Co-expression in CHO cells of two muscle proteins involved in excitation-contraction coupling.
Ryanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and transverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules. CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the alpha 2, beta and gamma subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cell's plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.
Takekura
H
H
Department of Cell and Developmental Biology, University of Pennsylvania, School of Medicine, Philadelphia 19143-6058, USA.
Takeshima
H
H
Nishimura
S
S
Takahashi
M
M
Tanabe
T
T
Flockerzi
V
V
Hofmann
F
F
Franzini-Armstrong
C
C
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D016466:CHO Cells / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D002110:Caffeine / Q000494:pharmacology; D015220:Calcium Channels / Q000032:analysis / Q000235:genetics*; D020746:Calcium Channels, L-Type; D002148:Calmodulin-Binding Proteins / Q000032:analysis / Q000235:genetics*; D002458:Cell Fractionation; D006224:Cricetinae; D004594:Electrophysiology; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000648:ultrastructure; D005614:Freeze Fracturing; D015870:Gene Expression / Q000502:physiology; D007150:Immunohistochemistry; D007365:Intercellular Junctions / Q000737:chemistry; D008854:Microscopy, Electron; D009119:Muscle Contraction / Q000502:physiology*; D009124:Muscle Proteins / Q000032:analysis / Q000235:genetics*; D018482:Muscle, Skeletal / Q000737:chemistry; D011485:Protein Binding / Q000502:physiology; D019837:Ryanodine Receptor Calcium Release Channel
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D002148:Calmodulin-Binding Proteins; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine