The downward movement of the head domain can be followed by the approach of H120 to H213. Nagaya et al. (2005) showed Birinapant solubility dmso that these residues contributed to an intersubunit zinc binding site and that, when H120 and H213 are both replaced by cysteine, a new intersubunit disulfide can form which inhibits channel opening. The intimate approximation of the head domain (chain A) with the dorsal fin (chain B) appears to be in itself sufficient to lead to channel opening in a suitably mutated “reporter” receptor (Jiang et al., 2012). The movements

of the left flipper (chain A) and dorsal fin (chain B) exert tension on the β sheeted wall of the lower body of the same subunit, causing it to flex outward by a slight separation of its constituent β sheets and

increasing its circumference by a progressively greater amount as it passes down toward the outer surface of the membrane (Roberts et al., 2012). Here, at the outer end of TM2 the “diameter” increases from 18 to 32 Å (Figure 3C). This widening of the lower body causes the transmembrane helices MK-2206 in vitro to separate at their outer ends, and expands the pore like a three-leafed iris (Figure 3F). The foregoing interpretation and description gives a compelling account of how a P2X receptor can bind ATP and transform from a closed to an open state. Yet there are caveats and limitations. The first is that the determination of a single open structure does not preclude the existence of other stable forms, and obviously it does not address the movements that occur in the most flexible parts of the protein. Studies using normal mode analysis and molecular dynamic simulations (Du et al., 2012; Florfenicol Jiang et al., 2012) are likely to be most informative in providing insight into conformational dynamics. Additionally, the zebrafish P2X4 receptor used for crystallography lacked intracellular N and C termini, which will likely have an effect on channel properties. Likewise, the membrane proximal regions of these N- and

C-terminal domains contain conserved motifs in which minor substitutions can impair receptor function (North, 2002). There is also evidence that these membrane proximal intracellular regions are involved in desensitization (Werner et al., 1996), and the molecular rearrangements underlying desensitization remain unclear. Further, P2X receptors have three ATP binding sites (Bean et al., 1990), and the present structures provide little insight into the molecular basis of the observed cooperativity (Ding and Sachs, 1999; North, 2002). Finally, the stationary snapshots and nanosecond simulations leave us with much to learn about the kinetics of receptor activation and how these proteins may be better suited to respond to longer-lasting diffusing signals than to the short, sharp pulses of a fast transmitter. There has been considerable activity in the drug development world focused on P2X receptors, and this has been well reviewed (Coddou et al.