title: Impact of the whole-cell patch-clamp configuration on the pharmacological assessment of the hERG channel: trazodone as a case example.
abstract: BACKGROUND
  Voltage- and state-dependent blocks are important mechanisms by which drugs affect voltage-gated ionic channels. However, spontaneous (i.e. drug-free) time-dependent changes in the activation and inactivation of hERG and Na(+) channels have been reported when using conventional whole-cell patch-clamp in HEK-293 cells.
  
  
  METHODS
  hERG channels were heterologously expressed in HEK-293 cells and in Xenopus laevis oocytes. hERG current (IhERG) was recorded using both conventional and perforated whole-cell patch-clamp (HEK-293 cells), and two microelectrode voltage-clamp (Xenopus oocytes) in drug-free solution, and in the presence of the drug trazodone.
  
  
  RESULTS
  In conventional whole-cell setup, we observed a spontaneous time-dependent hyperpolarizing shift in the activation curve of IhERG. Conversely, in perforated patch whole-cell (HEK-293 cells) or in two microelectrode voltage-clamp (Xenopus oocytes) activation curves of IhERG were very stable for periods ~50min. Voltage-dependent inactivation of IhERG was not significantly altered in the three voltage clamp configurations tested. When comparing voltage- and state-dependent effects of the antidepressant drug trazodone on IhERG, similar changes between the three voltage clamp configurations were observed as under drug-free conditions.
  
  
  CONCLUSIONS
  The comparative analysis performed in this work showed that only under conventional whole-cell voltage-clamp conditions, a leftward shift in the activation curve of IhERG occurred, both in the presence and absence of drugs. These spontaneous time-dependent changes in the voltage activation gate of IhERG are a potential confounder in pharmacological studies on hERG channels expressed in HEK-293 cells.
authors.affiliation: Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico.
authors.forename: Aldo A
authors.initials: AA
authors.lastname: Rodriguez-Menchaca
authors.affiliation: Centro Universitario de Investigaciones Biomédicas, Universidad de Colima, Colima, Col., Mexico.
authors.forename: Tania
authors.initials: T
authors.lastname: Ferrer
authors.affiliation: Centro Universitario de Investigaciones Biomédicas, Universidad de Colima, Colima, Col., Mexico.
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Navarro-Polanco
authors.affiliation: Centro Universitario de Investigaciones Biomédicas, Universidad de Colima, Colima, Col., Mexico.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Sanchez-Chapula
authors.affiliation: Centro Universitario de Investigaciones Biomédicas, Universidad de Colima, Colima, Col., Mexico. Electronic address: eloy@ucol.mx.
authors.forename: Eloy G
authors.initials: EG
authors.lastname: Moreno-Galindo
mesh_terms: D000818:Animals; D018687:Antidepressive Agents, Second-Generation; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000187:drug effects* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods*; D013997:Time Factors; D014196:Trazodone / Q000494:pharmacology*; D014982:Xenopus laevis
chemical_list: D018687:Antidepressive Agents, Second-Generation; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D014196:Trazodone
keywords: HEK-293 cells; Methods; Patch clamp; Trazodone; Whole-cell; hERG channels