Two serial filters determine P2X7R cation selectivity, Ser342 in the central pore and lateral acidic residues at the cytoplasmic interface

The human P2X7R (hP2X7R) is a homotrimeric cell surface receptor gated by extracellular ATP4- 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 ATP4- 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 Xenopus laevis 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 ATP4--gated receptor. We conclude that the ion selectivity of wild-type hP2X7R is determined by two sequential filters in one single pathway: (1) a primary size filter, S342, in the membrane center and (2) three cation filters lateral to the channel axis, one per subunit interface, consisting of a total of nine acidic residues at the cytoplasmic interface. SignificancePore size and electrostatic interactions are key to the permeation selectivity of ion channels. Previous cysteine scanning mutagenesis identified a tri-serine-342 ring located halfway across the membrane as the gate and selectivity filter of the P2X7 receptor channel, accessible from the inside to cationic but not anionic reagents. Consistent with a downstream cation filter, we could now switch P2X7R from cation to anion selectivity by lysine substitution of acidic residues at the cytoplasmic interface. Our data show that two sequential selectivity filters control the cation selectivity of the P2X7R channel, a dynamic tri-serine-342 size filter and three conformationally static cation filters of three acidic residues each. We propose that the ion selectivity of P2X receptors involves the mechanism described here.