Identification of Amino Acid Residues Contributing to the ATP-binding Site of a Purinergic P2X Receptor

Abstract

P2X receptor subunits have intracellular N and C termini, two membrane-spanning domains, and an extracellular loop of about 280 amino acids. We expressed the rat P2X 2 receptor in human embryonic kidney cells, and used alanine-scanning mutagenesis on 30 residues with polar side chains conserved among the seven rat P2X receptor subunits. This identified a region proximal to the first transmembrane domain which contained 2 lysine residues that were critical for the action of ATP (Lys 69 and Lys 71 ). We substituted cysteines in this region (Asp 57 to Asp 71 ) and found that for S65C and I67C ATPevoked currents were inhibited by methanethiosulfonates. At I67C, the inhibition by negatively charged ethylsulfonate and pentylsulfonate derivatives could be overcome by increasing the ATP concentration, consistent with a reduced affinity of ATP binding. The inhibitory action of the methanethiosulfonates was prevented by pre-exposure to ATP, suggesting occlusion of the binding site. Finally, introduction of negative charges into the receptor by mutagenesis at this position (I67E and I67D) also gave receptors in which the ATP concentration-response curve was right-shifted. The results suggest that residues close to Ile 67 contribute to the ATP-binding site.P2X receptors are ligand-gated ion channels in the plasma membrane (1). They are homomeric or heteromeric proteins, formed by assembly of subunits named P2X 1 -P2X 7 . Current evidence suggests that three subunits form a channel (or a multiple of three) (2, 3). The ligand for the P2X receptors is ATP, acting from the extracellular milieu. In this way ATP released from cells functions as a synaptic transmitter, an autocrine or paracrine signal, in a wide range of mammalian tissues (4). Other membrane proteins which bind ATP from the extracellular aspect include G-protein-coupled P2Y receptors, and degradative enzymes such as ectoATPase (4).The individual subunits of the P2X receptor seem unrelated in amino acid sequence to other ion channels, and indeed to other proteins. The proteins range in length from 379 to 595 amino acids. The P2X 2 receptor is normally glycosylated (Asn 183 , Asn 239 , and Asn 298 ), and experiments with the introduction of additional glycosylation sites by mutagenesis indicate that most of the protein is extracellular (the ectodomain: residues 50 -330) (5, 6). The N terminus and C terminus lie within the cell, and the residues 30 -50 and 330 -352 form membrane-spanning domains. Three kinds of approaches have been made to determine which parts of the molecule contribute to which of the functional properties of the channel, but none of these have addressed the ATP-binding site. First, the substituted cysteine accessibility method has been used to show that residues in and around the second membrane-spanning domain contribute to the formation of permeation pathway for cations (7,8); in particular, Thr 336 appears to lie in the outer vestibule of the pore and Asn 349 is internal to the gate of the pore (7). Second, desensitization is profound in the ca...

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

“…The large extracellular loop is believed to be responsible for ligandbinding and is quite conserved among the P2X receptors, although the fine structure of the ligand-binding pocket is still uncertain. Conserved positively charged amino acid residues are important for nucleotide binding (K64, K66, R294, R307, and K311) [5,57,[73][74][75]. Aromatic amino acids are associated with recognition of adenine nucleotides in many ATP-binding proteins and are proposed to bind the adenine ring [5,75].…”

Amino acid variations resulting in functional and nonfunctional zebrafish P2X1 and P2X5.1 receptors

“…One contributing factor is that this subunit lacks two highly conserved lysine residues believed to form part of the ATP binding site of P2X receptors [37,38]. However, there must be other differences from mammalian P2X 2 receptors as well, since substitution of EMR with KMK failed to result in ATP-induced currents above background.…”

P2X7 Receptor Cell Surface Expression and Cytolytic Pore Formation Are Regulated by a Distal C-terminal Region

“…The extracellular loop contains conserved cysteine, lysine, and glycine residues along with a number of potential N-linked glycosylation sites, all of which contribute to the structural constraints required for ATP binding (11)(12)(13). The C terminus of the P2X7R is 120 amino acids longer than any of the other P2X members.…”