Identification of amino acids within the P2X(2) receptor C-terminus that regulate desensitization

1. The ATP-activated PBX2(a) and P2X(2(b)) receptor splice variants, which differ only in their C-terminal sequences, desensitize at different rates. We used mutational analysis to investigate the involvement of the C-termina l region in receptor desensitization. Rat wild type and mutant P2X(2) recep tors were expressed in Xenopus oocytes and currents were measured using the two-electrode voltage-clamp technique. 2. Truncating P2X(2) at the Lys369 splice site increased the rate of desens itization by >100-fold. Recovery from desensitization was slowed by similar to 5-fold. 3. Addition of Val370 onto the C-terminus of the truncated receptor slowed desensitization by similar to 70-fold. Point mutations that substituted eit her smaller or larger hydrophobic amino acids for Va1370, within the P2X(2( a)) splice variant, had profound effects on the rate of desensitization. Th e rate decreased with increasing hydrophobicity but was not dependent upon the precise structure of the side group. 4. A mutant receptor, with only nine amino acids, Val-Asp-Pro-Lys-Gly-Leu-A la-Gln-Leu, beyond the Lys369 splice site, desensitized at a similar rate t o P2X(2(a)). Injection of the peptide of this sequence into oocytes express ing P2X(2(a)) increased the rate of desensitization, whereas the eight-resi due peptide lacking the valine had no effect. 5. Neutralizing lysines in the vicinity of the splice site increased the ra te of receptor desensitization. Substituting glutamine for Lys365 produced the greatest effect (similar to 30-fold increase), whereas mutating lysines that were further upstream or downstream of this position had progressivel y less of an effect. 6. We conclude that the C-terminal splice site of the P2X(2) receptor is lo cated within a region that is critically involved in regulating the rate of receptor desensitization. The valine at position 370 interacts with an int racellular hydrophobic site to slow the rate of desensitization. Nearby lys ines may facilitate this interaction.