ATP and related adenine nucleotides are thought to play an important regulatory role as activators of the cardiac ryanodine receptor (RyR). We have previously demonstrated that the phosphate groups of ATP provide most of the energy required to activate the channel (Chan et al. 2000) but it has been assumed that the adenine moiety of ATP is essential for recognition and binding to the adenine nucleotide site. To examine if the triphosphate group requires the adenine (and ribose) group for activity at the cardiac RyR we have therefore investigated if triphosphate (PPPi) alone can activate cardiac RyR channels.
Native RyR from sheep hearts obtained from an abattoir were incorporated into bilayers under voltage-clamp conditions and single-channel recordings were monitored with Ca2+ as the permeant ion as previously described (Chan et al. 2000). [3H]ryanodine binding to isolated heavy SR was performed as previously described (Kermode et al. 1998). In the presence of 10 mM Ca, the amount of [3H]ryanodine bound to the heavy SR membrane fraction was 0.232 ± 0.020 pmol (mg protein)-1 (mean ± S.D.; n = 3). ATP (10 mM), PPPi (10 mM), adenosine (5 mM), pyrophosphate (PPi) (10 mM) and inorganic phosphate (Pi) (10 mM) stimulated ryanodine binding to 163, 140, 112, 102 and 108 % of control, respectively (n = 3). In agreement with the binding studies, the open probability (Po) of single RyR channels incorporated into bilayers was increased in a concentration-dependent manner by PPPi. For example, in a typical channel, Po was 0.059 in the presence of 10 mM cytosolic Ca2+ alone and was 0.126 and 0.66 after cytosolic addition of 1 and 10 mM PPPi, respectively.
In summary we have shown that PPPi can activate the cardiac RyR but not as effectively as ATP. The adenine moiety is therefore not essential for activation of the channel but is required to realise the full activating potential of the PPPi group.
This work was supported by the British Heart Foundation.