Cross-linking analysis of the ryanodine receptor and alpha1-dihydropyridine receptor in rabbit skeletal muscle triads.
In mature skeletal muscle, excitation-contraction (EC) coupling is thought to be mediated by direct physical interactions between the transverse tubular, voltage-sensing dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR) Ca2+ release channel of the sarcoplasmic reticulum (SR). Although previous attempts at demonstrating interactions between purified RyR and alpha1-DHPR have failed, the cross-linking analysis shown here indicates low-level complex formation between the SR RyR and the junctional alpha1-DHPR. After cross-linking of membranes highly enriched in triads with dithiobis-succinimidyl propionate, distinct complexes of more than 3000 kDa were detected. This agrees with numerous physiological and electron-microscopic findings, as well as co-immunoprecipitation experiments with triad receptors and receptor domain-binding studies. However, a distinct overlap of immunoreactivity between receptors was not observed in crude microsomal preparations, indicating that the triad complex is probably of low abundance. Disulphide-bonded, high-molecular-mass clusters of triadin, the junctional protein proposed to mediate interactions in triads, were confirmed to be linked to the RyR. Calsequestrin and the SR Ca2+-ATPase were not found in cross-linked complexes of the RyR and alpha1-DHPR. Thus, whereas recent studies indicate that the two receptors exhibit temporal differences in their developmental inductions and that receptor interactions are not essential for the formation and maintenance of triads, this study supports the signal transduction hypothesis of direct physical interactions between triad receptors in adult skeletal muscle.
Murray
B E
BE
Department of Pharmacology, University College Dublin, Belfield, Dublin 4, Ireland.
Ohlendieck
K
K
D000818:Animals; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D046911:Macromolecular Substances; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D046911:Macromolecular Substances; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C070048:triadin