Allosteric modulators of ligand-gated receptor channels induce conformational changes of the entire protein that alter potencies and efficacies for orthosteric ligands, expressed as the half maximal effective concentration (EC50) and maximum current amplitude, respectively. Here, we studied the influence of allostery on channel pore dilation, an issue not previously addressed. Experiments were done using the rat P2X4 receptor expressed in human embryonic kidney 293T cells and gated by adenosine 5'-triphosphate (ATP) in the presence and absence of ivermectin (IVM), an established positive allosteric regulator of this channel. In the absence of IVM, this channel activates and deactivates rapidly, does not show transition from open to dilated states, desensitizes completely with a moderate rate, and recovers only fractionally during washout. IVM treatment increases the efficacy of ATP to activate the channel and slows receptor desensitization during sustained ATP application and receptor deactivation after ATP washout. The rescue of the receptor from desensitization temporally coincides with pore dilation, and the dilated channel can be reactivated after washout of ATP. Experiments with vestibular and transmembrane domain receptor mutants further established that IVM has distinct effects on opening and dilation of the channel pore, the first accounting for increased peak current amplitude and the latter correlating with changes in the EC50 and kinetics of receptor deactivation. The corresponding kinetic (Markov state) model indicates that the IVM-dependent transition from open to dilated state is coupled to receptor sensitization, which rescues the receptor from desensitization and subsequent internalization. Allosterically induced sensitization of P2X4R thus provides sustained signaling during prolonged and repetitive ATP stimulation.

Department of Cellular and Molecular Neuroendocrinology Institute of Physiology Academy of Sciences of the Czech Republic Prague Czech Republic

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Bobanovic LK, Royle SJ, Murrell-Lagnado RD. P2X receptor trafficking in neurons is subunit specific. J Neurosci. 2002;22(12):4814–4824. doi:22/12/4814 [pii] PubMed PMC

Brandle U, Spielmanns P, Osteroth R, Sim J, Surprenant A, Buell G, Ruppersberg JP, Plinkert PK, Zenner HP, Glowatzki E. Desensitization of the P2X(2) receptor controlled by alternative splicing. FEBS Lett. 1997;404(2–3):294–298. PubMed

Browne LE, Compan V, Bragg L, North RA. P2X7 receptor channels allow direct permeation of nanometer-sized dyes. J Neurosci. 2013;33(8):3557–3566. doi:10.1523/JNEUROSCI.2235-12.2013. PubMed PMC

Coddou C, Yan Z, Obsil T, Huidobro-Toro JP, Stojilkovic SS. Activation and regulation of purinergic P2X receptor channels. Pharmacol Rev. 2011;63(3):641–683. doi:10.1124/pr.110.003129. PubMed PMC

Compan V, Ulmann L, Stelmashenko O, Chemin J, Chaumont S, Rassendren F. P2X2 and P2X5 subunits define a new heteromeric receptor with P2X7-like properties. J Neurosci. 2012;32(12):4284–4296. doi:10.1523/JNEUROSCI.6332-11.2012. PubMed PMC

Fischer W, Wirkner K, Weber M, Eberts C, Koles L, Reinhardt R, Franke H, Allgaier C, Gillen C, Illes P. Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293-hP2X3 cells and their inhibition by ethanol and trichloroethanol. J Neurochem. 2003;85(3):779–790. PubMed

He ML, Gonzalez-Iglesias AE, Stojilkovic SS. Role of nucleotide P2 receptors in calcium signaling and prolactin release in pituitary lactotrophs. J Biol Chem. 2003;278(47):46270–46277. doi:10.1074/jbc.M309005200. PubMed

He ML, Zemkova H, Koshimizu TA, Tomic M, Stojilkovic SS. Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels. Am J Physiol Cell Physiol. 2003;285(2):C467–479. doi:10.1152/ajpcell.00042.2003. PubMed

Jacobson KA, Gao ZG, Goblyos A, Ijzerman AP. Allosteric modulation of purine and pyrimidine receptors. Adv Pharmacol. 2011;61:187–220. doi:10.1016/B978-0-12-385526-8.00007-2. PubMed PMC

Jelinkova I, Vavra V, Jindrichova M, Obsil T, Zemkova HW, Zemkova H, Stojilkovic SS. Identification of P2X(4) receptor transmembrane residues contributing to channel gating and interaction with ivermectin. Pflugers Arch. 2008;456(5):939–950. doi:10.1007/s00424-008-0450-4. PubMed

Jelinkova I, Yan Z, Liang Z, Moonat S, Teisinger J, Stojilkovic SS, Zemkova H. Identification of P2X4 receptor-specific residues contributing to the ivermectin effects on channel deactivation. Biochem Biophys Res Commun. 2006;349(2):619–625. doi:10.1016/j.bbrc.2006.08.084. PubMed

Jindrichova M, Vavra V, Obsil T, Stojilkovic SS, Zemkova H. Functional relevance of aromatic residues in the first transmembrane domain of P2X receptors. J Neurochem. 2009;109(3):923–934. PubMed PMC

Khadra A, Tomic M, Yan Z, Zemkova H, Sherman A, Stojilkovic SS. Dual Gating Mechanism and Function of P2X7 Receptor Channels. Biophys J. 2013;104(12):2612–2621. doi:10.1016/j.bpj.2013.05.006. PubMed PMC

Khadra A, Yan Z, Coddou C, Tomic M, Sherman A, Stojilkovic SS. Gating properties of the P2X2a and P2X2b receptor channels: experiments and mathematical modeling. J Gen Physiol. 2012;139(5):333–348. doi:10.1085/jgp.201110716. PubMed PMC

