Luminal Mg2+, a key factor controlling RYR2-mediated Ca2+ release:: Cytoplasmic and luminal regulation modeled in a tetrameric channel

In cardiac muscle, intracellular Ca2+ and Mg2+ are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca2+] in the SR ([Ca2+](L)) stimulates the Ca2+ release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg2+, which has not been regarded as an important regulator of Ca2+ release. The effects of luminal Ca2+ and Mg2+ on sheep RYR2 were measured in lipid bilayers. Cytoplasmic and luminal Ca2+ produced a synergistic increase in the opening rate of RYRs. A novel, high affinity inhibition of RYR2 by luminal Mg2+ was observed, pointing to an important physiological role for luminal Mg2+ in cardiac muscle. At diastolic [Ca2+](C), luminal Mg2+ inhibition was voltage independent, with K-i = 45 mu M at luminal [Ca2+] ([Ca2+](L)) = 100 mu M. Luminal and cytoplasmic Mg2+ inhibition was alleviated by increasing [Ca2+](L) or [Ca2+](C). Ca2+ and Mg2+ on opposite sides of the bilayer exhibited competitive effects on RYRs, indicating that they can compete via the pore for common sites. The data were accurately fitted by a model based on a tetrameric RYR structure with four Ca2+-sensing mechanisms on each subunit: activating luminal L-site (40-mu M affinity for Mg2+ and Ca2+), cytoplasmic A-site (1.2 mu M for Ca2+ and 60 mu M for Mg2+), inactivating cytoplasmic I-I-site (similar to 10 mM for Ca2+ and Mg2+), and I-2-site ( 1.2 mu M for Ca2+). Activation of three or more subunits will cause channel opening. Mg2+ inhibition occurs primarily by Mg2+ displacing Ca2+ from the L- and A-sites, and Mg2+ fails to open the channel. The model predicts that under physiological conditions, SR load-dependent Ca2+ release (1) is mainly determined by Ca2+ displacement of Mg2+ from the L- site as SR loading increases, and (2) depends on the properties of both luminal and cytoplasmic activation mechanisms.