Control of Diastolic Activity of the RyR2 Channel by Luminal Calcium and ATP

Abstract

The mechanism of activation of the cardiac ryanodine receptor (RyR2) by luminal Ca2+ in the presence of ATP was examined in planar lipid bilayers. The dose response of the RyR2 channel to ATP was characterized at a range of cytosolic (100-400 nM) and luminal (0.01 - 53 mM) Ca2+ concentrations. Luminal Ca2+ markedly increased the maximal open probability in the presence of ATP (POmax) and markedly decreased EC50 for ATP (EC50ATP). Cytosolic Ca2+, without substantially activating the RyR2 channel in the absence of ATP, greatly amplified the effects of 1 mM [Ca2+]L on POmax and EC50ATP. An allosteric model of RyR2 interaction with ATP showed that the increase in RyR2 open probability by luminal Ca2+ in the presence of ATP is induced by a decrease of the ATP dissociation constant (KATP) at low [Ca2+]L, by an increase in the positive allosteric effect of ATP on channel opening (decrease of fATP) at intermediate [Ca2+]L, and by an increase in the stability of the ATP-free RyR2 open state (decrease of KO0) at the highest [Ca2+]L. Increasing [Ca2+]C did not affect KATP but led to a parallel decrease of KO0 and fATP. These results suggest that the allosteric effect of ATP might be mediated by the energy of the ATP-free open state, i.e., indirectly by the effect of the occupancy of the channel by Ca2+ at the cytosolic and luminal sites on the stability of the open state. The increase of RyR2 open probability at diastolic levels of cytosolic Ca2+ by elevated luminal Ca2+ may play a role in the calcium overload induced Ca2+ release.Supported by APVV-0721-10, APVV-0628-10, VEGA2/0118/09 and VEGA 2/0190/10