Acidic Amino Acids Impart Enhanced Ca²⁺ Permeability and Flux in Two Members of the ATP-gated P2X Receptor Family

P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca²⁺ permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca²⁺ permeability of the P2X₂ receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca²⁺ currents (Pf%) of the rat and human P2X₁ and P2X₄ subtypes. We used patch-clamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X₁ receptor but had no effect on the lower Pf% of the P2X₂ receptor, suggesting that ionized side chains regulate the Ca²⁺ flux of some family members. Removing the fixed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X₁ and P2X₄ receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X₂ receptor. Taken together, the data suggest that COO⁻ side chains provide an electrostatic force that interacts with Ca²⁺ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X₁ and P2X₄ receptors is conserved in the P2X₃ receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His⁴⁵ increased Pf% of the P2X₃ channel, suggesting that this positive charge masks the facilitation of Ca²⁺ flux by the neighboring Glu⁴⁶. The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca²⁺ permeability and flux of some P2X receptors.