Effects of imipramine on the cloned P2X2 receptor/channel and its mutants expressed in Xenopus oocytes were examined. Imipramine (100 μM) partially blocked an ionic current mediated through the wild-type P2X2 receptor/channel. With a higher concentration (300 μM) of imipramine, the current block was attenuated, suggesting that the second, lower affinity, 'unblocking' binding-site for imipramine exists in addition to the 'blocking' binding-site. These profiles of the modulation by imipramine were influenced by the substitution of negatively charged or polarized amino acid residues near the outer mouth of the channel pore (Asp315, Thr330 and Asn333) with neutral amino acid residues (Val or lle). With the neutralization of Asp315, the current 'block' by 100 μM imipramine was attenuated. With the neutralization of Thr330 the current 'block' by 100 μM imipramine was enhanced. With the neutralization of Asn333, the 'unblock' by 300 μM imipramine disappeared. The results suggest that imipramine modulates P2X2 receptor/channels by interacting these amino acid residues.
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