Identification of the amino acid residues in the extracellular domain of rat P2X7 receptor involved in functional inhibition by acidic pH

Background and purpose: P2X(7) receptors are potently inhibited by extracellular acidification. The underlying molecular basis remains unknown. This study aimed to examine the role of extracellular histidine, lysine, aspartic acid and glutamic acid residues in the functional inhibition of rat P2X(7) receptors by acidic pH. Experimental approach: We introduced point mutations into rat P2X(7) receptor by site-directed mutagenesis, expressed wild type (WT) and mutant receptors in human embryonic kidney (HEK293) cells and, using patch clamp recording, characterized the effects of acidic pH on BzATP [2'-3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate]-evoked ionic currents. Key results: Reducing extracellular pH, that is, increasing extracellular proton concentrations, inhibited BzATP-evoked currents in cells expressing WT P2X(7) receptors, with IC50 value (half-maximal antagonist or inhibitor concentration) for protons of 0.2 mu mol.L-1. The major effect of acidification was suppression of the maximal current response without altering the agonist sensitivity. Five residues in the receptor extracellular domain (His(85), Lys(110), Lys(137), Asp(197) and His(219)) were mutated to alanine and current inhibition by protons assessed. Compared with WT, the H85A, H219A, K137A mutants were two- to threefold more sensitive, whereas the K110A and D197A mutants were 2.5- and 9-fold less sensitive. Double-alanine substitution of Lys(110) and Asp(197) resulted in 23-fold decreased sensitivity to inhibition by protons. Furthermore, charge neutralization (K110M, K110F, D197N and D197F), but not charge conserving mutation (K110R and D197E), attenuated the inhibition of currents by protons. Conclusions and implications: Functional inhibition of rat P2X(7) receptors by acidic pH was variably affected by the extracellular His(85), Lys(110), Lys(137), Asp(197) and His(219) residues, with the Asp(197) residue being most critical for this inhibition.