title: Diadenosine pentaphosphate is a potent activator of cardiac ryanodine receptors revealing a novel high-affinity binding site for adenine nucleotides.
abstract: OBJECTIVE
  Diadenosine polyphosphates are normally present in cells at low levels, but significant increases in concentrations can occur during cellular stress. The aim of this study was to investigate the effects of diadenosine pentaphosphate (Ap5A) and an oxidized analogue, oAp5A on the gating of sheep cardiac ryanodine receptors (RyR2).
  
  
  METHODS
  RyR2 channel function was monitored after incorporation into planar bilayers under voltage-clamp conditions.
  
  
  RESULTS
  With10 micromol.L(-1) cytosolic Ca2+, a significant 'hump' or plateau at the base of the dose-response relationship to Ap5A was revealed. Open probability (Po) was significantly increased to a plateau of approximately 0.2 in the concentration range 100 pmol x L(-1)-10 micromol x L(-1). High Po values were observed at >10 micromol x L(-1) Ap5A, and Po values close to 1 could be achieved. Nanomolar levels of ATP and adenosine also revealed a hump at the base of the dose-response relationships, although GTP did not activate at any concentration, indicating a common, high-affinity binding site on RyR2 for adenine-based compounds. The oxidized analogue, oAp5A, did not significantly activate RyR2 via the high-affinity binding site; however, it could fully open the channel with an EC(50) of 16 micromol.L(-1) (Ap5A EC(50) = 140 micromol x L(-1)). Perfusion experiments suggest that oAp5A and Ap5A dissociate slowly from their binding sites on RyR2.
  
  
  CONCLUSIONS
  The ability of Ap5A compounds to increase Po even in the presence of ATP and their slow dissociation from the channel may enable these compounds to act as physiological regulators of RyR2, particularly under conditions of cellular stress.
authors.affiliation: Department of Physiology & Pharmacology, University of Bristol, School of Medical Sciences, University Walk, Bristol, UK.
authors.forename: L
authors.initials: L
authors.lastname: Song
authors.forename: S M
authors.initials: SM
authors.lastname: Carter
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: R
authors.initials: R
authors.lastname: Sitsapesan
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002120:Calcium Channel Agonists / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D008024:Ligands; D008051:Lipid Bilayers; D009206:Myocardium / Q000378:metabolism*; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D010504:Periodic Acid / Q000737:chemistry; D010743:Phospholipids; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D012756:Sheep
chemical_list: D000227:Adenine Nucleotides; D002120:Calcium Channel Agonists; D015226:Dinucleoside Phosphates; D008024:Ligands; D008051:Lipid Bilayers; D010743:Phospholipids; D019837:Ryanodine Receptor Calcium Release Channel; D010504:Periodic Acid; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; C009288:metaperiodate