P2X receptor channels open in response to the binding of extracellular ATP, a property that is essential for purinergic sensory signaling. Apo and ATP-bound X-ray structures of the detergentsolubilized zebrafish P2X4 receptor provide a blueprint for receptor mechanisms but unexpectedly showed large crevices between subunits within the transmembrane (TM) domain of the ATPbound structure. Here we investigate both intersubunit and intrasubunit interactions between TM helices of P2X receptors in membranes using both computational and functional approaches. Our results suggest that intersubunit crevices found in the TM domain of the ATP-bound crystal structure are not present in membraneembedded receptors but substantiate helix interactions within individual subunits and identify a hot spot at the internal end of the pore where both the gating and permeation properties of P2X receptors can be tuned. We propose a model for the structure of the open state that has stabilizing intersubunit interactions and that is compatible with available structural constraints from functional channels in membrane environments.pore-opening mechanism | transmembrane intersubunit crevices P 2X receptor channels are a family of trimeric cation-selective channels that are activated by extracellular ATP (1, 2). These ligand-gated ion channels are expressed in many tissues, including the central and peripheral nervous systems and the immune system, where they play a range of important roles in sensory signaling and inflammation (1, 3).Recent X-ray structures of the zebrafish P2X4 (zfP2X4) receptor in apo and ATP-bound forms (Protein Data Bank ID codes 4DW0 and 4DW1, respectively) have revealed the molecular design of these proteins (2, 4, 5) and have provided valuable information on how ATP binding triggers the opening of the transmembrane (TM) pore ( Fig. 1 A and B). ATP binds to a cleft between subunits within the large extracellular domain, inducing cleft closure and an accompanying lateral flexing of the β-sheet connecting the extracellular domain to the TM domain (5). The apo structure of the zfP2X4 receptor reveals that the TM1 helix is positioned peripheral to the TM2 helix and that the TM2 helix occludes the pore at the central axis within the outer half of the membrane (4, 5). In accord with this structure, accessibility studies show that the TM2 helix lines the aqueous pore and that the residues forming the gate are positioned within the occlusion in the apo structure (6-8). Lateral fenestrations within the extracellular domain provide a path for ions to enter and exit the extracellular vestibule positioned above this TM2 occlusion, and these fenestrations are thought to change conformation in response to ATP binding (9, 10). The ATP-bound structure shows that pore opening involves widening of the extracellular vestibule and an iris-like expansion of the pore (5). Intersubunit interactions within the TM domain in the apo structure are limited to the gate region of TM2 (5), and thus the pore expansion observed in the ATP-bound struc...