Functional sensitivity of the native skeletal Ca(2+)-release channel to divalent cations and the Mg-ATP complex.
Sarcoplasmic reticulum (SR) vesicles, prepared from rabbit skeletal muscle, were characterized by functional and binding assays and incorporated into planar lipid bilayers. Single-channel activity was recorded in an asymmetric calcium buffer system and studied under voltage clamp conditions. Under these experimental conditions, a large conductance (100 pS in 50 mM Ca2+ trans) divalent cation selective channel displaying high ruthenium red and low Ca2+ sensitivity was identified. This pathway has been previously described as the Ca(2+)-release channel of the SR of skeletal muscle. We now report that in the presence of a Mg-ATP complex, the Ca2+ sensitivity of the open probability of this channel is increased. Furthermore, we show that micromolar cis Sr2+ concentrations also activated the Ca(2+)-release channel. The open probability of the Sr(2+)-activated channel was increased in the presence of a 2 mM Mg-ATP complex and adenine nucleotides on the cytoplasmic face of the Ca(2+)-release channel. These results were confirmed by isotopic flux measurements using passively 45Ca(2+)-loaded vesicles. In the latter case, the presence of extravesicular AMP-PCP (the nonhydrolysable ATP analog) enhanced the percentage of 45Ca2+ release induced either by Ca2+ or Sr2+ activation. In conclusion our findings emphasize the fact that the divalent cation activation of the Ca(2+)-release channel may be induced by Ca2+ and Sr2+, but not by Ba2+, in the presence of adenine nucleotides. Furthermore, they support the view that in situ Ca2+ and Mg-ATP complexes are involved in modulating the gating mechanism of this specific pathway.
Rousseau
E
E
Département de physiologie et biophysique, Faculté de médecine, Université de Sherbrooke, Que., Canada.
Pinkos
J
J
Savaria
D
D
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D015220:Calcium Channels / Q000187:drug effects*; D002132:Calcium Radioisotopes; D002413:Cations, Divalent / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009132:Muscles / Q000187:drug effects / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D013324:Strontium / Q000378:metabolism; D013395:Sucrose
D015220:Calcium Channels; D002132:Calcium Radioisotopes; D002413:Cations, Divalent; D011506:Proteins; D013395:Sucrose; D000255:Adenosine Triphosphate; D013324:Strontium