Abstract
Cardiac ryanodine receptor (RyR2) function is modulated by Ca2+ and Mg2+. To better characterize Ca2+ and Mg2+ binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M2+: Mg2+, Ca2+, Sr2+, Ba2+) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M2+ binding to high affinity activating sites at the cytosolic channel surface, specific for Ca2+ or Sr2+. This activation was interfered by Mg2+ and Ba2+ acting at low affinity M2+-unspecific binding sites. When testing the effects of luminal M2+ as current carriers, all M2+ increased maximal RyR2 open probability (compared to Cs+), suggesting the existence of low affinity activating M2+-unspecific sites at the luminal surface. Responses to M2+ vary from channel to channel (heterogeneity). However, with luminal Ba2+or Mg2+, RyR2 were less sensitive to cytosolic Ca2+ and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca2+or Sr2+). Kinetics of RyR2 with mixtures of luminal Ba2+/Ca2+ and additive action of luminal plus cytosolic Ba2+ or Mg2+ suggest luminal M2+ differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca2+/Sr2+-specific sites, which stabilize high Po mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca2+ activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M2+ binding sites (specific for Ca2+ and unspecific for Ca2+/Mg2+) that dynamically modulate channel activity and gating status, depending on SR voltage.
Highlights
During excitation-contraction coupling in the heart, calcium ions (Ca2+) are mobilized from the sarcoplasmic reticulum (SR) to the cytosol through ryanodine receptor Ca2+ release channels (RyR isoform 2, RyR2), located at the terminal cisternae of the SR [1,2,3,4]
We studied the modulation of RyR2 by alkaline earth cations (M2+: Mg2+, Ca2+, Sr2+ or Ba2+) added either to the cytosolic or luminal channel surface
RyR2 did not activate when cytosolic Mg2+ or Ba2+ levels were increased. This confirms that cytosolic M2+ activating sites are selective for Ca2+ and Sr2+
Summary
During excitation-contraction coupling in the heart, calcium ions (Ca2+) are mobilized from the sarcoplasmic reticulum (SR) to the cytosol through ryanodine receptor Ca2+ release channels (RyR isoform 2, RyR2), located at the terminal cisternae of the SR [1,2,3,4]. Previous research has shown that this massive intracellular Ca2+ release in cardiac muscle depends on extracellular Ca2+ entry through the L-type Ca2+ channels (reviewed in [5,6]). Ca2+ induces maximal activation, whereas 1–10 mM Ca2+ is inhibitory [1,2,3,4,10]. This suggests the existence of two different types of cytosolic Ca2+ binding sites: activating sites with high affinity (micromolar) and inhibitory sites with low affinity (millimolar). It is thought that Mg2+ inhibition of RyR2 function involves both competition of Mg2+ with Ca2+ binding to cytosolic activating sites and Mg2+ binding to additional inhibitory cytosolic Mg2+ binding sites [11,12].
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