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.
Department of Molecular Pharmacology, Medical Faculty of the RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany. Electronic address: rhausmann@ukaachen.de.
Ralf
R
Hausmann
Grünenthal GmbH, Global Drug Discovery, Department of Molecular Pharmacology, Zieglerstrasse 6, 52078 Aachen, Germany.
Gregor
G
Bahrenberg
Department of Molecular Pharmacology, Medical Faculty of the RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany.
Daniel
D
Kuhlmann
Department of Molecular Pharmacology, Medical Faculty of the RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany.
Michaela
M
Schumacher
Department of Molecular Pharmacology, Medical Faculty of the RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany.
Ursula
U
Braam
Grünenthal GmbH, Global Drug Discovery, Department of Molecular Pharmacology, Zieglerstrasse 6, 52078 Aachen, Germany.
Dagmar
D
Bieler
Grünenthal GmbH, Global Drug Discovery, Department of Molecular Pharmacology, Zieglerstrasse 6, 52078 Aachen, Germany.
Ilka
I
Schlusche
Department of Molecular Pharmacology, Medical Faculty of the RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany.
Günther
G
Schmalzing
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D057166:High-Throughput Screening Assays; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D009154:Mutation; D061848:Optical Imaging; D018408:Patch-Clamp Techniques; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D051381:Rats; D058476:Receptors, Purinergic P2X2 / Q000235:genetics / Q000378:metabolism; D058477:Receptors, Purinergic P2X3 / Q000235:genetics* / Q000378:metabolism*; D014982:Xenopus laevis
D058920:Purinergic P2X Receptor Antagonists; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D000255:Adenosine Triphosphate; D002118:Calcium
Calcium imaging; Desensitization; Drug screening; Mechanism of antagonism; P2X3 receptor