Khakh BS, Bao XR, Labarca C, Lester HA. Neuronal P2X transmitter-gated cation channels change their ion selectivity in seconds. Nature neuroscience. 1999;2(4):322–330. PubMed

Khakh BS, Lester HA. Dynamic selectivity filters in ion channels. Neuron. 1999;23(4):653–658. PubMed

Khakh BS, North RA. Neuromodulation by extracellular ATP and P2X receptors in the CNS. Neuron. 2012;76(1):51–69. doi:10.1016/j.neuron.2012.09.024. PubMed PMC

Khakh BS, Proctor WR, Dunwiddie TV, Labarca C, Lester HA. Allosteric control of gating and kinetics at P2X(4) receptor channels. J Neurosci. 1999;19(17):7289–7299. PubMed PMC

Klapperstuck M, Buttner C, Schmalzing G, Markwardt F. Functional evidence of distinct ATP activation sites at the human P2X(7) receptor. J Physiol. 2001;534(Pt 1):25–35. PubMed PMC

Koshimizu T, Tomic M, Van Goor F, Stojilkovic SS. Functional role of alternative splicing in pituitary P2X2 receptor-channel activation and desensitization. Mol Endocrinol. 1998;12(7):901–913. PubMed

Norenberg W, Illes P. Neuronal P2X receptors: localisation and functional properties. Naunyn-Schmiedeberg's archives of pharmacology. 2000;362(4–5):324–339. PubMed

North RA. Molecular physiology of P2X receptors. Physiol Rev. 2002;82(4):1013–1067. PubMed

Popova M, Trudell J, Li K, Alkana R, Davies D, Asatryan L. Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors. Purinergic Signal. 2013;9(4):621–632. doi:10.1007/s11302-013-9373-4. PubMed PMC

Priel A, Silberberg SD. Mechanism of ivermectin facilitation of human P2X4 receptor channels. J Gen Physiol. 2004;123(3):281–293. PubMed PMC

Qureshi OS, Paramasivam A, Yu JC, Murrell-Lagnado RD. Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis. J Cell Sci. 2007;120(Pt 21):3838–3849. doi:jcs.010348 [pii] 10.1242/jcs.010348. PubMed

Roger S, Pelegrin P, Surprenant A. Facilitation of P2X7 receptor currents and membrane blebbing via constitutive and dynamic calmodulin binding. J Neurosci. 2008;28(25):6393–6401. PubMed PMC

Rokic MB, Stojilkovic SS. Two open states of P2X receptor channels. Frontiers in cellular neuroscience. 2013;7:215. doi:10.3389/fncel.2013.00215. PubMed PMC

Rokic MB, Stojilkovic SS, Vavra V, Kuzyk P, Tvrdonova V, Zemkova H. Multiple roles of the extracellular vestibule amino acid residues in the function of the rat P2X4 receptor. PLoS One. 2013;8(3):e59411. doi:10.1371/journal.pone.0059411. PubMed PMC

Schachter JB, Sromek SM, Nicholas RA, Harden TK. HEK293 human embryonic kidney cells endogenously express the P2Y1 and P2Y2 receptors. Neuropharmacology. 1997;36(9):1181–1187. PubMed

Silberberg SD, Li M, Swartz KJ. Ivermectin Interaction with transmembrane helices reveals widespread rearrangements during opening of P2X receptor channels. Neuron. 2007;54(2):263–274. PubMed

Stojilkovic SS, Yan Z, Obsil T, Zemkova H. Structural insights into the function of P2X4: an ATP-gated cation channel of neuroendocrine cells. Cell Mol Neurobiol. 2010;30(8):1251–1258. doi:10.1007/s10571-010-9568-y. PubMed PMC

Surprenant A, Rassendren F, Kawashima E, North RA, Buell G. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7) Science. 1996;272(5262):735–738. PubMed

Toulme E, Soto F, Garret M, Boue-Grabot E. Functional properties of internalization-deficient P2X4 receptors reveal a novel mechanism of ligand-gated channel facilitation by ivermectin. Mol Pharmacol. 2006;69(2):576–587. PubMed

Virginio C, MacKenzie A, North RA, Surprenant A. Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor. J Physiol. 1999;519(Pt 2):335–346. PubMed PMC

Virginio C, MacKenzie A, Rassendren FA, North RA, Surprenant A. Pore dilation of neuronal P2X receptor channels. Nature neuroscience. 1999;2(4):315–321. PubMed

Yan Z, Khadra A, Li S, Tomic M, Sherman A, Stojilkovic SS. Experimental characterization and mathematical modeling of P2X7 receptor channel gating. J Neurosci. 2010;30(42):14213–14224. doi:10.1523/JNEUROSCI.2390-10.2010. PubMed PMC

Yan Z, Li S, Liang Z, Tomic M, Stojilkovic SS. The P2X7 receptor channel pore dilates under physiological ion conditions. J Gen Physiol. 2008;132(5):563–573. doi:10.1085/jgp.200810059. PubMed PMC

Zemkova H, Yan Z, Liang Z, Jelinkova I, Tomic M, Stojilkovic SS. Role of aromatic and charged ectodomain residues in the P2X(4) receptor functions. J Neurochem. 2007;102(4):1139–1150. doi:10.1111/j.1471-4159.2007.04616.x. PubMed

Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models

Identification of functionally important residues of the rat P2X4 receptor by alanine scanning mutagenesis of the dorsal fin and left flipper domains