Functional sensitivity of the native skeletal Ca2+-release channel to divalent cations and the Mg–ATP complex

Abstract

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 Ca2+-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 Ca2+-release channel. The open probability of the Sr2+-activated channel was increased in the presence of a 2 mM Mg–ATP complex and adenine nucleotides on the cytoplasmic face of the Ca2+-release channel. These results were confirmed by isotopic flux measurements using passively 45Ca2+-loaded vesicles. In the latter case, the presence of extra vesicular AMP-PCP (the nonhydroly sable 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 Ca2+-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.Key words: Ca2+ release, sarcoplasmic reticulum, planar lipid bilayer, strontium.