Effects of Divalent Current Carriers on Voltage-Dependence of RyR2 Channels

Abstract

Voltage-dependent modulation of cardiac ryanodine receptors (RyR2) was studied in planar lipid bilayers. Different earth alkaline cations (M2+: Mg2+, Ca2+, Sr2+, Ba2+) were utilized as current carriers. When added to the cytosolic surface of the channels, Ca2+ (EC50 ∼2μM) or Sr2+ (EC50 ∼20μM) interacted with cytosolic high affinity (HA) sites and activated the channels. Neither Mg2+ nor Ba2+ activated RyR2 but only Mg2+ interfered with Ca2+/Sr2+ action. Fully activated RyR2 (100-200μM cytosolic Ca2+) were inhibited with low affinity (LA) by all M2+ (similar IC50, ranging 3.6-5.7mM) suggesting that LA inhibitory sites do not distinguish M2+ identity. We found that RyR2 were much more active with lumenal Ca2+/Sr2+ versus lumenal Ba2+/Mg2+. However increasing lumenal→cytosolic M2+ flux by increasing lumenal holding voltage (Vm) never increased open probability (Po). In contrast, increasing Vm decreased Po in half of the RyR2 exposed to lumenal Ca2+/Sr2+ and in all RyR2 exposed to lumenal Ba2+/Mg2. This suggests that lumenal flux does not reach HA but LA sites. An effect of Vm is evident in all channels displaying modal gating (low and high Po mode) but not in RyR2 where high Po dominates. Indeed, high Po mode is much more abundant with lumenal Ca2+/Sr2+. Moreover, increasing cytosolic Ca2+ activated all channels and removed the effect of Vm. In summary, modulation of RyR2 gating by M2+ flux is complex and seems to reflect lumenal M2+-dependent stabilization of high Po and low Po mode (the latter being voltage-dependent). Supported by NIH R01 GM078665 to JAC.