Voltage‐Dependent Mobilization of Intracellular Calcium in Skeletal Muscle

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.

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Sustained subthreshold-for-twitch depolarization in rat single ventricular myocytes causes sustained calcium channel activation and sarcoplasmic reticulum calcium release.

Talo

Stern

Spurgeon

et al. 1990

The Journal of General Physiology

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Single rat ventricular myocytes, voltage-clamped at -50 to -40 mV, were depolarized in small steps in order to define the mechanisms that govern the increase in cytosolic [Ca ~+] (Ca i) and contraction, measured as a reduction in myocyte length. Small (3-5 mV), sustained (seconds) depolarizations that caused a small inward or no detectable change in current were followed after a delay by small (<2% of the resting length), steady reductions in cell length measured via a photodiode array, and small, steady increases in Ca i measured by changes in Indo-1 fluorescence. Larger (greater than -30 and less than -20 mV), sustained depolarizations produced phasic Ca 2+ currents, Ca i transients, and twitch contractions, followed by a steady current and a steady increase in Ca~ and contraction. Nitrendipine (or Cd, verapamil, or Ni) abolished the steady contraction and always produced an outward shift in steady current. The steady, nitrendipine-sensitive current and sustained increase in Ca i and contraction exhibited a similar voltage dependence over the voltage range between -40 and -20 mV. 2/zM ryanodine in the presence of intact Ca 2+ channel activity also abolished the steady increase in Cal and contraction over this voltage range. We conclude that when a sustained depolarization does not exceed about -20 mV, the resultant steady, graded contraction is due to SR Ca 2+ release graded by a steady ("window") Ca 2 § current. The existence of appreciable, sustained, graded Ca 2 § release in response to Ca ~+ current generated by arbitrarily small depolarizations is not compatible with any model of Ca2+-induced Ca ~+ release in which the releasing effect of the Ca 2+

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Voltage‐Dependent Mobilization of Intracellular Calcium in Skeletal Muscle

Cited by 3 publications

References 30 publications

Voltage‐Dependent Mechanisms for Raising Intracellular Free Calcium Concentration: Calcium Channels

Voltage‐Dependent Mechanisms for Raising Intracellular Free Calcium Concentration: Calcium Channels

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

Sustained subthreshold-for-twitch depolarization in rat single ventricular myocytes causes sustained calcium channel activation and sarcoplasmic reticulum calcium release.

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