N-Terminal Regulation of hERG1 K+ Channel Deactivation


Sunday, February 16, 2014 15a
receptor X were transfected into CHO cells. The CHO transfectants were char-
acterized by FACS and then scaled up for KinExA binding studies. KinExA has
been used to measure binding affinity of Adnectin-A to the cell surface ex-
pressed receptor X to measure the effect of avidity of the multivalent adnectin
binding to receptor clusters. As controls for the functional activity of the
Adnectin-A and the affinity of the monovalent interaction, the same KinExa
assay was used, substituting the soluble receptor X extracellular domain for
the transfected CHO cells. The binding avidity measured by KinExA for
CHO expressed receptor is 14 pM for both species of receptor X. However,
the affinity of Adnectin-A for monovalent soluble Receptor X was quite
different between the species suggesting that avidity due to receptor clustering
equilizes the functional avidity at the cell surface.

Platform: Voltage-gated K Channels: Activation/
Inactivation Mechanisms

92-Plat
A-Type Kv4 Channel Closed-State Inactivation is Modulated by the
Tetramerization Domain Interacting with Auxilary KChIP4a
Yi-Quan Tang1, Fan Yang2, Jingheng Zhou1, Jie Zheng2, KeWei Wang1.
1Peking University, Beijing, China, 2University of California at Davis, Davis,
CA, USA.
A-type Kv4 potassium channels undergo a conformational change towards a
non-conductive state at negative membrane potentials, a dynamic process
known as closed-state inactivation (CSI). CSI causes inhibition of channel ac-
tivity without prerequisite of channel opening, thus providing a dynamic regu-
lation of neuronal excitability, dendritic signal integration and synaptic
plasticity. However, the structural determinants underlying Kv4 CSI remain
largely unknown. We have recently demonstrated that auxiliary KChIP4a sub-
unit contains an N-terminal Kv4 inhibitory domain (KID) that directly interacts
with Kv4.3 channels to enhance CSI. In this study, we utilized the FRET two-
hybrid mapping and BiFC-based screening combined with electrophysiology,
and identified the intracellular tetramerization (T1) domain that functions to
suppress CSI and serves as a receptor for the binding of KID. Disrupting
Kv4.3 T1-T1 interaction by mutating C110A within the C3H1 motif of T1
domain facilitated CSI, and ablated the KID-mediated enhancement of CSI.
Furthermore, replacing the characteristic C3H1 motif of Kv4.3 T1 domain
with the T1 domain from Kv1.4 without the C3H1 motif or Kv2.1 with the
C3H1 motif resulted in channels functioning with enhanced or suppressed
CSI, respectively. Taken together, our findings reveal a novel role of the T1
domain in suppressing Kv4 CSI with the C3H1 motif functioning to stabilize
the channel activation gate; and KChIP4a KID directly interacts with the T1
domain to relieve the stabilization, leading to facilitation of CSI and inhibition
of channel function.

93-Plat
Two-in-One: Activation and Inactivation at the Intracellular Gate of a Kv
Channel
Manuel Covarrubias1, Jeffrey D. Fineberg2.
1
Neuroscience, Jefferson Medical College of Thomas Jefferson University,
Philadelphia, PA, USA,

2
Physiology and Molecular Biophysics, Jefferson

Medical College of Thomas Jefferson University, Philadelphia, PA, USA.
N-type and P/C-type inactivation are firmly established mechanisms of inac-
tivation in voltage-gated Kþ (Kv channels). However, Kv4.x channel com-
plexes, which undergo fast preferential closed-state inactivation (CSI;
Fineberg et al., 2012, JGP 140.5:513-527), appear to use a distinct but un-
known inactivation mechanism. Previously, we hypothesized that a weak
interaction between the voltage sensing domain and the intracellular activa-
tion gate underlies CSI (Bähring & Covarrubias, 2011, J Physiol 589:461-
79). Thus, CSI is essentially governed by the intracellular activation gate,
which fails to open and adopts an inactivated conformation. To directly test
this hypothesis, we investigated the heterologously expressed Kv4.1 ternary
channel complex including accessory subunits KChIP1 and DPP6, and ex-
ploited the ‘‘trap-door’’ paradigm of the activation gate. The results show
that Kv4.1 inactivation traps intracellularly applied quaternary ammonium
blockers (bTBuA and TBuA) inside the channel’s pore. The trapped blockers
can only escape if the channels are opened again by subsequent depolariza-
tions. By contrast, inactivation cannot trap TEA, whose binding kinetics is
faster than that of channel gating. Moreover, under identical conditions, a
Shaker Kv channel (ShB-T449K) known to exhibit fast P/C-type inactivation
cannot trap bTBuA. These findings conclusively suggest that the intracellular
activation gate of the Kv4.1 ternary channel complex plays a novel dual role,
controlling both activation and inactivation. Supported in part by NIH grant
R01 NS032337 (MC).
94-Plat
Development and Validation Studies of Universal Pharmacophore Models
for hERG Channel Openers
Serdar Durdagi1,2, Matthew Patterson2, Sergei Y. Noskov2.
1Department of Biophysics, Bahcesehir University, Faculty of Medicine,
Istanbul, Turkey,

