Desensitization of the P2X₂ receptor controlled by alternative splicing

Abstract

P2X receptors are ion channels gated by extracellular ATP. We report here cloning of a P2X 2 receptor splice variant (P2X2_2) carrying a 207 bp deletion in the intracellular Cterminus and the analysis of the corresponding genomic structure of the P2X 2 gene. P2X 2 _2 is as highly expressed as the original P2X 2 sequence in various tissues. ATP-activated currents mediated by heterologous expressed P2X 2 or P2X2_ 2 receptors showed significant differences in desensitization time constants and steady-state currents in the continuous presence of ATP. These results imply functional differences between cells differentially expressing these P2X 2 isoforms.

Properties of ATP-gated ion channels assembled from P2X2 subunits in mouse cochlear Reissner’s membrane epithelial cells

“…This is in contrast to guinea pig RM epithelial cells which express the P2X2a mRNA transcript, but not the P2X2b isoform [8]. The P2X2b isoform lacks a series of C-terminal 69 amino acids and forms a functional homomeric ATP-gated channel which desensitises more rapidly than the full-length P2X2a isoform [15,16,29,32]. This P2X2b splice variant and others has been detected in various guinea pig and rat cochlear tissues [15,16,22].…”

“…The 499-bp PCR product was the full-length P2X 2 (P2X 2-1 ; P2X2a; NM153400). The 293-bp PCR product was the P2X 2-2 (P2X2b; AB094664) splice variant previously detected in guinea pig and rat cochlea [15,16,22] with a 207-bp deletion in the C-terminal region. These results were confirmed by sequencing and alignment with the published mouse sequences.…”

“…The majority of heterologous expression studies of P2X 2 receptors report none or slow (tens of seconds) desensitisation; however, it is evident that alternative splicing influences this, along with expression level, cell type and experimental conditions [1,17,27,28]. A feature of this native mouse RM epithelial cell P2X 2 receptor was its desensitisation properties.…”

“…Studies have demonstrated that alternative splicing of the P2X 2 receptor subunit generates subunit isoforms that contribute faster desensitisation than the full-length P2X 2 receptor (P2X2a) [16,[28][29][30][31]. As a result, the presence of splice variants were investigated in mouse RM epithelial cells.…”

“…However, native ATPinduced responses in many cochlear cells have not correlated well with known recombinant P2X receptor combinations [8][9][10][11][12][13]. This has been mainly attributed to heteromeric expression, with multiple P2X 2 splice variant combinations possible [14][15][16], as demonstrated in rat primary auditory neurons expressing a novel P2X 2-3 / 3 heteromer [17]. Such has been the case for the P2X receptor in Reissner's membrane (RM), a two-cell-thick partition that separates the endolymphatic (high K + , low Na + ) and perilymphatic (high Na + , low K + ) fluid compartments.…”