Cloning and pharmacological characterization of the dog P2X7 receptor.
OBJECTIVE
Human and rodent P2X7 receptors exhibit differences in their sensitivity to antagonists. In this study we have cloned and characterized the dog P2X7 receptor to determine if its antagonist sensitivity more closely resembles the human or rodent orthologues.


METHODS
A cDNA encoding the dog P2X7 receptor was isolated from a dog heart cDNA library, expressed in U-2 OS cells using the BacMam viral expression system and characterized in electrophysiological, ethidium accumulation and radioligand binding studies. Native P2X7 receptors were examined by measuring ATP-stimulated interleukin-1beta release in dog and human whole blood.


RESULTS
The dog P2X7 receptor was 595 amino acids long and exhibited high homology (>70%) to the human and rodent orthologues although it contained an additional threonine at position 284 and an amino acid deletion at position 538. ATP possessed low millimolar potency at dog P2X7 receptors. 2'-&3'-O-(4benzoylbenzoyl) ATP had slightly higher potency but was a partial agonist. Dog P2X7 receptors possessed relatively high affinity for a number of selective antagonists of the human P2X7 receptor although there were some differences in potency between the species. Compound affinities in human and dog blood exhibited a similar rank order of potency as observed in studies on the recombinant receptor although absolute potency was considerably lower.


CONCLUSIONS
Dog recombinant and native P2X7 receptors display a number of pharmacological similarities to the human P2X7 receptor. Thus, dog may be a suitable species for assessing target-related toxicity of antagonists intended for evaluation in the clinic.
Roman
S
S
Neurology Centre of Excellence for Drug Discovery, GlaxoSmithKline Research & Development Limited, New Frontiers Science Park, Harlow, Essex, UK. shilina.2.roman@gsk.co.uk
Cusdin
F S
FS
Fonfria
E
E
Goodwin
J A
JA
Reeves
J
J
Lappin
S C
SC
Chambers
L
L
Walter
D S
DS
Clay
W C
WC
Michel
A D
AD
D000255:Adenosine Triphosphate / Q000008:administration & dosage; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004285:Dogs; D004594:Electrophysiology; D004996:Ethidium / Q000378:metabolism; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D011869:Radioligand Assay; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058486:Receptors, Purinergic P2X7; D012377:Rodentia; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D053583:Interleukin-1beta; C546468:P2RX7 protein, human; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D000255:Adenosine Triphosphate; D004996:Ethidium