Functional role of alternative splicing in pituitary P2X2 receptor-channel activation and desensitization

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 P2X(2)R subtype in anterior pituitary cells. Our results indicate that the primary P2X(2)R 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 p2X(2)R and the spliced isoform P2X(2-2)R, which lacks a stretch of carboxyl-terminal amino acids (Val(370)-Gln(438)). 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 P2X(2)R and P2X(2-2)R 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+], 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 P2X(2)R. The ligand-selectivity profile of native channels was consistent with the presence of P2X(2)R in somatotrophs. Finally, the desensitization rate of P2X(2)R 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 P2X(2)R and coexpression of P2X(2)R and P2X(2-2)R subunits provide effective mechanisms for controlled cationic influx in somatotrophs.