title: Structural requirements of human ether-a-go-go-related gene channels for block by bupivacaine.
abstract: BACKGROUND
  Local anesthetics interact with human ether-a-go-go-related gene (HERG) channels via the aromatic amino acids Y652 and F656 in the S6 region. This study aimed to establish whether the residues T623, S624, and V625 residing deeper within the pore are also involved in HERG channel block by bupivacaine. In addition, the study aimed to further define the role of the aromatic residues Y652 and F656 in bupivacaine inhibition by mutating these residues to threonine.
  
  
  METHODS
  Alanine and threonine mutants were generated by site-directed mutagenesis. Electrophysiologic and pharmacologic properties of wild-type and mutant HERG channels were established using two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing HERG channels.
  
  
  RESULTS
  Tail currents at -120 mV through HERG wild-type channels were inhibited with an IC50 value of 132 +/- 22 microm (n = 33). Bupivacaine (300 microm) inhibited wild-type tail currents by 62 +/- 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 microm) was reduced by all mutations (P < 0.001). The effect was largest for F656A (inhibition 5 +/- 2%, n = 6) in the lower S6 region and for T623A (inhibition 13 +/- 4%, n = 9) near the selectivity filter. Introducing threonine at positions 656 and 652 significantly reduced inhibition by bupivacaine compared with HERG wild type (P < 0.001).
  
  
  CONCLUSIONS
  The authors' results indicate that not only the aromatic residues Y652 and F656 but also residues residing deeper within the pore and close to the selectivity filter of HERG channels are involved in inhibition of HERG channels by the low-affinity blocker bupivacaine.
authors.affiliation: Department of Anesthesiology and Institute for Neural Signal Transduction, University Medical Center Hamburg-Eppendorf, Germany.
authors.forename: Cornelia C
authors.initials: CC
authors.lastname: Siebrands
authors.forename: Patrick
authors.initials: P
authors.lastname: Friederich
mesh_terms: D019943:Amino Acid Substitution; D000779:Anesthetics, Local / Q000494:pharmacology; D000818:Animals; D002045:Bupivacaine / Q000494:pharmacology*; D000072237:ERG1 Potassium Channel; D004594:Electrophysiology / Q000379:methods; D051638:Ether-A-Go-Go Potassium Channels / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D005260:Female; D018014:Gene Transfer Techniques; D006801:Humans; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques / Q000379:methods; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013997:Time Factors; D014982:Xenopus laevis
chemical_list: D000779:Anesthetics, Local; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D026902:Potassium Channel Blockers; D002045:Bupivacaine