Modulation of Cardiac Ryanodine Receptor Channels by Alkaline Earth Cations

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 P_o 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.