title: P2X receptor chimeras highlight roles of the amino terminus to partial agonist efficacy, the carboxyl terminus to recovery from desensitization, and independent regulation of channel transitions.
abstract: P2X receptor subtypes can be distinguished by their sensitivity to ATP analogues and selective antagonists. We have used chimeras between human P2X1 and P2X2 receptors to address the contribution of the extracellular ligand binding loop, transmembrane segments (TM1 and TM2), and intracellular amino and carboxyl termini to the action of partial agonists (higher potency and efficacy of BzATP and Ap5A at P2X1 receptors) and antagonists. Sensitivity to the antagonists NF449, suramin, and PPADS was conferred by the nature of the extracellular loop (e.g. nanomolar for NF449 at P2X1 and P2X2-1EXT and micromolar at P2X2 and P2X1-2EXT). In contrast, the effectiveness of partial agonists was similar to P2X1 levels for both of the loop transfers, suggesting that interactions with the rest of the receptor played an important role. Swapping TM2 had reciprocal effects on partial agonist efficacy. However, TM1 swaps increased partial agonist efficacy at both chimeras, and this was similar for swaps of both TM1 and 2. Changing the amino terminus had no effect on agonist potency but increased partial agonist efficacy at P2X2-1N and decreased it at P2X1-2N chimeras, demonstrating that potency and efficacy can be independently regulated. Chimeras and point mutations also identified residues in the carboxyl terminus that regulated recovery from channel desensitization. These results show that interactions among the intracellular, transmembrane, and extracellular portions of the receptor regulate channel properties and suggest that transitions to channel opening, the behavior of the open channel, and recovery from the desensitized state can be controlled independently.
authors.affiliation: From the Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom.
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Allsopp
authors.affiliation: From the Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom.
authors.forename: Louise K
authors.initials: LK
authors.lastname: Farmer
authors.affiliation: From the Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom.
authors.forename: Alistair G
authors.initials: AG
authors.lastname: Fryatt
authors.affiliation: From the Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom. Electronic address: rje6@le.ac.uk.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Evans
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D005110:Extracellular Space / Q000737:chemistry; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry; D015640:Ion Channel Gating / Q000187:drug effects*; D008954:Models, Biological; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058911:Purinergic P2X Receptor Agonists / Q000494:pharmacology*; D058920:Purinergic P2X Receptor Antagonists; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D058474:Receptors, Purinergic P2X1 / Q000737:chemistry* / Q000378:metabolism*; D058476:Receptors, Purinergic P2X2 / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013997:Time Factors; D014982:Xenopus laevis
chemical_list: D058911:Purinergic P2X Receptor Agonists; D058920:Purinergic P2X Receptor Antagonists; D058474:Receptors, Purinergic P2X1; D058476:Receptors, Purinergic P2X2; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate
keywords: ATP; Electrophysiology; Ion Channels; Mutagenesis; Pharmacology