title: Block of native and cloned vanilloid receptor 1 (TRPV1) by aminoglycoside antibiotics.
abstract: Vanilloid receptor 1 (TRPV1) is a Ca2+ permeable non-specific cation channel located at the peripheral nerve terminals and functions as a polymodal nociceptor. Neomycin, an aminoglycoside antibiotic induces analgesia in various animal models. However, the mechanism of action of neomycin has not been fully understood. In this study, we have determined the effect of neomycin on native TRPV1 in cultured embryonic DRG neurons and cloned TRPV1 heterologously expressed in Xenopus oocytes using patch clamp, double electrode voltage clamp, and Ca2+ fluorescence imaging techniques. Here, we show that neomycin potently (IC50 approximately 400 nM) blocks TRPV1-mediated membrane currents in DRG neurons and the block is unrelated to capsaicin concentrations used to evoke currents, suggesting a non-competitive block. Similarly, capsaicin- and proton-induced currents are blocked in oocytes, but to a lesser extent. Increases in capsaicin-induced intracellular Ca2+ levels are also reduced by neomycin. Single-channel current analyses reveal that single-channel conductance is unaffected by neomycin and there is no indication of open channel block. The predominant effect is to lower, the open probability (Po) at both, negative and positive potentials. Kinetic analyses reveal that the number of exponential components required to fit the open time distributions remains the same or reduced, however, the longest open time constant and the area of distribution are shortened at negative and positive potentials, respectively. The area of distribution of longest closed-time constants were significantly prolonged at negative and positive potentials. We conclude that neomycin inhibits TRPV1 channel activity by allosteric binding and altering channel gating.
authors.affiliation: Department of Pharmacology, Southern Illinois University School of Medicine, 801 N Rutledge, Springfield, IL 62702, USA.
authors.forename: Manish
authors.initials: M
authors.lastname: Raisinghani
authors.forename: Louis S
authors.initials: LS
authors.lastname: Premkumar
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002211:Capsaicin / Q000494:pharmacology; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004622:Embryo, Mammalian; D004625:Embryo, Nonmammalian; D005727:Ganglia, Spinal; D005786:Gene Expression Regulation / Q000187:drug effects*; D015640:Ion Channel Gating / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D009474:Neurons / Q000187:drug effects*; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D011336:Probability; D051381:Rats; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014982:Xenopus laevis
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D011955:Receptors, Drug; D002211:Capsaicin; D002118:Calcium