|
|
|
|
|
|
|
||
J. Cell Biol.,
Volume 140, Number 4, February 23, 1998 843-851
§
* Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California; Abstract. CCS embryonic stem (ES) cells possessing
two mutant alleles (ry1r Department
of Anesthesia, Brigham and Women's Hospital, Boston, Massachusetts; and § Department of Cardiology, Children's Hospital,
Boston, Massachusetts
/ry1r) for the skeletal muscle
ryanodine receptor (RyR) have been produced and injected subcutaneously into severely compromised immunodeficient mice to produce teratocarcinomas in which Ry1R 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 µg/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, Ry1R (skeletal),
Ry2R (cardiac), nor Ry3R (brain), were detected in differentiated 1B5 cells. Measurements of intracellular
Ca2+ by ratio fluorescence imaging of fura-2-loaded
cells revealed that differentiated 1B5 cells exhibited no
responses to K+ (40 mM) depolarization, ryanodine
(50-500 µM), or caffeine (20-100 mM). Transient
transfection of the 1B5 cells with the full-length rabbit
Ry1R cDNA restored the expected responses to K+ depolarization, caffeine, and ryanodine. Depolarization-induced Ca2+ release was independent of extracellular
Ca2+, consistent with skeletal-type excitation-contraction coupling. Wild-type Ry1R 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 Ry1R
structure relates to function.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
