Isoquinolines as Antagonists of the P2X7 Nucleotide Receptor: High Selectivity for the Human versus Rat Receptor Homologues
- 1Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 (B.D.H., J.R., G.R.D.), and 2Glaxo Institute for Molecular Biology, Plan-les-Ouates, 1228 Geneva, Switzerland (C.V., A.S.)
Abstract
1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) andN-[1-[N-methyl-p-(5 isoquinolinesulfonyl)benzyl]-2-(4 phenylpiperazine)ethyl]-5-isoquinolinesulfonamide (KN-04) potently inhibit the human lymphocyte P2Z receptor, an ATP-gated cation channel [Br J Pharmacol 120:1483–1490 (1997)]. Although the molecular identity of the lymphocyte P2Z receptor has not been established, it shares many functional characteristics with the cloned P2X7 nucleotide receptor. We have tested whether these isoquinolines inhibit P2X receptor function in human embryonic kidney 293 cells that stably express the human or rat recombinant P2X7 receptors. ATP activation of cation currents and uptake of the organic dye ethidium were potently inhibited by KN-62 and KN-04 in human embryonic kidney cells expressing the human P2X7R but not the rat P2X7R, even though these species homologues share 80% amino acid identity. Introduction of the first 335 amino acids of the human P2X7R sequence conferred KN-62 sensitivity to the rat P2X7R; this suggests that isoquinolines interact with residues in the amino-terminal half (containing the large extracellular loop) of the human P2X7R. KN-62 and KN-04 also potently inhibited ATP-gated Ca2+ influx and ethidium uptake in several leukocyte cell lines (THP-1, BAC1.2f5, and BW5147) that natively express the human or murine P2X7R mRNA. The ability of isoquinoline sulfonamides to potently inhibit human and murine P2X7R signaling will be a useful tool for identifying P2Z/P2X7 functional responses in other cell types. The substantial differences in pharmacological sensitivity between rat and human P2X7R may also indicate structural domains important in channel/pore activation.
Footnotes
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Send reprint requests to: Dr. George R. Dubyak, Department of Physiology & Biophysics, School of Medicine E565, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4970. E-mail: gxd3{at}po.cwru.edu
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This work was supported in part by National Institutes of Health Grant GM36387 (G.R.D.).
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Portions of this work were presented at a meeting for the American Society of Biology and Molecular Biology (San Francisco, CA) and at Purines ’97 (New Orleans, LA).
- Abbreviations:
- P2X7R
- P2X7receptor
- rP2X7
- rat P2X7
- hP2X7
- human P2X7
- HEK
- human embryonic kidney
- CamKII
- calcium/calmodulin-dependent protein kinase II
- Bz
- 3′-O-(4-benzoyl)benzoyl
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- IFN
- interferon-γ
- LPS
- lipopolysaccharide
- IL-1β
- interleukin-1β
- CLL
- chronic lymphocytic leukemia
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
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- Received January 14, 1998.
- Accepted March 11, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
