Aminoglycoside block of P2X2 receptors heterologously expressed in Xenopus laevis oocytes

Eva-Verena Bongartz; Jürgen Rettinger; Ralf Hausmann

doi:10.1007/s11302-010-9204-9

Purinergic Signalling

December 2010, Volume 6, Issue 4, pp 393–403

Aminoglycoside block of P2X2 receptors heterologously expressed in Xenopus laevis oocytes

Authors
Authors and affiliations

Eva-Verena Bongartz
Jürgen Rettinger
Ralf HausmannEmail author

Original Article

First Online:: 16 November 2010

Received:: 25 February 2009
Accepted:: 01 November 2010

DOI: 10.1007/s11302-010-9204-9

Cite this article as:: Bongartz, EV., Rettinger, J. & Hausmann, R. Purinergic Signalling (2010) 6: 393. doi:10.1007/s11302-010-9204-9

4 Citations
50 Downloads

Abstract

Aminoglycosides are polycationic antibiotics that have been shown to block a variety of cation channels. The inhibitory effect of externally applied aminoglycosides on P2X2 receptor currents was examined after heterologous expression in Xenopus laevis oocytes using the two-electrode voltage-clamp technique. All of the aminoglycosides tested inhibited the ATP-evoked responses with potencies ranging from 71 μM to 2 mM (IC₅₀ values). The ranked order of potency was streptomycin > gentamicin > neomycin > paromomycin > kanamycin. The inhibition of P2X receptor currents was independent of the ATP concentration used for the activation, which is compatible with a noncompetitive mechanism. The inhibition was voltage-dependent and was reduced at more positive membrane potentials. To examine whether the current block was dependent on the receptor conformation, the aminoglycoside effect on a non-desensitizing P2X2-X1 receptor chimera was analyzed. The results from these measurements suggest that inhibition is caused by an open pore block that locks the P2X receptor chimera in an open nonconducting state from which the agonist dissociation is slow. We also demonstrate that the P2X2-X1 chimera can serve as a tool to directly test whether an antagonist acts competitively or not.

