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J Biol Chem, Vol. 273, Issue 21, 12853-12857, May 22, 1998

Identification of Amino Acid Residues Contributing to Desensitization of the P2X₂ Receptor Channel

Taka-aki Koshimizu, Melanija Tomi, Miharu Koshimizu, and Stanko S. Stojilkovic

From the Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892

The P2X₂ receptor (P2X₂R) 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 P2X₂R transcript accounts for the existence of several transcript types, one of which (P2X_2-2R) encodes a functional channel. P2X_2-2R lacks a stretch of cytoplasmic C-terminal amino acids (Val³⁷⁰-Gln⁴³⁸) and exhibits rapid and complete desensitization, whereas P2X₂R desensitizes slowly and incompletely. The role of the C terminus in P2X₂R 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 Arg³⁷¹-Ile³⁹¹ segment of the P2X₂R 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 Arg³⁷¹-Thr³⁷²-Pro³⁷³ and Lys³⁷⁴-His³⁷⁵-Pro³⁷⁶, had an enhanced rate of desensitization. Single amino acid deletions in the P2X₂R C terminus and a series of insertions of wild-type sequences into the corresponding spliced site identified four residues, Pro³⁷³-Lys³⁷⁴-His³⁷⁵-Pro³⁷⁶, required for sustained Ca²⁺ influx through agonist-occupied wild-type channels. Thus, it is likely that the Pro³⁷³-Pro³⁷⁶ sequence of P2X₂R 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.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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