Ca²⁺ and voltage dependence of cardiac ryanodine receptor channel block by sphingosylphosphorylcholine

Abstract.

The effect of sphingosylphosphorylcholine (SPC) on the cytoplasmic Ca²⁺ and voltage dependence of channel gating by cardiac ryanodine receptors (RyR) was examined in lipid bilayer experiments. Micromolar concentrations of the lysosphingolipid SPC added to cis solutions rapidly and reversibly decreased the single-channel open probability (P _o) of reconstituted RyR channels. The SPC-induced decrease in P _o was marked by an increase in mean closed time and burst-like channel gating. Gating kinetics during intraburst periods were unchanged from those observed in the absence of the sphingolipid, although SPC induced a long-lived closed state that appeared to explain the observed decrease in channel P _o. SPC effects were observed over a broad range of cis [Ca²⁺] but were not competitive with Ca²⁺. Interestingly, the sphingolipid-induced, long-lived closed state displayed voltage-dependent kinetics, even though other channel gating kinetics were not sensitive to voltage. Assuming SPC effects represent channel blockade, these results suggest that the blocking rate is independent of voltage whereas the unblocking rate is voltage dependent. Together, these results suggest that SPC binds directly to the cytoplasmic side of the RyR protein in a location in or near the membrane dielectric, but distinct from cytoplasmic Ca²⁺ binding sites on the protein.