A TRANSGENIC MYOGENIC CELL-LINE LACKING RYANODINE RECEPTOR PROTEIN FOR HOMOLOGOUS EXPRESSION STUDIES - RECONSTITUTION OF RY(1)R PROTEIN ANDFUNCTION

CCS embryonic stem (ES) cells possessing two mutant alleles (ry(1)r-/r y(1)r-) for the skeletal muscle ryanodine receptor (RyR) have been pro duced and injected subcutaneously into severely compromised immunodefi cient mice to produce teratocarcinomas in which Ry(1)R expression is a bsent. Several primary fibroblast cell lines were isolated and subclon ed 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 growt h inhibited, do not exhibit drastic morphological change upon serum re duction, and possess the normal complement of chromosomes, Four of the se fibroblast clones were infected with a retrovirus containing the cD NA encoding myoD and a puromycin selection marker, Several (1-2 mu g/m l) puromycin-resistant subclones from each initial cell line were expa nded and examined for their ability to express myoD and to form multin ucleated myotubes that express desmin and myosin upon removal of mitog ens, One of these clones (1B5 cells) was selected on this basis for fu rther study, These cells, upon withdrawal of mitogens for 5-7 d, were shown by Western blot analysis to express key triadic proteins, includ ing skeletal triadin, calsequestrin, FK506-binding protein, 12 kD, sar co(endo)plasmic reticulum calcium-ATPase1, and dihydropyridine recepto rs, Neither RyR isoform protein, Ry(1)R (skeletal), Ry(2)R (cardiac), nor Ry(3)R (brain), were detected in differentiated 1B5 cells. Measure ments of intracellular Ca2+ by ratio fluorescence imaging of fura-2-lo aded cells revealed that differentiated 1B5 cells exhibited no respons es to K+ (40 mM) depolarization, ryanodine (50-500 mu M), or caffeine (20-100 mM). Transient transfection of the 1B5 cells with the full-len gth rabbit Ry(1)R cDNA restored the expected responses to K+ depolariz ation, caffeine, and ryanodine. Depolarization-induced Ca2+ release wa s independent of extracellular Ca2+, consistent with skeletal-type exc itation-contraction coupling, Wild-type Ry(1)R expressed in 1B5 cells were reconstituted into bilayer lipid membranes and found to be indist inguishable from channels reconstituted from rabbit sarcoplasmic retic ulum with respect to unitary conductance, open dwell times, and respon ses to ryanodine and ruthenium red, The 1B5 cell line provides a power ful and easily managed homologous expression system in which to study how Ry(1)R structure relates to function.