Purified ryanodine receptor from rabbit skeletal muscle is the calcium-release channel of sarcoplasmic reticulum.

Abstract

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified as a single 450,000-dalton polypeptide from CHAPS-solubilized triads using immunoaffinity chromatography. The purified receptor had a [aH]ryanodine-binding capacity (Bm~,) of 490 pmol/mg and a binding affinity (Kd) of 7.0 nM. Using planar bilayer recording techniques, we show that the purified receptor forms cationic channels selective for divalent ions. Ryanodine receptor channels were identical to the Ca-release channels described in native sarcoplasmic reticulum using the same techniques. In the present work, four criteria were used to establish this identity: (a) activation of channels by micromolar Ca and millimolar ATP and inhibition by micromolar ruthenium red, (b) a main channel conductance of 110 +__ 10 pS in 54 mM tram Ca, (c) a long-term open state of lower unitary conductance induced by ryanodine concentrations as low as 20 nM, and (d) a permeability ratio Pc~/Pxn, ~-14. In addition, we show that the purified ryanodine receptor channel displays a saturable conductance in both monovalent and divalent cation solutions (3~m~ for K and Ca = 1 nS and 172 pS, respectively). In the absence of Ca, channels had a broad selectivity for monovalent cations, but in the presence of Ca, they were selectively permeable to Ca against K by a permeability ratio Pc~/Px ~ 6. Receptor channels displayed several equivalent conductance levels, which suggest an oligomeric pore structure. We conclude that the 450,000-dalton polypeptide ryanodine receptor is the Ca-release channel of the sarcoplasmic reticulum and is the target site of ruthenium red and ryanodine.

Properties of Ca2+ release induced by clofibric acid from the sarcoplasmic reticulum of mouse skeletal muscle fibres

“…Ryanodine receptor (RyR)-mediated Ca 2+ release is one of the most important mechanisms in the regulation of cytosolic Ca 2+ concentrations (Berridge, 1993). Ca 2+ -induced Ca 2+ release (CICR), a release mechanism from the sarcoplasmic reticulum (SR) extensively studied in skinned ®bres (Endo, 1977; and in isolated SR vesicles (Meissner, 1984;Meissner et al, 1986;Smith et al, 1986), has been shown to be a function of the RyR (Imagawa et al, 1987;Lai et al, 1988;Hymel et al, 1988;Smith et al, 1988).…”

Calcium inactivation of calcium release in frog cut muscle fibers that contain millimolar EGTA or Fura-2.

“…Although the flux through a single SR Ca channel has not been measured inside a frog cut muscle fiber, it has been measured in bilayers that contain either nadve channels (Smith, Coronado, and Meissner, 1986) or channels from purified ryanodine receptor protein (Smith, Imagawa, Ma, Fill, Campbell, and Coronado, 1988) from rabbit skeletal muscle. With 53 mM Ca on the side of the bilayer that corresponds to the lumen of the SR, the single-channel current is ~3 pA at 0 mV, 90-99~ As far as we are aware, currents in single channels from skeletal muscle have not been measured with concentrations of Ca as low as 1-9 mM, the estimated free concentration inside the SR (Hasselbach and Oetliker, 1983).…”

“…There is considerable evidence, however, that the single-channel current is not directly proportional to free [Ca] between 1-9 and 53 mM. Smith et al (1988) studied single channels in bilayers with the same concentration of Ca on both sides of the membrane. They found that the relation between conductance at 0 mV and [Ca] could be fitted by a 1:1 binding isotherm with Km --3 mM.…”

Identification and characterization of proteins in sarcoplasmic reticulum from normal and failing human left ventricles

“…Among these are a 105 000-Da Ca 2+ -transporting ATPase (Brandl et al, 1986(Brandl et al, , 1987; phospholamban, a 27000-29 000-Da pentamer whose phosphorylation is associated with stimulation of Ca 2+ uptake (Kirchberger et al, 1974;Tada et al, 1974); calsequestrin, a 55 000-Da protein that binds Ca 2+ within the sarcoplasmic reticulum with low affinity and high capacity ; two glycoproteins, 53000-Da and 130 000-Da, whose functions remain unknown but which may be involved in intraluminal Ca 2+ -binding and regulation of Ca 2+ uptake ; Campbell and M a c L e n n o n 1 9 8 1 , C a m p b e l l a n d Pepper et al, 1985;Helmke and Howard, 1987;Leberer et al, 1989a;Leberer et al, 1989b;Kutchai and Campbell, 1989); and a 400000-Da ryanodine-sensitive Ca 2+ channel (Lai et al, 1988;Innui et al, 1987;Campbell et al, 1987;Smith et al, 1988). The Ca 2+ -transporting ATPase of cardiac sarcoplasmic reticulum, like phospholamban, is the product of a gene expressed in cardiac and slow-twitch skeletal muscle but not in fast-twitch skeletal muscle (whose sarcoplasmic reticulum Ca 2+ ATPase is the product of a separate gene; Brandl et al, 1986).…”