Zinc and copper are atypical modulators of ligand-gated ionic channels in the central nervous system. We sought to identify the amino acids of the rat P2X 4 Extracellular ATP and structurally related nucleotides act as novel cell messengers through the activation of P2X receptors, which belong to the family of ligand-gated ionic channels. In addition, nucleotides, and particularly pyrimidine nucleotides, such as UTP and UDP, act on metabotropic P2Y receptors, members of the G protein-coupled receptor family. Seven subtypes of P2X channels have been identified and have been shown to be involved in a variety of neuronal pathways including pain transmission, the urination reflex, vas deferens contraction favoring sperm migration, etc. (1). These receptors are unique among ligand-gated ionic channels because each receptor subunit has only two transmembrane domains, with both the C and N termini facing the cytosol (2, 3). Moreover, recent studies using atomic force microscopy (4) provided topological evidence of the channel conformation and established that the functional P2X receptor channels are trimers, composed of either homo or heterotrimeric subunits (4 -6). Site-directed mutagenesis has provided pivotal information about specific P2X properties: the channel pore, agonist binding residues, receptor desensitization and allosteric modulation (7-16). As with other ligand-gated ionic channels, the P2X receptors are modulated by divalent metals including trace metals although the nature of the modulation and the magnitude of these effects vary among the different P2X subunits (12-17). The role of divalent trace metals as neuromodulators is of interest as zinc and copper are both novel and atypical brain transmitters (18,19) and novel intracellular second messengers (20). The notion that zinc and copper are stored in neurons and are released upon electrical depolarization further highlights their importance in brain excitability with ample physiological and pharmacological implications (21,22).The P2X 4 receptor is an interesting model of an ionic channel differentially modulated by divalent trace metals. In a series of studies, and Coddou et al. (23,24) reported that zinc potentiated the ATP-evoked currents, whereas copper exerts an inhibitory effect on the activity of this receptor. Furthermore, single site-directed mutagenesis of each of the three extracellular histidine residues of the P2X 4 receptor revealed that only histidine 140 plays a key role in the inhibitory modulation by copper and high zinc concentrations (13). The replacement of His 140 by an alanine (H140A mutant) was not only resistant to the copper-induced inhibition of the ATPgated receptor activity but evidenced a dramatic increase in the zinc-induced potentiation. Zinc potentiated more than 20-fold the ATP-evoked currents in the H140A mutant; the metal evidenced in this mutant a sigmoid concentration-response dependence instead of the bell-shaped zinc curve described in the wild-type receptor. This finding brought forth the hypoth-* This resea...