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  • Title: Conducting and voltage-dependent behaviors of the native and purified SR Ca2+-release channels from the canine diaphragm.
    Author: Picher M, Decrouy A, Proteau S, Rousseau E.
    Journal: Biochim Biophys Acta; 1997 Sep 04; 1328(2):243-60. PubMed ID: 9315621.
    Abstract:
    The ryanodine-sensitive Ca2+-release channel of the canine diaphragm sarcoplasmic reticulum (SR) was characterized using biochemical assays and the planar lipid bilayer technique. Diaphragm SR membranes have a [3H]ryanodine-binding capacity (Bmax) of 1.2 pmol/mg protein and a binding affinity (K(D)) of 6.3 nM. The conductance of the native channel was 330 pS in 50 mM/250 mM trans/cis CsCH3SO3 and was reduced to 71 pS by 10 mM Ca2+ trans. The Ca2+-release channel was purified as a 400 kDa protein on SDS-PAGE and displayed a conductance of 715 pS in 200 mM KCl. The native and purified Ca2+ channels were activated by micromolar Ca2+ and ATP and inhibited by Mg2+, ryanodine and ruthenium red. Although diaphragm muscle contraction was shown to depend on extracellular Ca2+ like cardiac muscles, we provide evidence that the diaphragm SR Ca2+-release channel may be classified as a skeletal ryanodine receptor isoform. First, the IC50 for [3H]ryanodine binding was in the same range as estimated for skeletal SR, with 20 nM. Second, the channel was maximally activated by 10-30 microM cytoplasmic Ca2+ and inhibited at higher concentrations. Third, ryanodine binding to the diaphragm SR was less sensitive to Ca2+ than cardiac SR, with EC50, values of 50 and 1 microM, respectively. Finally, Ca2+-release activity and [3H]ryanodine binding capacity of the diaphragm and skeletal SR were similarly more sensitive to Mg2+ than cardiac SR. Together, these results suggest a predominantly skeletal-type of excitation-contraction coupling in the diaphragm.
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