Functional interactions between cytoplasmic domains of the skeletal muscle Ca2+ release channel.
The skeletal muscle Ca2+ release channel (RYR1), which plays a critical role in excitation-contraction coupling, is a homotetramer with a subunit molecular mass of 565 kDa. Oxidation of the channel increases its activity and produces intersubunit cross-links within the RYR1 tetramer (Aghdasi, B., Zhang, J., Wu, Y., Reid, M. B., and Hamilton, S. L. (1997) J. Biol. Chem. 272, 3739-3748). Alkylation of hyperreactive sulfhydryls on RYR1 with N-ethylmaleimide (NEM) inhibits channel function and blocks the intersubunit cross-linking. We used calpain and tryptic cleavage, two-dimensional SDS-polyacrylamide gel electrophoresis, N-terminal sequencing, sequence-specific antibody Western blotting, and [14C]NEM labeling to identify the domains involved in these effects. Our data are consistent with a model in which 1) diamide, an oxidizing agent, simultaneously produces an intermolecular cross-link between adjacent subunits within the RYR1 tetramer and an intramolecular cross-link within a single subunit; 2) all of the cysteines involved in both cross-links are in either the region between amino acids approximately 2100 and 2843 or the region between amino acids 2844 and 4685; 3) oxidation exposes a new calpain cleavage site in the central domain of the RYR1 (in the region around amino acid 2100); 4) sulfhydryls that react most rapidly with NEM are located in the N-terminal domain (between amino acids 426 and 1396); 5) alkylation of the N-terminal cysteines completely inhibits the formation of both inter- and intrasubunit cross-links. In summary, we present evidence for interactions between the N-terminal region and the putatively cytoplasmic central domains of RYR1 that appear to influence subunit-subunit interactions and channel activity.
Wu
Y
Y
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
Aghdasi
B
B
Dou
S J
SJ
Zhang
J Z
JZ
Liu
S Q
SQ
Hamilton
S L
SL
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism; D003432:Cross-Linking Reagents; D003958:Diamide; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D046911:Macromolecular Substances; D008961:Models, Structural; D008970:Molecular Weight; D009124:Muscle Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D009124:Muscle Proteins; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; D003958:Diamide; D012433:Ryanodine; D002154:Calpain; D005033:Ethylmaleimide