Two potential pathways for extracellular ions to access the transmembrane pore in P2X receptors. (A) Surface representation of the zfP2X4 receptor model in the closed state. Each color represents one of the three subunits. (B) A sagittal section of the closed-state model reveals two potential ion access pathways (central and lateral pathways; blue dashed arrows). Electrostatic potential surface calculated using the APBS tools (Baker et al., 2001) shows two acidic vestibules along these potential ion access pathways (upper and central vestibules). The surface is colored based on the potential, contoured from −6 kcal mol⁻¹ (red) to +6 kcal mol⁻¹ (blue). White denotes 0 kcal mol⁻¹. To complement the electrostatic contributions of the missing side chains in the crystal structure because of weak electron density, the side chains were added back to the model, and their geometry was optimized using the MODELLER software (Eswar et al., 2006). Structural representations in all figures were generated using PyMOL (Schrödinger, LLC).