Molecular Basis of Inhibitory Modulation of P2X2 Receptors by Zinc and Copper.

Abstract

P2X receptors are ion channels gated by ATP and modulated by the trace metals zinc and copper. Rat P2X2 (rP2X2) receptors show potentiation in the presence of a submaximal concentration of ATP but are inhibited by zinc at concentrations above 100 µM while human P2X2 (hP2X2) receptors are strongly inhibited by zinc over the range of 2-100 µM. Copper and zinc are released from the presynaptic terminals of some neurons but biological role of zinc modulation of P2X2 receptors is unknown in either species. In Chapter 2, I used the known 3D crystal structure of the zebrafish P2X4.1 receptor as a template to identify candidate residues that might be involved in zinc inhibition of hP2X2 receptors, and then tested the effect of these residues using site directed mutagenesis followed by biochemical and electrophysiological analysis. I demonstrated that a cluster of three histidine residues at the subunit interface controls zinc modulation. I also showed that the low affinity zinc inhibition of rP2X2 receptors can be converted to high affinity inhibition by a single residue change. I 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 Chapter 3, I showed that hP2X2 receptors are potently inhibited by copper. This high affinity inhibition has an extremely slow recovery after washout of copper. ATP was required for copper to reach its inhibitory binding site but was not essential for copper to leave the site. The first six cysteines were not required for normal copper inhibition. The same three histidine residues required for normal zinc inhibition were also required for normal copper inhibition. Furthermore, DTT, a reducing agent, dramatically accelerated recovery from copper inhibition. Humans with Wilson’s disease have excess amounts of copper accumulation in the brain. The copper sensitivity of hP2X2 suggests that these patients have non-functional P2X2 receptors.