was added to the patch pipette solution to measure intracellular Ca2+ release. Fluo-4 fluorescence was recorded using a PTI 814 photomultiplier system (PTI, S. Brunswick, NJ). Average fluorescence intensity (F) of a rectangular region on the patched myotube was recorded in 200-ms depolarizing test pulses from +80 to -50 mV in 10-mV increments with a holding potential of -80 mV. The average fluorescence was normalized to the resting fluorescence and expressed as ΔF/F0. The voltage dependence of charge movement (Q) and maximum intracellular Ca2+ release for each test potential were fitted according to the following Boltzmann distribution, A = Amax/{1 + exp [-(V - V1/2)/k]}(Eq. 1) where A is Q or ΔF/F0, V½ is the potential at which A = Amax/2, and k is a slope factor. Data were analyzed using ClampFit 9.0 and 10.0 (Axon Instruments) and SigmaPlot 9.0 and 10.0 (SPSS Science, Chicago, IL) software. Zygote Injection of in Vitro Synthesized RNA—For in vitro RNA synthesis 50 μg of all β subunit cDNAs and GFP cDNA were linearized with restriction enzymes XbaI and NotI, respectively, purified with phenol/chloroform and precipitated with 3 m NH4Ac in 70% EtOH and the pellet redissolved in RNase-free water. Linearized DNA templates were fidelity checked on an agarose gel. In vitro transcription was performed in a volume of 100 μl containing: 5 μg of linearized DNA template, 10 mm NTPs, with GTP supplemented by m7(5′)ppp(5′)G-cap (Roche Diagnostics), 100 units of T7-RNA Polymerase (Roche), and 200 units of RNase inhibitor (RNAsin; Roche), and incubated at 37 °C for 1 h. Template DNA was digested with 100 units of RNase-free DNase (Roche) for 15 min at 37 °C. After phenol/chloroform extraction and ethanol precipitation, the RNA pellet was redissolved in RNase-free water and aliquots frozen at -80 °C. RNA fidelity and concentration were checked on a 7% formaldehyde-agarose gel in MOPS running buffer. For RNA injection, eggs from heterozygous parental zebrafish in the one-cell stage were collected immediately after spawning and positioned in a 0.9-mm groove of an agarose tray to be microinjected within 20 min. Injection needles were pulled from heat-sterilized borosilicate glass capillaries (Harvard Instruments) and front-filled with RNA solution (0.2 μg/μl), containing 0.1% phenol red as an injection volume tracer (29Nüsslein-Volhard C. Dahm R. Nüsslein-Volhard C. Dahm R. Zebrafish. Oxford University Press, Oxford2002Google Scholar). Injection volume of RNA solution was ∼1/5 of total zygote volume (calculated 13 nl) and was injected using a motorized micromanipulator DC3001 and the pneumatic PicoPump PV830 (both WPI, Germany). Eight hours after injection, GFP fluorescence of healthy embryos was quantified using a PTI 814 photomultiplier system. Only proper developing injected embryos with a mean fluorescence signal exceeding 40% above uninjected control embryos were considered for freeze-fracture electron microscopy or digital motion analysis. Identification of Rescued Relaxed Larvae—Discrimination of the 25% of motility restored homozygous relaxed larvae used in motion analysis experiments from the injected normal siblings was done by keeping all injected larvae separated and thus identifiable for up to 5 days and by observing a gradual fallback to the paralyzed phenotype due to de

Referência(s)