Acidic Amino Acids Impart Enhanced Ca(2+) 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 Ca(2+) permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca(2+) permeability of the P2X(2) receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca(2+) currents (Pf%) of the rat and human P2X(1) and P2X(4) 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 P2X(1) receptor but had no effect on the lower Pf% of the P2X(2) receptor, suggesting that ionized side chains regulate the Ca(2+) 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 P2X(1) and P2X(4) receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X(2) receptor. Taken together, the data suggest that COO(−) side chains provide an electrostatic force that interacts with Ca(2+) in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X(1) and P2X(4) receptors is conserved in the P2X(3) receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His(45) increased Pf% of the P2X(3) channel, suggesting that this positive charge masks the facilitation of Ca(2+) flux by the neighboring Glu(46). The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca(2+) permeability and flux of some P2X receptors.