Two serial filters control P2X7 cation selectivity, Ser342 in the central pore and lateral acidic residues at the cytoplasmic interface

The human P2X7 receptor (hP2X7R) is a homotrimeric cell surface receptor gated by extracellular ATP^4- with two transmembrane helices per subunit, TM1 and TM2. A ring of three S342 residues, one from each pore-forming TM2 helix, located halfway across the membrane bilayer, functions to close and open the gate in the apo and ATP^4--bound open states, respectively. The hP2X7R is selective for small inorganic cations, but can also conduct larger organic cations such as Tris⁺. Here, we show by voltage-clamp electrophysiology in <i>Xenopus laevis</i> oocytes that mutation of S342 residues to positively charged lysines decreases the selectivity for Na⁺ over Tris⁺, but maintains cation selectivity. Deep in the membrane, laterally below the S342 ring are nine acidic residues arranged as an isosceles triangle consisting of residues E14, D352, and D356 on each side, which do not move significantly during gating. When the E14K mutation is combined with lysine substitutions of D352 and/or D356, cation selectivity is lost and permeation of the small anion Cl^- is allowed. Lysine substitutions of S342 together with D352 or E14 plus D356 in the acidic triangle convert the hP2X7R mutant to a fully Cl^--selective ATP^4--gated receptor. We conclude that the ion selectivity of wild-type hP2X7R is determined by two sequential filters in one single pathway: (i) a primary size filter, S342, in the membrane center and (ii) three cation filters lateral to the channel axis, one per subunit interface, consisting of a total of nine acidic residues at the cytoplasmic interface.