Programme Contents

Regulation of human RYR2 by intracellular Ca²⁺ and Mg²⁺

Heart failure is a complex disorder thah involves changes in Ca²⁺ handling protein expression, Ca²⁺ homeostasis, and tissue remodelling. The Ca²⁺ release channel (RyR2) activates and modulates heart function by controlling the Ca²⁺ release from SR. RyR2 is controlled by four different Ca²⁺/Mg²⁺-dependent mechanisms (Laver & Honen, 2008). Release of Ca²⁺ from the SR is stimulated by Ca²⁺ activation of RyR2 as a result of Ca²⁺ binding to either the cytoplasmic side (A-site, in the case of Ca²⁺ influx through voltage-dependent L-type channels) or luminal side of the channel (L-site, in the case of SR overload). In addition, there are two Ca²⁺ inactivation sites (I1 and I2) located on its cytoplasmic face. Intracellular Mg²⁺ (∼1 mmol/l) inhibits RyRs and acts as a ‘brake’ for Ca²⁺ release. In diastole, Mg²⁺ is a competitive antagonist for Ca²⁺ at the A- and L-sites and also inactivates RyR2 via the I1-site, which has similar affinity for both Ca²⁺ and Mg²⁺ (Laver, Baynes & Dulhunty, 1997). During systole, Mg²⁺ inhibition occurs mainly via the I1-site (Laver, Baynes & Dulhunty, 1997). This model is used here as a framework in which to understand how Ca²⁺ and Mg²⁺ regulate RyR2 in human heart and how it may differ from that of established animal models such as sheep and rat hearts.

RyR2 was isolated from failing human (Emery Dreifuss Muscular Dystrophy with cardiomyopathy, ischemic cardiomyopathy, and dilated cardiomyopathy), non-failing human, rat and sheep heart muscle as described previously for sheep RyRs (Laver et al., 1995). Human tissues were obtained with approval from the Ethics Committee, while animal tissues obtained with approval from the Animal Care and Ethics Committee of the University of Newcastle Australia. Channel gating was measured by single channel recording in the presence of ATP (2 mmol/l) and varying concentrations of Ca²⁺ and Mg²⁺. Initially, we compared the activity of RyRs from four non-failing human hearts in bilayer experiments using 100 nmol/l Ca²⁺ in the cytoplasm (diastolic [Ca²⁺]), and 0.1 mmol/l Ca²⁺ in the lumen. Under these conditions, RyRs from all hearts showed similar gating activity. RyRs from failing hearts were significantly higher in activity compared to healthy heart. Western blots of RyR2 showed higher phosphorylation at PS2808 and PS2814 in failing hearts, consistent with the proposal that in failing hearts, RyR2 activity is increased due to hyperphosphorylation as a result of upregulation of CaMKII and PKA (Marx et al., 2000).

Regulation by intracellular Ca²⁺ and Mg²⁺ from human RyR2 was also compared to that seen in two commonly used animal models for RyR function, rat and sheep. We found that cytoplasmic Ca²⁺ dependence of RyRs P_o from sheep, rat, and human showed similar bell-shaped responses to cytoplasmic Ca²⁺ with half-activating concentrations (K_a) of 1-3 μmol/l Ca²⁺ and half-inhibiting concentration (K_i) of ∼1 mmol/l (Ca²⁺ binding to the A and I1-site, respectively). All species were similarly inhibited by cytoplasmic Mg²⁺ in the presence of 100 nmol/l Ca²⁺ in the cytoplasm (diastolic [Ca²⁺]). However, at high cytoplasmic Ca²⁺, RyR2 from rat was 10-fold more sensitive to cytoplasmic Mg²⁺ than sheep and human. RyRs from the three species could be activated by luminal Ca²⁺. RyRs showed a single, hyperbolic dependence on luminal Ca²⁺ with maximum opening rate of 2/s, 4/s, and 18 for rat, sheep, and human, respectively. Human hearts were 10-fold more sensitive to luminal Ca²⁺ than those from rat and sheep, corresponding to the Ca²⁺ affinity of the L-site. However, human RyR2 is 4-5 fold less sensitive to luminal Mg²⁺ at low luminal [Ca²⁺] in comparison to rat and sheep. These differences in the regulation between human, sheep and rat RyRs may reflect differences in excitation-contraction coupling in species with very different basal heart rates.

Laver DR, Baynes TM, Dulhunty AF. (1997) Magnesium inhibition of ryanodine-receptor calcium channels: evidence for two independent mechanisms. Journal of Membrane Biology 156: 213-229.

Laver DR, Honen BN. (2008) Luminal Mg²⁺, a key factor controlling RYR2-mediated Ca²⁺ release: cytoplasmic and luminal regulation modeled in a tetrameric channel. Journal of General Physiology 132: 429-446.

Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR, Dulhunty AF (1995) Cytoplasmic Ca²⁺ inhibits the ryanodine receptor from cardiac muscle. Journal of Membrane Biology 147: 7-22.

Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. (2000) PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 101: 365-376.