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Human P2X2 receptors (hP2X2) are strongly inhibited by zinc over the range of 2-100 μM, whereas rat P2X2 receptors (rP2X2) are strongly potentiated over the same range, and then inhibited by zinc over 100 μM. However, the biological role of zinc modulation is unknown in either species. To identify candidate regions controlling zinc inhibition in hP2X2 a homology model based on the crystal structure of zebrafish P2X4.1 was made. In this model, His-204 and His-209 of one subunit were near His-330 of the adjacent subunit. Cross-linking studies confirmed that these residues are within 8 Å of each other. Simultaneous mutation of these three histidines to alanines decreased the zinc potency of hP2X2 nearly 100-fold. In rP2X2, one of these histidines is replaced by a lysine, and in a background in which zinc potentiation was eliminated, mutation of Lys-197 to histidine converted rP2X2 from low potency to high potency inhibition. We explored whether the zinc-binding site lies within the vestibules running down the central axis of the receptor. Elimination of all negatively charged residues from the upper vestibule had no effect on zinc inhibition. In contrast, mutation of several residues in the hP2X2 middle vestibule resulted in dramatic changes in the potency of zinc inhibition. In particular, the zinc potency of P206C could be reversibly shifted from extremely high (∼10 nM) to very low (>100 μM) by binding and unbinding MTSET. These results suggest that the cluster of histidines at the subunit interface controls access of zinc to its binding site.

The response of P2X(2) receptors to submaximal concentrations of ATP is potentiated by low levels of extracellular zinc. Histidines 120 and 213 have previously been shown to be essential in binding zinc across an intersubunit binding site. We tested the flexibility of the zinc-binding site by making mutations that had the effect of shifting the two essential histidines up to 13 residues upstream or downstream from their original positions and then testing the ability of the mutated receptors to respond to zinc. Using this method, we were able to explore potential orientations of the two regions relative to one another. Our data are consistent with a moderately flexible zinc-binding site and inconsistent with parallel and anti-parallel orientations of the regions surrounding histidines 120 and 213.

The efficiency of synaptic transmission between nerve and muscle depends on the number and density of acetylcholinesterase molecules (AChE) at the neuromuscular junction. However, little is known about the way this density is maintained and regulated in vivo. By using time lapse and quantitative fluorescence imaging assays in living mice, we demonstrated that insertion of new AChEs occurs within hours of saturating pre-existing AChEs with fasciculin2, a snake toxin that selectively labels AChE. In the absence of muscle postsynaptic activity or evoked nerve presynaptic neurotransmitter release, AChE insertion was decreased significantly, whereas direct stimulation of the muscle completely restored AChE insertion to control levels. This activity-dependent AChE insertion is mediated by intracellular calcium. In muscle stimulated in the presence of a Ca2+ channel blocker or calcium-permeable Ca2+ chelator, AChE insertion into synapses was significantly decreased, whereas ryanodine or ionophore A12387 treatment of blocked and unstimulated synapses significantly increased AChE insertion. These results demonstrated that synaptic activity is critical for AChE insertion and indicated that a rise in intracellular calcium either through voltage-gated calcium channels or from intracellular stores is critical for proper AChE insertion into the adult synapse.

P2X receptors are ATP-gated ion channels made up of three similar or identical subunits. It is unknown whether ligand binding is intersubunit or intrasubunit, either for agonists or for allosteric modulators. Zinc binds to rat P2X2 receptors and acts as an allosteric modulator, potentiating channel opening. To probe the location of this zinc binding site, P2X2 receptors bearing mutations of the histidines at positions 120 and 213 were expressed in Xenopus oocytes. Studies of H120C and H213C mutants produced five lines of evidence consistent with the hypothesis that the residues in these positions bind zinc. Mixing of subunits containing the H120A or H213A mutation generated receptors that showed zinc potentiation, even though neither of these mutant receptors showed zinc potentiation on its own. Furthermore, expression of trimeric concatamers with His --> Ala mutations at some but not all six positions showed that zinc potentiation correlated with the number of intersubunit histidine pairs. These results indicate that zinc potentiation requires an interaction across a subunit interface. Expression of the H120C/H213C double mutant resulted in the formation of ectopic disulfide bonds that could be detected by changes in the physiological properties of the receptors after treatment with reducing and oxidizing agents. Immunoblot analysis of H120C/H213C protein separated under nonreducing conditions demonstrated that the ectopic bonds were between adjacent subunits. Taken together, these data indicate that His120 and His213 sit close to each other across the interface between subunits and are likely to be key components of the zinc binding site in P2X2 receptors.