title: Voltage- and time-dependent properties of the recombinant rat vanilloid receptor (rVR1).
abstract: Whole-cell voltage-clamp techniques were used to investigate the capsaicin-, voltage- and time-dependent properties of the rat vanilloid receptor (rVR1) stably expressed in human embryonic kidney (HEK) 293 cells. At a holding potential of -70 mV, application of capsaicin (0.03-30 microM) to HEK 293 cells expressing the rVR1 receptor led to the appearance of inward currents (EC50, 497 nM; Hill coefficient, nH, 2.85) which were reversibly antagonized by 10 microM capsazepine. Current-voltage relationships, determined using depolarizing or hyperpolarizing voltage ramps, had reversal potentials close to 0 mV, exhibited substantial outward rectification and possessed a region of negative slope conductance at holding potentials negative to around -70 mV. Further experiments indicated that the outward rectification and the region of negative slope conductance did not result from external block of the channel by either Ba2+, Ca2+ or Mg2+. During our characterization of rVR1, it became apparent that the rectification behaviour of this receptor was not entirely instantaneous as might be expected for a ligand-gated ion channel, but rather displayed clear time-dependent components. We characterized the kinetics of these novel gating properties in a series of additional voltage-step experiments. The time-dependent changes in rVR1-mediated conductance due to membrane depolarization or repolarization occurred with bi-exponential kinetics. On depolarization to +70 mV the time-dependent increase in outward current developed with mean time constants of 6.7 +/- 0.7 and 51.8 +/- 18.4 ms, with the faster time constant playing a dominant role (64.4 +/- 3.8 %). Similar kinetics also described the decay of 'tail currents' observed on repolarization. Furthermore, these time-dependent changes appeared to be unaffected by the removal of extracellular divalent cations and were not significantly voltage dependent. Our data reveal that rVR1 exhibits substantial time- and voltage-dependent gating properties that may have significance for the physiology of sensory transduction of nociceptive signals.
authors.affiliation: Neuroscience Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (North), Harlow, Essex CM19 5AW, UK. Martin_J_Gunthorpe@sbphrd.com
authors.forename: M J
authors.initials: MJ
authors.lastname: Gunthorpe
authors.forename: M H
authors.initials: MH
authors.lastname: Harries
authors.forename: R K
authors.initials: RK
authors.lastname: Prinjha
authors.forename: J B
authors.initials: JB
authors.lastname: Davis
authors.forename: A
authors.initials: A
authors.lastname: Randall
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D001464:Barium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002211:Capsaicin / Q000031:analogs & derivatives / Q000494:pharmacology; D002478:Cells, Cultured; D005727:Ganglia, Spinal / Q000166:cytology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009474:Neurons / Q000737:chemistry* / Q000166:cytology / Q000502:physiology*; D009619:Nociceptors / Q000502:physiology; D018408:Patch-Clamp Techniques; D051381:Rats; D011919:Rats, Inbred Strains; D011955:Receptors, Drug / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D011955:Receptors, Drug; D011994:Recombinant Proteins; D001464:Barium; D008274:Magnesium; C071423:capsazepine; D002211:Capsaicin; D002118:Calcium