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Abstract
P2X receptor function in the CNS is poorly understood, and currently available data are partly inconsistent. In the presented study, we investigated P2X3 receptors stably expressed in HEK293 cells. Non-stationary noise analysis of whole cell currents and rapid ATP application through flash photolysis allowed for assessing the single channel conductance (6.6 pS) and the fast activation kinetics of the receptor (20 ms). The characteristics of channel desensitization and pharmacological properties matched previous findings. The properties of wild type receptors were compared with P2X3 constructs carrying a fluorescent tag (ECFP or DsRed2) at the C-terminus. These fluorescently labeled subunits formed functional receptors, with neither the affinity of the ligand binding site nor channel properties (ion selectivity, gating kinetics, single channel conductance) differing from wild type. We conclude that both fusion proteins tested here are suitable for generating transgenic mice, which can be expected to promote understanding of the physiological role of P2X3 receptors in CNS signaling.
| Acronyms | ||
| human embryonic kidney cell line 293 | = | HEK293 |
| enhanced cyan fluorescent protein | = | ECFP |
| (enhancend) green fluorescent protein | = | (E)GFP |
| Discosoma sp. red fluorescent protein | = | DsRed2 |
| wild type | = | wt |
| untranslated region | = | UTR |
| neomycine phosphotransferase gene expression cassette | = | neo |
| coding sequence | = | CDS |
| adenosine 5′-triphosphate, P3-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) ester | = | DMNPE-caged ATP |
| Acronyms | ||
| human embryonic kidney cell line 293 | = | HEK293 |
| enhanced cyan fluorescent protein | = | ECFP |
| (enhancend) green fluorescent protein | = | (E)GFP |
| Discosoma sp. red fluorescent protein | = | DsRed2 |
| wild type | = | wt |
| untranslated region | = | UTR |
| neomycine phosphotransferase gene expression cassette | = | neo |
| coding sequence | = | CDS |
| adenosine 5′-triphosphate, P3-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) ester | = | DMNPE-caged ATP |