NS1643 Interacts around L529 of hERG to Alter Voltage Sensor Movement on the Path to Activation
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NS1643
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Amsterdam University College (AUC)
NS1643 Interacts around L529 of hERG to Alter Voltage Sensor Movement
on the Path to Activation
Jiqing Guo,1Yen May Cheng,2James P. Lees-Miller,1Laura L. Perissinotti,3Tom W. Claydon,2Christina M. Hull,2
Samrat Thouta,2 Daniel E. Roach,1 Serdar Durdagi,3 Sergei Y. Noskov,1,3,* and Henry J. Du...
1400 Biophysical Journal Volume 108 March 2015 1400–1413
Article
NS1643 Interacts around L529 of hERG to Alter Voltage Sensor Movement
on the Path to Activation
Jiqing Guo,1 Yen May Cheng,2 James P. Lees-Miller,1 Laura L. Perissinotti,3 Tom W. Claydon,2 Christina M. Hull,2
Samrat Thouta,2 Daniel E. Roach,1 Serdar Durdagi,3 Sergei Y. Noskov,1,3,* and Henry J. Duff1,*
1
Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada; 2Department of Biomedical Physiology and
Kinesiology, Molecular Cardiac Physiology Group, Simon Fraser University, Burnaby, British Columbia, Canada; and 3Centre for Molecular
Simulations, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada
ABSTRACT Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous
studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643,
the V1/2 of activation of L529I ( 34 5 4 mV) is similar to wild-type (WT) ( 37 5 3 mV; P > 0.05). WT and L529I showed no
difference in the slope factor in the absence of NS1643 (8 5 0 vs. 9 5 0) but showed a difference in the presence of
NS1643 (9 5 0.3 vs. 22 5 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the
voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood.
NS1643 shifts the V1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials ( 131 5 4 mV for K525C
and 120 5 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643
(18 5 2 vs. 34 5 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 5 5 to 71 5 10
(P < 0.01) whereas for K525C the slope factor did not change (18 5 2 at baseline and 16 5 2 for NS1643). At baseline,
K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to
24 5 4 vs. 9 5 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix,
altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by
the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic prin-
ciple governing the action of some activators of hERG1 channels is proposed.
INTRODUCTION
Well-orchestrated opening and closing or gating of ion chan- the cooperative interactions between subunits that preceded
nels in cardiac myocytes controls cardiac electrical excita- a final opening step. The general involvement of uncharged
tion and relaxation that is directly coupled to the normal residues in model systems can be extended to clinically rele-
functioning of the heart (1). A component of this mechanism vant human proteins such as a family of hERG1 channels.
is the presence of potassium channels that shape signaling by The hERG1 gene (also referred to as KCNH2) encodes
selective permeation of Kþ and voltage-dependent gating the a-subunit of an ion channel (Kv11.1, sometimes simply
(2). Previous studies of voltage-dependent gating in Kþ chan- denoted as hERG1) underlying the rapid component in
nels have reported substitution of three nonpolar residues of the delayed rectified potassium current (IKr) in cardiac
the S4 of the Shaw channel into Shaker (V369I, I372L, myocytes (7). In the human heart, modulation of hERG1
S376T; ILT motif) reproduced the voltage-dependent proper- currents reportedly has both therapeutic and proarrhythmic
ties of activation of the Shaw channel (3–6). These amino- consequences (8,9). Wang and Rasmusson (10) have
acid substitutions were quite conservative, suggesting that modeled the state-dependent changes in activation of
physiologically very important changes can be achieved hERG1 channels. Compared to the activation of Shaker,
with rather subtle modifications of nonpolar amino acids the activation kinetics of the ionic hERG1 current are very
within the S4. Moreover, the Aldrich laboratory (5,6) pro- slow. Gating current kinetics indicate that voltage-sensor
vided direct evidence that the slowed activation was achieved movements in the pathway toward activation of the hERG
by changing cooperative transitions late in the pathway to- channel are also slow, although components of charge that
ward opening of the channel. Their studies suggested that move quickly have also been reported (10–13). In general,
noncharged residues in the S4 could play a pivotal role in voltage-sensor movements reported from fluorescence
studies mirror the movement of the majority of charge
measured using gating currents (14).
Submitted April 24, 2014, and accepted for publication December 8, 2014.
While the best-characterized feature of hERG1 is drug
*Correspondence: hduff@ucalgary.ca or snoskov@ucalgary.ca
interaction with its promiscuous intracavity blocking site
Serdar Durdagi’s current address is Department of Biophysics, School of
by a variety of drugs, a rapidly emerging strategy focuses
Medicine, Bahcesehir University, Istanbul, Turkey.
on channel activation by small molecules—i.e., channel
Editor: Randall Rasmusson.
