Interactions of H562 in the S5 Helix with T618 and S621 in the Pore Helix Are Important Determinants of hERG1 Potassium Channel Structure and Function

Abstract

hERG1 is a member of the cyclic nucleotide binding domain family of K(+) channels. Alignment of cyclic nucleotide binding domain channels revealed an evolutionary conserved sequence HwX(A/G)C in the S5 domain. We reasoned that histidine 562 in hERG1 could play an important structure-function role. To explore this role, we created in silica models of the hERG1 pore domain based on the KvAP crystal structure with Rosetta-membrane modeling and molecular-dynamics simulations. Simulations indicate that the H562 residue in the S5 helix spans the gap between the S5 helix and the pore helix, stabilizing the pore domain, and that mutation at the H562 residue leads to a disruption of the hydrogen bonding to T618 and S621, resulting in distortion of the selectivity filter. Analysis of the simulated point mutations at positions 562/618/621 showed that the reciprocal double mutations H562W/T618I would partially restore the orientation of the 562 residue. Matching hydrophobic interactions between mutated W562 residue and I618 partially compensate for the disrupted hydrogen bonding. Complementary in vitro electrophysiological studies confirmed the results of the molecular-dynamics simulations on single mutations at positions 562, 618, and 621. Experimentally, mutations of the H562 to tryptophan produced a functional channel, but with slowed deactivation and shifted V(1/2) of activation. Furthermore, the double mutation T618I/H562W rescued the defects seen in activation, deactivation, and potassium selectivity seen with the H562W mutation. In conclusion, interactions between H562 in the S5 helix and amino acids in the pore helix are important determinants of hERG1 potassium channel function, as confirmed by theory and experiment.

Pore Helices Play a Dynamic Role as Integrators of Domain Motion during Kv11.1 Channel Inactivation Gating

“…4 are consistent with the notion that there is tight energetic coupling between the pore helices and S5 helices. Two recent studies have postulated direct interactions between Thr-618 of the pore helix and either His-562 (44) or Trp-568 (35) on the S5 helix. The discrepancy between these two studies highlights the uncertainty in aligning the S5 helix of Kv11.1 channels with that of other Kv channel proteins due, in part, to a low degree of homology within this region (45,46).…”

“…The discrepancy between these two studies highlights the uncertainty in aligning the S5 helix of Kv11.1 channels with that of other Kv channel proteins due, in part, to a low degree of homology within this region (45,46). Mutation of His-562 (44,45,47), or 45,47) produced mostly nonexpressing/nonfunctional channels, or in a few cases, abolished the inactivation process entirely (35). One exception to this is H562W; however, this mutation produced an insufficient shift in ⌬log(K eq,0 ) to gain an accurate -value (supplemental Table 1).…”

Structure-Guided Topographic Mapping and Mutagenesis to Elucidate Binding Sites for the Human Ether-a-Go-Go-Related Gene 1 Potassium Channel (KCNH2) Activator NS1643

“…Micropipettes were pulled from borosilicate glass capillary tubes on a programmable horizontal puller (Sutter Instrument Company, Novato, CA). Standard patch-clamp methods were used to measure the whole-cell currents of hERG1 mutants expressed in HEK 293 cells by using the Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) (Lees-Miller et al, 2009). The pipette solution contained the following: 10 mM KCl, 110 mM K-aspartate, 5 mM MgCl 2 , 5 mM Na 2 ATP, 10 mM ethylene glycol-bis(␤-aminoethyl ether)-N,N,NЈ,NЈtetraacetic acid, 5 mM HEPES, and 1 mM CaCl 2 .…”

“…In addition, their model does not include the S5 pore linker (turret), which is formed by approximately 40 amino acids. One of the key conclusions of a hERG1 modeling article published previously (Lees-Miller et al, 2009) was that de novo folding of the missing elements [traditionally avoided in a plethora of homology models including the one in Grunnet et al (2011)] may have a critical impact on residue packing. This may affect the quality of the docking studies.…”

“…Missing elements were modeled with the Rosetta de novo protein-modeling suite (http://www.rosettacommons.org/) and further refined with molecular dynamics simulations . We partially validated this model against available experimental information ( Lees-Miller et al, 2009). …”