IDENTIFICATION OF AMINO-ACID-RESIDUES CONTRIBUTING TO DESENSITIZATIONOF THE P2X(2) RECEPTOR-CHANNEL

The P2X(2) receptor (P2X(2)R) is a member of the ATP-gated ion channel s that mediate Ca2+ entry in several tissues, including the brain, adr enal medulla, and pituitary. Alternative usage of cryptic splice sites in the primary P2X(2)R transcript accounts for the existence of sever al 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, where as P2X(2)R desensitizes slowly and incompletely, The role of the C ter minus in P2X(2)R desensitization was studied by generating several cha nnel mutants and monitoring intracellular free Ca2+ changes in transfe cted single GT1-7 neurons. Deletion studies indicated that the Arg(371 )-Ile(391) segment of the P2XR(2)R is required for sustained Ca2+ infl ux. To identify the important residues within this segment, three cont iguous amino acids were sequentially changed to alanine, Only two of t hese replacement mutants, at Arg(371)-Thr(372)-pro(373) and Lys(374)-H is(375)-Pro(376), had, enhanced rate of desensitization. Single amino acid deletions in the PBX,R C terminus and a series of insertions of w ild-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 funct ional motif that is critical for the development of the slow desensiti zation profile observed in these channels. Consequently, deletion of t his motif by alternative splicing provides an effective mechanism for generating a channel with controlled Ca2+ influx.