Conducting and voltage-dependent behaviors of the native and purified SR Ca2+-release channels from the canine diaphragm.
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.
Picher
M
M
Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, QC, Canada.
Decrouy
A
A
Proteau
S
S
Rousseau
E
E
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D003964:Diaphragm / Q000378:metabolism*; D004285:Dogs; D004553:Electric Conductivity; D015640:Ion Channel Gating; D008051:Lipid Bilayers; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials; D008962:Models, Theoretical; D009124:Muscle Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D009206:Myocardium / Q000737:chemistry; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D015220:Calcium Channels; D008051:Lipid Bilayers; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium