Identification of amino acid residues contributing to desensitization of the P2X2 receptor channel

The P2X(2) receptor (P2X(2)R) is a member of the ATP-gated ion channels that mediate Ca2+ entry in several tissues, including the brain, adrenal medulla, and pituitary. Alternative usage of cryptic splice sites in the primary P2X(2)R transcript accounts for the existence of several transcript types, one of which (PSX2R) encodes a functional channel. P2X(2-2)R lacks a stretch of cytoplasmic C-terminal amino acids (Val(370)-Gln(438)) and exhibits rapid and complete desensitization, whereas P2X(2)R desensitizes slowly and incompletely, The role of the C terminus in P2X(2)R desensitization was studied by generating several channel mutants and monitoring intracellular free Ca2+ changes in transfected single GT1-7 neurons. Deletion studies indicated that the Arg(371)-Ile(391) segment of the P2XR(2)R is required for sustained Ca2+ influx. To identify the important residues within this segment, three contiguous amino acids were sequentially changed to alanine, Only two of these replacement mutants, at Arg(371)-Thr(372)-pro(373) and Lys(374)-His(375)-Pro(376), had, enhanced rate of desensitization. Single amino acid deletions in the PBX,R C terminus and a series of insertions of wild-type sequences into the corresponding spliced site identified four residues, Pro(373)-Lys(374)-His(375)-Pro(376), required for sustained Ca2+ influx through agonist-occupied wild-type channels. Thus, it is likely that the Pro(373)-Pro(376) sequence of PSX2R represents a functional motif that is critical for the development of the slow desensitization profile observed in these channels. Consequently, deletion of this motif by alternative splicing provides an effective mechanism for generating a channel with controlled Ca2+ influx.