Activation of Trimeric P2X2 Receptors by Fewer than Three ATP Molecules

Abstract

P2X receptors are trimeric membrane proteins. When they bind extracellular ATP, a conformational change occurs that opens a transmembrane ion channel. The ATP-binding pocket is formed in a cleft between two subunits, and a critical amino acid residue for ATP contact is Lys 69 (P2X2 numbering). In the present work, we sought to determine whether the binding of fewer than three ATP molecules could open the ion channel. We expressed eight concatenated cDNAs in human embryonic kidney cells, which encoded three serially joined, epitopetagged, subunits with either Lys or Ala at position 69 (denoted as KKK, KKA, KAK, AKK, KAA, AKA, AAK, and AAA). Western blotting of surface-biotinylated proteins indicated that breakdown of concatemers to individual subunits was minimal. Recording of membrane currents in response to ATP (whole cell and excised outside-out patch) showed that all formed functional channels except AAK, AKA, and AAA. There was no difference in the kinetics of activation and deactivation among KKK, KKA, KAK, and AKK channels, and amplitude of the unitary conductances was in all cases not different from that found after expression of a single wild-type subunit. Currents through KKA and KAK receptors were larger than those observed for AKK receptors. The results indicate that trimeric P2X receptors containing only two intact binding sites can be readily activated by ATP.

Optical control of trimeric P2X receptors and acid-sensing ion channels

“…P329 is oriented toward other P329 residues in adjacent subunits and is solvent-exposed. We found that bridging two of the three cysteines was sufficient to open and close trimeric P2X2 receptors, consistent with the finding that only two intact agonist-binding sites (and by inference only two ATP molecules) are required for P2X receptor activation (26). It is noteworthy that at rest (the closed channel state) the BMA was in its higher-energy cis isomer, suggesting it is constrained in this form by the protein to which it is conjugated.…”

“…Two of the three forms with a single P329C substitution also gave small responses to light. This was most notable for the form with cysteine in the first of the three concatenated subunits and may reflect aberrant ("heads-up") channel formation from three N-terminal subunits or from minimal breakdown of the concatemers as observed and discussed previously (26).…”

“…The homotrimeric P2X receptor presents three identical ATP binding sites, but occupancy of only two of these is sufficient to drive channel opening (26). We constructed concatenated cDNAs that encoded three joined subunits and introduced the P329C mutation into the first and/or second and/or third subunit.…”

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

“…(1) The binding sites of ATP are contributed by domains from different subunits, namely, the head and LF domains from the same subunit, and the DF and upper body domains from a neighbor subunit ( Figure 4A). Therefore, there are three ATP binding sites in a three-fold symmetric mode, although a recent study showed that ATP binding at only two of the three sites is sufficient for channel opening [107] . (2) The body domains of the three subunits intertwine with each other, forming the fundamental core of the P2X receptors ( Figure 4B), which is surrounded by the three ATP binding sites.…”

P2X receptors

“…Several lines of evidence indicated that the functional protein was a trimer: indeed, three ATP-binding sites had been suggested by Bean [14] on the basis of his ATP dose-response curves from bullfrog sensory neurons. This evidence included biochemical approaches using blue native polyacrylamide gel electrophoresis [30] and functional approaches with co-expression and concatenation of subunits carrying reporter mutations [31][32][33][34][35]. The demonstration that P2X receptors were trimers set them in clear distinction from the tetrameric glutamate-gated ion channels and the pentameric nicotinic superfamily (which also includes channels gated by glycine, g-aminobutryic acid and 5-hydroxytryptamine; [22]).…”