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 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.

K201 (JTV519) is a Ca2+-Dependent Blocker of SERCA and a Partial Agonist of Ryanodine Receptors in Striated Muscle

“…However, at a SR voltage of 260 mV (similar as in Almassy et al, 2008), we observed flicker block and intermediate states of conductance (which could be attributed to short-lived substates) in RyR2 induced by K201. The blocking effect of K201 is intriguing, but the role of voltage dependence in RyRs has unknown physiologic significance as channels are thought to operate at a nearly constant SR voltage of ∼0 mV Diaz-Sylvester et al, 2011).…”

“…It is well known that RyRs activity heavily depends on luminal Ca 21 levels (Sitsapesan and Williams, 1998;Fill and Copello, 2002;Terentyev et al, 2002;Diaz-Sylvester et al, 2011;Porta et al, 2012). Although the effects of K201 on SR Ca 21 content appear to be mild under control conditions (Loughrey et al, 2007), the drug could have more significant effects under pathologic situations as in ischemia.…”

“…Closed times were Effects of K201 on RyRs and SERCA visibility of any K201-induced changes in conductance. Even minor K201 effects would be visible when minimizing noise from flicker short openings/closures, which is found under control conditions in most other RyRs Diaz-Sylvester et al, 2011). We used Cs 1 (instead of Tris 1 ) in our cytosolic (cis) solution to increase RyR2 channel amplitude at 0 and 215 mV, as well as to generate measurable cis-totrans Cs 1 currents (i.e., 2.3 pA cytosol to lumen) at the SR voltage (V m ) of 260 mV.…”

“…Channel recordings (4-8 minutes in duration in each condition) were filtered at 1 kHz, digitized at 20 kHz with a Digidata 1360 (Molecular Devices), stored, and analyzed using pClamp10 software (Axon Instruments) as described before Diaz-Sylvester et al, 2011). The presence and abundance of substates and/or flicker block were estimated as previously described (Porta et al, 2008).…”

Eudistomin D and Penaresin Derivatives as Modulators of Ryanodine Receptor Channels and Sarcoplasmic Reticulum Ca2+ATPase in Striated Muscle

“…This heterogeneity results from the fact that some channels can dwell more often in high-open-probability (P o ) mode, characterized by long openings and short closures, while others display frequent low-P o mode, distinguished by frequent and brief flickering openings . RyRs dwelling in the high-P o mode were found to display increased sensitivity to physiologic agonists, lower sensitivity to blockers, and increased ability to associate with neighboring channels for coupled gating (Gyorke and Fill, 1993;Zahradnikova et al, 1999;Copello et al, 2002;Diaz-Sylvester et al, 2011;Porta et al, 2012). Thus, the domains in the RyR molecule involved in the stabilization of high-and low-P o modes have a huge significance for the synchronous gating of the channels.…”