2
Biological Sciences, Institute for Biocomplexity and

Informatics, University of Calgary, Calgary, AB, Canada.
The intra-cavitary drug blockade of hERG channel, a common off-target for
many drugs, have been extensively studied both experimentally and theoreti-
cally. Structurally diverse ligands inadvertent blockade of rapid component
of delayed rectifying Kþ currents are potentially pro-arrhythmic and may
lead to drug-induced long QT syndrome-LQTS. There are a number of natural
strategies for rational drug design; one dubbed the ‘‘passive’’ approach avoids
block of hERG1 whereas the ‘‘proactive’’ strategy designs treatments to acti-
vate the channel. While ‘‘passive’’ approach has been developed for decades,
studies of structural mechanisms of hERG channel activation by small mole-
cules are truly novel. Accordingly, design of the hERG openers or current ac-
tivators may offer a momentum for modern anti-arrhythmia drug development.
Significant number of small molecules with capacity for hERG activation was
identified in mandatory hERG screens. To establish possible correlation be-
tween activators structure and reactivity, we attempted to construct a universal
pharmacophore model for hERG channel openers using PHASE protocol. The
biochemical data on 38 Kþ channel activators are used in training and test sets.
These compounds span a wide range of structurally different chemotypes with
~10

^
5-fold variances in binding affinity, which is sufficient for statistically

sound model. A developed five sites AAHHR (A, hydrogen-bond accepting,
H, hydrophobic, R, aromatic) pharmacophore model has showed reasonable
high statistical results compared to other constructed models and was selected
for steric and electrostatic contour maps analysis. The predictive power of the
model was also tested with 6 external test-set (as true unknowns) compounds.
Pharmacophore model is also combined with previously developed receptor-
based homology model of hERG K channel and novel activators are generated
and screened. The developed ligand-based models may serve as a basis for the
synthesis of novel potential therapeutic hERG activators.

95-Plat
N-Terminal Regulation of hERG1 KD Channel Deactivation
Steven J. Thomson, Angela Hansen, Michael C. Sanguinetti.
Nora Eccles Harrison Cardiovascular Research and Training Institute,
University of Utah, Salt Lake City, UT, USA.
Slow deactivation of hERG1 (Kv 11.1) potassium channels maintains IKr dur-
ing final repolarization of the cardiac action potential and opposes asynchro-
nous early depolarization. Inherited point mutations in hERG1 that accelerate
deactivation of IKr cause long QT syndrome (LQTS), a disorder of ventricular
repolarization that increases the risk of lethal cardiac arrhythmia.
The intracellular N-terminal domain of hERG1 is known to be essential for
slow deactivation. Deletion of the entire (~350 residues) or just the initial 16
residues of the N-terminus accelerates deactivation 10-fold. The same effect
is achieved by neutralization of the charged residues, Arg4 or Arg5. How
many of the 4 N-termini are required to slow channel deactivation is unknown.
hERG1, like other Kv channels, is a homotetramer. By repeatedly linking the
C-terminal of one subunit to the N-terminal of the next subunit we constructed
concatenated hERG1 tetramers. A variety of homomeric and heteromeric
concatenated tetramers were characterized (i.e., WTn/R4A:R5A(4-n); where
n = 1 to 4). The concatenated channel containing a single R4A/R5A subunit
and 3 wild-type subunits deactivated as fast as the concatenated channel con-
taining only R4A/R5A subunits. The LQTS-associated mutation R56Q, located
in the N-terminal of hERG1 was also studied. Again, a concatenated tetramer
containing a single mutant subunit deactivated as fast as channels with R56Q
mutations in all four subunits. Our results show that all 4 N-termini are required
to mediate slow deactivation in wild-type hERG1 channels.

96-Plat
Lipid Affinity to the Voltage-Gated Potassium Channel KvAP
Elise Faure1, Christine Thompson2, Rikard Blunck1,2.
1
Physiology, Université de Montréal, GÉPROM, Montréal, QC, Canada,

2
Physique, Université de Montréal, GÉPROM, Montréal, QC, Canada.
Voltage-gated potassium channels (KV) are formed by a central conducting
pore surrounded by four voltage sensor domains. Functional studies have re-
vealed that biophysical properties of lipid molecules in the channels environ-
ment can have strong effects on the activity of KV channels. Here, we
investigated the influence of different lipids as well as their affinity to KvAP
channels. We carried out electrophysiology measurements by fusing vesicles
containing purified channels into planar lipid bilayers with varied lipid compo-
sitions. We found that KvAP properties are mainly determined by the lipid


	A-Type Kv4 Channel Closed-State Inactivation is Modulated by the Tetramerization Domain Interacting with Auxilary KChIP4a
	Two-in-One: Activation and Inactivation at the Intracellular Gate of a Kv Channel
	Development and Validation Studies of Universal Pharmacophore Models for hERG Channel Openers
	N-Terminal Regulation of hERG1 K+ Channel Deactivation
	Lipid Affinity to the Voltage-Gated Potassium Channel KvAP