Prediction of hERG Channel Inhibition Using In Silico Techniques - Ion Channels and Their Inhibitors - page 223

Using alanine scanning, Kamiya et al. [ 9 ] showed that amino acids Thr623,
Ser624, Tyr652 and Phe656 play an important role in hERG inhibition by
terfenadine and cisapride.
Finally, Hosaka et al. [ 95 ] applied a site-directed mutagenesis to analyze the
interactions between nifekalant and amino acids in the pore region of hERG. The
mutation of Thr623, Val625, Gly648, Tyr652 and Phe656 to alanine abolished
the block of the hERG channel. The mutant Val625Ala disrupted the K þ selectivity.
This indicates that the side chain of Val625 might be essential for the stability of the
selectivity filter structure, hence the mutant Val625Ala might perturb the surface of
the inner cavity reducing indirectly the affinity for nifekalant. The homology model
of the hERG channel in the open state showed that Gly648 was not part of the pore
channel, so the reduction of the hERG block was due to an indirect action.
5.5 Role of Gly648
Siebrands et al. [ 112 ] created the hERG mutants T623A, S624A, V625A, G648A,
Y652A, Y652T, F656A and F656T by site-direct mutagenesis to analyze the
interactions between bupivacaine and the hERG channel. All the mutants abolished
the channel block by bupivacaine. The mutation of Gly648 to alanine might lead to
a reorientation of the amino acids in the S6 domain, thus indirectly reducing the
affinity for the blocker.
5.6 Which Subunits Are Involved in Drug Binding?
Recently it was investigated which subunits of the hERG channel are involved in
the interaction with the blockers [ 113 , 114 ]. Myokai et al. [ 113 ] constructed seven
tandem dimers with single or double mutations (Y652A and/or P656A) to test
which mutations affect the binding of cisapride. The results brought to light that
the binding site of cisapride consists of several subunits. Combining the voltage
dependence of the cisapride block, the steady-state block, mutagenesis and kinetic
data, it was suggested that cisapride binds at first to the low-affinity binding site
formed by the two Phe656 of opposite subunits. Only when the voltage-dependent
conformational changes reorient the residues in the pore, cisapride binds to the
high-affinity binding site constituted by the two Tyr652 of adjacent subunits and
Phe656.
Imai et al. [ 114 ] constructed the tandem dimers mutating the amino acids
Ser624, Tyr652, and Phe656 of opposite subunits to investigate the binding site
of cisapride, E-4031 and terfenadine. The inhibition curves of the mutant channels
revealed that cisapride and E-4031 interact with Tyr652 and Phe656 of adjacent
subunits and with Ser624, while terfenadine interacts with Tyr652 and Phe656 of