title: Acidic amino acids impart enhanced Ca2+ permeability and flux in two members of the ATP-gated P2X receptor family.
abstract: 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.
authors.affiliation: Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
authors.forename: Damien S K
authors.initials: DS
authors.lastname: Samways
authors.forename: Terrance M
authors.initials: TM
authors.lastname: Egan
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D008954:Models, Biological; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4; D016415:Sequence Alignment; D014162:Transfection; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000378:metabolism
chemical_list: D024342:Amino Acids, Acidic; C548226:P2RX4 protein, human; C548228:P2rx4 protein, rat; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D029961:Zebrafish Proteins; D000255:Adenosine Triphosphate; D002118:Calcium