Abstract
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.
Highlights
Zinc inhibits human P2X2 with high potency
Homology Model for Human P2X2 receptors (hP2X2) Suggests That His-204, His-209, and His-330 Lie Close Together Across the Subunit Interface— In rP2X2, the zinc-binding site that causes potentiation includes two histidines on opposite sides of the subunit interface
When the sequence of hP2X2 was used to create a homology model based on the structure of zP2X4.1, His-330 from one subunit was predicted to lie close to His-204 and His-209 across the interface between adjacent subunits (Fig. 1A), raising the possibility that this histidine cluster might contribute to the zinc-binding site responsible for the high potency zinc inhibition seen in hP2X2 receptors
Summary
Zinc inhibits human P2X2 with high potency. Results: Site-directed mutations of P2X2 with a 100,000-fold range in the potency of zinc inhibition were made. It is possible that the movements associated with channel opening might allow access to the upper vestibule These vestibules are potential locations for the high potency inhibitory zinc-binding site of hP2X2. Our goal for this study was to use a homology model of hP2X2 based on the structure of zP2X4.1 to suggest candidate residues for involvement in inhibitory zinc binding, with the hope of identifying mutations that dramatically enhance or attenuate zinc inhibition of hP2X2 Identification of such mutants might provide a framework for understanding the molecular basis of zinc inhibition, and offer the possibility of using them as tools to probe the role of P2X receptors in vivo
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