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 μm (n = 33). Bupivacaine (300 μm) inhibited wild-type tail currents by 62 ± 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 μm) 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.