Keywords

Aminoglycoside antibiotics P2X Xenopus oocytes Open pore block

References

1.
Nicke A, Bäumert HG, Rettinger J et al (1998) P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. EMBO J 17:3016–3028CrossRefPubMedGoogle Scholar
2.
Aschrafi A, Sadtler S, Niculescu C et al (2004) Trimeric architecture of homomeric P2X2 and heteromeric P2X1+2 receptor subtypes. J Mol Biol 342:333–343CrossRefPubMedGoogle Scholar
3.
Jasti J, Furukawa H, Gonzales EB et al (2007) Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. Nature 449:316–323CrossRefPubMedGoogle Scholar
4.
Kawate T, Michel JC, Birdsong WT et al (2009) Crystal structure of the ATP-gated P2X4 ion channel in the closed state. Nature 460:592–598CrossRefPubMedGoogle Scholar
5.
Jarvis MF (2010) The neural–glial purinergic receptor ensemble in chronic pain states. Trends Neurosci 33:48–57CrossRefPubMedGoogle Scholar
6.
Cao L, Broomhead HE, Young MT et al (2009) Polar residues in the second transmembrane domain of the rat P2X2 receptor that affect spontaneous gating, unitary conductance, and rectification. J Neurosci 29:14257–14264CrossRefPubMedGoogle Scholar
7.
Kracun S, Chaptal V, Abramson J et al (2010) Gated access to the pore of a P2X receptor: structural implications for closed–open transitions. J Biol Chem 285:10110–10121CrossRefPubMedGoogle Scholar
8.
Jiang R, Martz A, Gonin S et al (2010) A putative extracellular salt bridge at the subunit interface contributes to the ion channel function of the ATP-gated P2X2 receptor. J Biol Chem 285:15805–15815CrossRefPubMedGoogle Scholar
9.
Keceli B, Kubo Y (2009) Functional and structural identification of amino acid residues of the P2X2 receptor channel critical for the voltage- and [ATP]-dependent gating. J Physiol 587:5801–5818CrossRefPubMedGoogle Scholar
10.
Wildman SS, King BF, Burnstock G (1999) Modulatory activity of extracellular H+ and Zn2+ on ATP-responses at rP2X1 and rP2X3 receptors. Br J Pharmacol 128:486–492CrossRefPubMedGoogle Scholar
11.
Virginio C, Church D, North RA et al (1997) Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor. Neuropharmacology 36:1285–1294CrossRefPubMedGoogle Scholar
12.
Khakh BS, Proctor WR, Dunwiddie TV et al (1999) Allosteric control of gating and kinetics at P2X4 receptor channels. J Neurosci 19:7289–7299PubMedGoogle Scholar
13.
Miller KJ, Michel AD, Chessell IP et al (1998) Cibacron blue allosterically modulates the rat P2X4 receptor. Neuropharmacology 37:1579–1586CrossRefPubMedGoogle Scholar
14.
North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82:1013–1067PubMedGoogle Scholar
15.
Schatz A, Bugie E, Waksman SA (2005) Streptomycin, a substance exhibiting antibiotic activity against gram-positive and gram-negative bacteria. 1944. Clin Orthop Relat Res 437:3–6CrossRefPubMedGoogle Scholar
16.
Rybak LP, Whitworth CA (2005) Ototoxicity: therapeutic opportunities. Drug Discov Today 10:1313–1321CrossRefPubMedGoogle Scholar
17.
Selimoglu E (2007) Aminoglycoside-induced ototoxicity. Curr Pharm Des 13:119–126CrossRefPubMedGoogle Scholar
18.
Blanchet C, Erostegui C, Sugasawa M et al (2000) Gentamicin blocks ACh-evoked K+ current in guinea-pig outer hair cells by impairing Ca2+ entry at the cholinergic receptor. J Physiol 525(Pt 3):641–654CrossRefPubMedGoogle Scholar
19.
Schacht J (1993) Biochemical basis of aminoglycoside ototoxicity. Otolaryngol Clin North Am 26:845–856PubMedGoogle Scholar
20.
Vital BO, Prado-Franceschi J (1969) The nature of neuromuscular block produced by neomycin and gentamicin. Arch Int Pharmacodyn Thér 179:78–85Google Scholar
21.
Ohmori H (1985) Mechano-electrical transduction currents in isolated vestibular hair cells of the chick. J Physiol 359:189–217PubMedGoogle Scholar
22.
Winegar BD, Haws CM, Lansman JB (1996) Subconductance block of single mechanosensitive ion channels in skeletal muscle fibers by aminoglycoside antibiotics. J Gen Physiol 107:433–443CrossRefPubMedGoogle Scholar
23.
Okamoto T, Sumikawa K (1991) Antibiotics cause changes in the desensitization of ACh receptors expressed in Xenopus oocytes. Brain Res Mol Brain Res 9:165–168CrossRefPubMedGoogle Scholar
24.
Amici M, Eusebi F, Miledi R (2005) Effects of the antibiotic gentamicin on nicotinic acetylcholine receptors. Neuropharmacology 49:627–637PubMedGoogle Scholar
25.
