Type 1 ryanodine receptor in cardiac mitochondria: Transducer of excitation–metabolism coupling

Abstract

Mitochondria in a variety of cell types respond to physiological Ca(2+) oscillations in the cytosol dynamically with Ca(2+) uptakes. In heart cells, mitochondrial Ca(2+) uptakes occur by a ruthenium red-sensitive Ca(2+) uniporter (CaUP), a rapid mode of Ca(2+) uptake (RaM) and a ryanodine receptor (RyR) localized in the inner mitochondrial membrane (IMM). Three subtypes of RyRs have been described and cloned, however, the subtype identity of the mitochondrial ryanodine receptor (mRyR) is unknown. Using subtype specific antibodies, we characterized the mRyR in the IMM from rat heart as RyR1. These results are substantiated by the absence of RyR protein in heart mitochondria from RyR1 knockout mice. The bell-shape Ca(2+)-dependent [(3)H]ryanodine binding curve and its modulation by caffeine and adenylylmethylenediphosphonate (AMPPCP) give further evidence that mRyR functions pharmacologically like RyR1. Ryanodine prevents mitochondrial Ca(2+) uptake induced by raising extramitochondrial Ca(2+) to 10 microM. Similarly, ryanodine inhibits oxidative phosphorylation stimulated by 10 microM extramitochondrial Ca(2+). In summary, our results show that the mRyR in cardiac muscle has similar biochemical and pharmacological properties to the RyR1 in the sarcoplasmic reticulum (SR) of skeletal muscle. These results could also suggest an efficient mechanism by which mitochondria sequesters Ca(2+) via mRyR during excitation-contraction coupling to stimulate oxidative phosphorylation for ATP production to meet metabolic demands. Thus, the mRyR functions as a transducer for excitation-metabolism coupling.

Excitation-contraction coupling and mitochondrial energetics

“…Currently, there is no specific inhibitor that could differentiate between the mCU and mRyR1. Ruthenium red inhibits the mCU [124], but also the mRYR1 [19] and the RyR2 subtype [124]. In contrast, Ru360 inhibits the mCU, but not the RyR2 subtype [124].…”

“…6D; [12,117] . However, whether the mRyR1 is inhibited by Ru360 has not been determined [19]. Thus, it may be possible that the mRyR1 contributes to mitochondrial Ca 2+ -uptake, or may even resemble the mCU.…”

“…However, in cardiac mitochondria, the RaM required > 30 s to become fully reactivated at extramitochondrial [Ca 2+ ] < 100 nmol/L [30] and thus, cannot be responsible for beat-to-beat mitochondrial Ca 2+ uptake. Furthermore, Beutner et al [18,19] have proposed that mitochondrial RyR (RyR1 subtype) channels may mediate Ca 2+ flux across the inner mitochondrial membrane. The binding of [ 3 H]ryanodine to this mRyR1 has a bellshaped Ca 2+ -dependence, with an optimum between 10-30 µM [19], the latter value comparable to the reported K 0.5 of the mCU for Ca 2+ [54,77].…”

“…Furthermore, Beutner et al [18,19] have proposed that mitochondrial RyR (RyR1 subtype) channels may mediate Ca 2+ flux across the inner mitochondrial membrane. The binding of [ 3 H]ryanodine to this mRyR1 has a bellshaped Ca 2+ -dependence, with an optimum between 10-30 µM [19], the latter value comparable to the reported K 0.5 of the mCU for Ca 2+ [54,77]. Currently, there is no specific inhibitor that could differentiate between the mCU and mRyR1.…”

Physical Coupling Supports the Local Ca2+ Transfer between Sarcoplasmic Reticulum Subdomains and the Mitochondria in Heart Muscle

“…Although the majority of the RyR2 does not seem to directly face the mitochondria in the intact cardiac muscle, the RyR2s are sufficiently close (27-200 nm (9)) to the mitochondrial surface to expose the Ca 2ϩ uptake sites to an estimated 10 -20 M [Ca 2ϩ ] c during Ca 2ϩ release (36). Regarding the molecular mechanism of the Ca 2ϩ uptake by cardiac mitochondria, an interesting candidate is the mitochondrial mRyR1, recently described by Beutner, Sheu, and co-workers (22,37,38). The ruthenium derivative Ru360 has been reported highly specific to the MCU with no inhibitory effect on RyR2 in heart (21) or on RyR1 in skeletal muscle (39) (as opposed to ruthenium red that inhibits both MCU and RyR).…”