Ó 2015 by the Biophysical Society
0006-3495/15/03/1400/14 $2.00 http://dx.doi.org/10.1016/j.bpj.2014.12.055
, NS1643 Alters Voltage-Sensor Movement 1401
activators (openers) (15). An implemented screening of University, Burnaby, British Columbia, Canada, using protocols in accor-
novel drug candidates for their ability to attenuate hERG1 dance with animal care guidelines established by the Canadian Council
on Animal Care.
function has led to an identification of compounds capable
of hERG1 current enhancement. A thorough examination
of the effects of hERG1 activators in vitro and in intact S4 sequences alignment
cardiac tissue has not yet been completed. A drug-induced
Table S1 shows the alignment of S4 sequences highlighting the ILT
increase in the time-dependent IKr rather than its tail current
sequence identified by Aldrich’s laboratory in Shaker and Shaw as it relates
could truncate the action potential and cause the short to the equivalent hERG1 sequence (3–6). L529 in hERG1 appears to be
QT syndrome similar to that of gain-of-function mutations equivalent to I of the ILT motif. This is part of the rationale for creating
(16). One of the best-characterized hERG activators is L529I hERG1. A second part of the rationale is that we previously reported
NS1643 (17,18). It shifts the voltage-dependence of activa- that L529 has proximity to NS1643 when docked to a homology model of
hERG1 (18).
tion to hyperpolarized potentials, shifts the voltage-depen-
dence of C-type inactivation to depolarized potentials, and
increases tail-current amplitude. In a 2012 study, we used Structural model of hERG1
a structure-guided mutagenesis strategy to identify a num-
ber of novel amino acids that have proximity to NS1643 Our model of the S1–S6 domains of hERG1 channel has been previously
reported in Subbotina et al. (19) and Durdagi et al. (20) and has been inde-
docked to a model of the hERG1 channel (18). One amino
pendently validated by a number of laboratories (21,22). This homology
acid that was identified as potentially being involved in model was used to guide mutagenesis to define potential binding sites of
NS1643 binding was L529. However, the electrophysiolog- NS1643 to hERG1 (18). A number of putative binding sites were identified.
ical responses to NS1643 for that mutation were not exam- One of the key binding sites was the S4 domain of hERG1. In this study,
ined. The reported binding region in the S4 was relatively this homology model was used to help understand the potential molecular
interactions of NS1643 in the neighborhood of L529.
large, spanning from L523 to V535 and from L553 to W568.
In our original article (18), radial cavities (10 Å) were
created around a reference residue and then the reference Molecular dynamics on wild-type and
amino acid was iteratively shifted to examine the character- L529I hERG1
istics of multiple radial cavities. The docking scores were
obtained for each radial cavity to identify the most likely A series of molecular-dynamics (MD) simulations were performed to
test possible structural effects of L529I mutation and to refine binding
targets. We recognized that this approach had limitations pocket for future docking studies. All MD simulations were carried out
because of the flexibility of this domain and the homology using our previous homology models of hERG1 open and closed states
model itself. The L529 residue was identified as a potentially with the NAMD program (23) and the CHARMM-27/CHARMM-36
important residue but it was not highlighted as a key residue. force fields for proteins, ions, and phospholipids, and the TIP3P water
Even so, we identified other residues (18) that were important model (24). All simulations were carried out at 323 K and 1 atm using
periodic boundary conditions and the NPT ensemble. Similar to
determinants of pharmacologic response to NS1643. This previous MD simulations of Kþ channels, the particle-mesh Ewald algo-
study is based on the observation that L529 is equivalent to rithm was used for electrostatic interactions. Kþ ions at the selectivity
the hydrophobic isoleucine amino acid of the ILT domain filter were used in the S0:S2:S4 positions according to previous studies.
(Table S1 in the Supporting Material) described by the Each model was embedded into the DPPC membrane bilayer using the
Aldrich laboratory (5,6). Thus, a different experimental CHARMM-GUI membrane builder protocol. The simulation box con-
tained one protein, DPPC molecules, 3 Kþ ions, and pore water molecules
approach is being used to refine our understanding of the in the intracellular cavity, solvated by 0.15 M KCl aqueous salt solution.
importance of residues in the large and flexible NS1643 Structures were minimized and equilibrated with gradually decreasing
binding domain of hERG1. It is also tempting to propose a harmonic constraints (see CHARMM-GUI equilibration protocol for
possible coupling between an activator binding to the chan- details) for 2 ns and then subjected to a 50-ns production run. Langevin
nel and subsequent gating modification via perturbing coop- dynamics with very weak friction coefficient was used to keep the
temperature constant. The Langevin Nosé-Hoover method as implemented
erative movements of the voltage-sensor between subunits. in CHARMM 36b1 (24) was used to maintain the pressure at 1 atm.
Accordingly, this study focuses on the impact of substitutions The HELANAL module of the MDAnalysis software (25) was used to
of nonpolar amino acids at the L529 site in the S4 of hERG1 analyze the geometry of the helices on the basis of their Ca carbons
on the characteristic response to NS1643. We combined alone. The geometry of the a-helix is characterized by computing the
biochemical, spectroscopic, electrophysiological, and theo- local helix axes and local helix origins for four contiguous Ca atoms
(26). The softwares VMD (27) and PYMOL (28) were used to visualize
retical studies to gain deeper understanding of functional trajectories.
and structural factors governing activator action and its
dependence on nonpolar moieties in the S4 helix.
Molecular docking
MATERIALS AND METHODS The methodology used to dock the NS1643 activator has been previously
reported in Durdagi et al. (18). Briefly, the derived chemical coordinates
All animals were housed in the Animal Resource Centre of the Faculty of the drug were docked to the channel using GLIDE/INDUCED FIT
of Medicine, University of Calgary, or at the Alberta or Simon Fraser DOCKING programs.
Biophysical Journal 108(6) 1400–1413
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