Raisinghani M, Premkumar LS (2005) Block of native and cloned vanilloid receptor 1 (TRPV1) by aminoglycoside antibiotics. Pain 113:123–133CrossRefPubMedGoogle Scholar
26.
Masuko T, Kuno T, Kashiwagi K et al (1999) Stimulatory and inhibitory properties of aminoglycoside antibiotics at N-methyl-d-aspartate receptors. J Pharmacol Exp Ther 290:1026–1033PubMedGoogle Scholar
27.
Lin X, Hume RI, Nuttall AL (1993) Voltage-dependent block by neomycin of the ATP-induced whole cell current of guinea-pig outer hair cells. J Neurophysiol 70:1593–1605PubMedGoogle Scholar
28.
Ito K, Dulon D (2010) Purinergic signaling in cochleovestibular hair cells and afferent neurons. Purinergic Signal 6:201–209CrossRefPubMedGoogle Scholar
29.
Telang RS, Paramananthasivam V, Vlajkovic SM et al (2010) Reduced P2x(2) receptor-mediated regulation of endocochlear potential in the ageing mouse cochlea. Purinergic Signal 6:263–272CrossRefPubMedGoogle Scholar
30.
Housley GD, Greenwood D, Ashmore JF (1992) Localization of cholinergic and purinergic receptors on outer hair cells isolated from the guinea-pig cochlea. Proc Biol Sci 249:265–273CrossRefPubMedGoogle Scholar
31.
Szucs A, Szappanos H, Toth A et al (2004) Differential expression of purinergic receptor subtypes in the outer hair cells of the guinea pig. Hear Res 196:2–7CrossRefPubMedGoogle Scholar
32.
Zhao HB, Yu N, Fleming CR (2005) Gap junctional hemichannel-mediated ATP release and hearing controls in the inner ear. Proc Natl Acad Sci USA 102:18724–18729CrossRefPubMedGoogle Scholar
33.
Werner P, Seward EP, Buell GN et al (1996) Domains of P2X receptors involved in desensitization. Proc Natl Acad Sci USA 93:15485–15490CrossRefPubMedGoogle Scholar
34.
Rettinger J, Schmalzing G (2004) Desensitization masks nanomolar potency of ATP at the P2X1 receptor. J Biol Chem 279:6426–6433CrossRefPubMedGoogle Scholar
35.
Miledi R, Parker I, Woodward RM (1989) Membrane currents elicited by divalent cations in Xenopus oocytes. J Physiol 417:173–195PubMedGoogle Scholar
36.
Weber WM (1999) Endogenous ion channels in oocytes of Xenopus laevis: recent developments. J Membr Biol 170:1–12CrossRefPubMedGoogle Scholar
37.
Evans RJ, Lewis C, Buell G et al (1995) Pharmacological characterization of heterologously expressed ATP-gated cation channels (P_2x purinoceptors). Mol Pharmacol 48:178–183PubMedGoogle Scholar
38.
Ding S, Sachs F (2000) Inactivation of P2X₂ purinoceptors by divalent cations. J Physiol 522:199–214CrossRefPubMedGoogle Scholar
39.
Rettinger J, Schmalzing G (2003) Activation and desensitization of the recombinant P2X1 receptor at nanomolar ATP concentrations. J Gen Physiol 121:451–461CrossRefPubMedGoogle Scholar
40.
International Union of Pharmacology, Khakh BS, Burnstock G, Kennedy C et al (2001) XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits. Pharmacol Rev 53:107–118PubMedGoogle Scholar
41.
Rettinger J, Braun K, Hochmann H et al (2005) Profiling at recombinant homomeric and heteromeric rat P2X receptors identifies the suramin analogue NF449 as a highly potent P2X1 receptor antagonist. Neuropharmacology 48:461–468CrossRefPubMedGoogle Scholar
42.
Grosman C, Auerbach A (2001) The dissociation of acetylcholine from open nicotinic receptor channels. Proc Natl Acad Sci USA 98:14102–14107CrossRefPubMedGoogle Scholar
43.
Hausmann R, Rettinger J, Gerevich Z et al (2006) The suramin analog 4, 4′, 4″, 4‴-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (NF110) potently blocks P2X3 receptors: subtype selectivity is determined by location of sulfonic acid groups. Mol Pharmacol 69:2058–2067CrossRefPubMedGoogle Scholar

Copyright information

Authors and Affiliations

Eva-Verena Bongartz
- 1
Jürgen Rettinger
- 1
Ralf Hausmann
- 2
Email author

1.Department of Biophysical ChemistryMax Planck Institute of BiophysicsFrankfurtGermany
2.Molecular PharmacologyRWTH Aachen UniversityAachenGermany

Personalised recommendations

Article actions

Unlimited access to the full article
Instant download
Include local sales tax if applicable

Subscribe to Journal

Get Access to

Purinergic Signalling

for the whole of 2017

Aminoglycoside block of P2X2 receptors heterologously expressed in Xenopus laevis oocytes

Abstract

Keywords

Copyright information

Authors and Affiliations

Personalised recommendations

Cookies