Purified ryanodine receptor of skeletal muscle sarcoplasmic reticulum forms Ca2+-activated oligomeric Ca2+ channels in planar bilayers.
The ryanodine receptor of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle has been purified and found by electron microscopy to be equivalent to the feet structures that are involved in situ in the junctional association of transverse tubules with terminal cisternae of SR. We now find that when the purified receptor is incorporated into vesicle-derived planar bilayers, it forms Ca2+-specific channels, which are dependent on submicromolar Ca2+ for activity. In the presence of 1 mM ATP, the channel shows essentially no activity at 10 nM Ca2+ but becomes highly activated at 50 nM Ca2+. At suboptimal Ca2+ levels (100 nM), the channel is strongly activated by 1 mM ATP and can be blocked by ruthenium red, both effects being prevented by higher Ca2+ levels (1 microM). Mg2+, added from the cis side at millimolar concentrations, blocks Ca2+ flux through the channel from trans to cis (equivalent to flux from luminal to myoplasmic compartment). Ryanodine stabilizes the open state of the channel and blocks the action of ruthenium red to close the channel. Thus, the purified ryanodine receptor incorporated into a bilayer has the Ca2+-channel characteristics consistent with the calcium release observed in isolated terminal cisternae vesicles. Furthermore, ryanodine induced the appearance of a sublevel gating mode characterized by long open conductance states, which were integral multiples of the smallest observed conductance, 3.8 pS in 50 mM Ca2+. The purified receptor consists essentially of a single-sized high molecular weight polypeptide (Mr. approximately equal to 360,000), which on reconstitution forms the square rectangles diagnostic of the feet structures. We conclude that the identity of the Ca2+-release channel of SR is the foot structure, which consists of an oligomer of the high molecular weight polypeptide.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology* / Q000502:physiology; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D008051:Lipid Bilayers; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000502:physiology*; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000502:physiology*; D012430:Ruthenium Red / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000502:physiology*
D007473:Ion Channels; D008051:Lipid Bilayers; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012430:Ruthenium Red; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium