A Transgenic Myogenic Cell Line Lacking Ryanodine Receptor Protein for Homologous Expression Studies: Reconstitution of Ry

CCS embryonic stem (ES) cells possessing two mutant alleles ( ry 1 r 2 / ry 1 r 2 ) for the skeletal muscle ryanodine receptor (RyR) have been produced and injected subcutaneously into severely compromised immunodeficient mice to produce teratocarcinomas in which Ry 1 R expression is absent. Several primary fibroblast cell lines were isolated and subcloned from one of these tumors that contain the knockout mutation in both alleles and exhibit a doubling time of 18–24 h, are not contact growth inhibited, do not exhibit drastic morphological change upon serum reduction, and possess the normal complement of chromosomes. Four of these fibroblast clones were infected with a retrovirus containing the cDNA encoding myoD and a puromycin selection marker. Several (1–2 m g/ml) puromycin-resistant subclones from each initial cell line were expanded and examined for their ability to express myoD and to form multinucleated myotubes that express desmin and myosin upon removal of mitogens. One of these clones (1B5 cells) was selected on this basis for further study. These cells, upon withdrawal of mitogens for 5–7 d, were shown by Western blot analysis to express key triadic proteins, including skeletal triadin, calsequestrin, FK506-binding protein, 12 kD, sarco(endo)plasmic reticulum calcium–ATPase1, and dihydropyridine receptors. Neither RyR isoform protein, Ry 1 R (skeletal), Ry 2 R (cardiac), nor Ry 3 R (brain), were detected in differentiated 1B5 cells. Measurements of intracellular Ca 2 1 by ratio fluorescence imaging of fura-2–loaded cells revealed that differentiated 1B5 cells exhibited no responses to K 1 (40 mM) depolarization, ryanodine (50–500 m M), or caffeine (20–100 mM). Transient transfection of the 1B5 cells with the full-length rabbit Ry 1 R cDNA restored the expected responses to K 1 depolarization, caffeine, and ryanodine. Depolarizationinduced Ca 2 1 release was independent of extracellular Ca 2 1 , consistent with skeletal-type excitation–contraction coupling. Wild-type Ry 1 R expressed in 1B5 cells were reconstituted into bilayer lipid membranes and found to be indistinguishable from channels reconstituted from rabbit sarcoplasmic reticulum with respect to unitary conductance, open dwell times, and responses to ryanodine and ruthenium red. The 1B5 cell line provides a powerful and easily managed homologous expression system in which to study how Ry 1 R structure relates to function. E lucidation of the molecular details of excitation– contraction (e–c) 1 coupling is a major area of research in muscle biology. The process by which depolarization of the sarcolemma causes the release of calcium from the sarcoplasmic reticulum (SR) to initiate crossbridge formation and, ultimately, muscle contraction is defined as e–c coupling (36). The ryanodine receptor (RyR) is the site of calcium release from SR in skeletal and cardiac muscle (28). Studies that have addressed the Address all correspondence to Dr. Isaac N. Pessah, Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616. Tel.: (530) 752-6696. Fax: (530) 752-4698. E-mail: inpessah@ ucdavis.edu Dr. Hanh Nguyen’s present address is Department of Medicine, Division of Cardiology, Albert Einstein College of Medicine, Bronx, NY. Joan Galceran’s present address is Department of Microbiology and Immunology, University of California, San Francisco, CA. 1. Abbreviations used in this paper : BLM, bilayer lipid membrane; a 1-DHPR, a -1 subunit of L-type voltage-dependent Ca 2 1 channel dihydropyridine receptor; e–c, excitation–contraction; ES, embryonic stem; FKBP12, FK506 binding protein, 12 kD; G418, neomyocin antibiotic analogue; HIHS, heat-inactivated horse serum; P o , open probability; Ry 1 R, Ry 2 R, and Ry 3 R, skeletal isoform of ryanodine receptor, cardiac isoform of ryanodine receptor, and brain isoform of ryanodine receptor; SCID, severely compromised immunodeficient; SERCA, sarco(endo)plasmic reticulum Ca 2 1 -ATPase; SR, sarcoplasmic reticulum. on July 8, 2017 jcb.rress.org D ow nladed fom