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Functional Role of Alternative Splicing in Pituitary P2X₂ Receptor-Channel Activation and Desensitization

Taka-aki Koshimizu¹, Melanija Tomi, Fredrick Van Goor and Stanko S. Stojilkovic

Endocrinology and Reproduction Research Branch National Institute of Child Health and Human Development National Intitutes of Health Bethesda, Maryland 20892

Although ATP-gated ion channel (P2XR) expression is high among anterior pituitary cells, identification of the receptor subtypes and their selective expression within subpopulations of cell types, as well as their physiological role(s), are incompletely characterized. In this study, we focused on the expression and activity of the P2X₂R subtype in anterior pituitary cells. Our results indicate that the primary P2X₂R gene transcript in pituitary cells undergoes extensive alternative splicing, with generation of six isoforms. Two of these isoforms encode functional channels when expressed in GT1 or HEK293 cells: the wild-type P2X₂R and the spliced isoform P2X_2-2R, which lacks a stretch of carboxyl-terminal amino acids (Val³⁷⁰-Gln⁴³⁸). Four other clones showed different alterations, including an interfered reading frame starting in the first transmembrane domain and a 27-amino acid deletion in the large extracellular loop. When expressed separately or in combination with wild-type channels, these clones were nonfunctional. In single cell Ca²⁺ current and cytosolic Ca²⁺ concentration ([Ca²⁺]_i) measurements, the P2X₂R and P2X_2-2R had similar EC₅₀ values for ATP and time courses for activation and recovery from desensitization but differed significantly in their desensitization rates. The spliced isoform exhibited rapid and complete desensitization, whereas the wild-type channel desensitized slowly and incompletely. The mRNAs for wild-type and spliced channels were identified in enriched somatotroph, but not gonadotroph or lactotroph fractions. Expression of a functional ATP-gated channel in somatotrophs was confirmed by the ability of ATP to increase the frequency of [Ca²⁺]_i spikes in spontaneously active cells or initiate spiking in quiescent cells. When voltage-gated Ca²⁺ influx was blocked, ATP increased [Ca²⁺]_i, with a similar profile and EC₅₀ to those observed in GT1 cells heterologously expressing wild-type or spliced P2X₂R. The ligand-selectivity profile of native channels was consistent with the presence of P2X₂R in somatotrophs. Finally, the desensitization rate of P2X₂R in a majority of somatotrophs was comparable to that observed in neurons coexpressing wild-type and spliced channels. These data indicate that alternative splicing of P2X₂R and coexpression of P2X₂R and P2X_2-2R subunits provide effective mechanisms for controlled cationic influx in somatotrophs.

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