BAC transgenic mice to study the expression of P2X2 and P2Y1 receptors.

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y1R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y1R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.

A hydrophobic residue in position 15 of the rP2X3 receptor slows desensitization and reveals properties beneficial for pharmacological analysis and high-throughput screening.

The homotrimeric P2X3 subtype, one of the seven members of the ATP-gated P2X receptor family, plays a role in sensory neurotransmission, including nociception. To overcome the bias resulting from fast desensitization of the P2X3 receptor in dose-response analyses, a non-desensitizing P2X2-X3 receptor chimera has been repeatedly used as a surrogate for the P2X3 receptor for functional analysis. Here, we show that only three of the P2X2-specific amino acid residues of the P2X2-X3 chimera, (19)P(21)V(22)I, are needed to confer a slowly desensitizing phenotype to the P2X3 receptor. The strongest delay in desensitization of the P2X3 receptor by a single residue was observed when (15)Ser was replaced by Val or another hydrophobic residue. Pharmacologically, the S(15)V-rP2X3 mutant behaved similarly to the wt-P2X3 receptor. Analysis of the S(15)V-rP2X3 receptor in 1321N1 astrocytoma cells by a common calcium-imaging-based assay showed 10-fold higher calcium transients relative to those of the wt-rP2X3 receptor. The S(15)V-rP2X3 cell line enabled reliable analysis of antagonistic potencies and correctly reported the mechanism of action of the P2X3 receptor antagonists A-317491 and TNP-ATP by a calcium-imaging assay. Together, these data suggest that the S(15)V-rP2X3 mutant may be suitable not only for automated fluorescence-based screening of molecule libraries for identification of lead compounds but also for facilitated pharmacological characterization of specific P2X3 receptor ligands. We suggest that the mechanism of desensitization of the P2X3 receptor may involve the movement of an N-terminal inactivation particle, in analogy to the "hinged-lid" or "ball and chain" mechanisms of voltage-gated NaV and Shaker KV channels, respectively.