Isoquinolines as antagonists of the P2X₇ nucleotide receptor: High selectivity for the human versus rat receptor homologues

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-met hyl-L-tyrosyl]-4- phenylpiperazine (KN-62) and N-[1-[N-methyl-p-(5 isoquinolinesulfonyl)benzyl]-2-(4 phenylpiperazine)ethyl]-5-isoquino- linesulfonamide (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 P2X₇ may also indicate structural domains important in channel/pore activation.