Bondarenko, Vladimir E.

DeSimone, Christopher V.

Morales, Michael J.

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inactivation process plays a critical role in controlling the length and frequency
of cardiac action potentials, as well as the firing patterns in neurons. The mo-
lecular process underlying the C-type inactivation mechanism remains unex-
plained despite the accumulation of experimental evidences showing the key
role played by the channels’ selectivity filter and some neighboring residues.
It’s been recently shown that the prokaryotic KcsA channel undergoes C-type
inactivation like its eukaryotic counterparts (Gao et al., PNAS, 102:17630
(2005)), establishing KcsA as a perfect prototypic model to study the structural
basis of the inactivation mechanism. An X-ray structure of the KcsA channel
obtained in presence of low Kþ concentration (Zhou et al., Nature 414:43
(2001), pdb code 1K4D) has since then been postulated to correspond to the
C-type inactivated state of the channel.While the structural analysis of this static
conformation suggests that pore lining amide hydrogens would prevent the per-
meation of ions, uncertainties remain about its stability under physiological con-
ditions and its ion occupancy state. These questions are of primary importance to
better understand the relevance of this structure to the physiological regulation
of ion permeation in Kþ channels. Using molecular dynamics simulations and
free energy calculations, we investigated on the stability, selectivity, and con-
ductance of the selectivity filter of KcsA in this putative inactivated state.
3397-Pos Board B444
Ancillary subunits and S3b amino acid substitutions alter the affinity of
HpTx2 for Kv4.3
Christopher V. DeSimone1, Vladimir E. Bondarenko2,Michael J. Morales1.
1University at Buffalo-SUNY, Buffalo, NY, USA, 2Georgia State University,
Atlanta, GA, USA.
HpTx2 is an ICK gating modifier toxin that selectively inhibits Kv4 channels,
making it an excellent probe for understanding Kv4 structure and function. To
characterize the molecular determinants of interaction, we performed alanine
scanning of Kv4.3 S3b linker. HpTx2-Kv4.3 interaction had a Kd ¼ 2.4 mM.
Two alanine mutants in Kv4.3 caused a dramatic increase in Kd: 6.4 mM for
V276A and 25 mM for L275A. LV275AA nearly eliminated toxin interaction.
Unlike HaTx and other well-characterized ICK toxins, HpTx2 binding does not
require a charged amino acid. To determine if the identity of the amino acids
altered HpTx2 specificity, we constructed LV275IF. This mutation decreased
the Kd to 0.54 mM, suggesting that the hydrophobic character of the binding
site is the most important property for interaction with HpTx2. Two of the al-
anine mutants, N280A and D282A caused stronger interaction of HpTx2 with
Kv4.3; the Kd for Kv4.3 [N280A] was 0.26 mM. We propose that these muta-
tions either remove a steric barrier to HpTx2 occupation of S3b, or that removal
of these polar side chains increases toxin affinity by increasing the hydrophobic
character of the binding site. To understand Kv4.3-based transient outward cur-
rents in native tissues, we tested the affinity of HpTx2 for Kv4.3 co-expressed
with KChIP2b. The toxin’s Kd for Kv4.3þKChIP2b was 0.95 mM. HpTx2
binds to the closed state of Kv4.3, which is stabilized by KChIP2b. We propose
the increased affinity is due to the increased stabilization of the closed state.
These data show that HpTx2 binding to Kv4.3 has aspects in common with
other ICK gating modifier toxins, but that the interventions that increase toxin
affinity suggests flexibility toward channel binding that belies its unusual spec-
ificity for Kv4 channels.
3398-Pos Board B445
HpTx2 Interaction With Kv4.3 and Kv4.1 Reveals Differences in Gating
Modification
Christopher V. DeSimone1, Vladimir E. Bondarenko2, Michael J. Morales1.
1University at Buffalo-SUNY, Buffalo, NY, USA, 2Georgia State University,
Atlanta, GA, USA.
HpTx2 is an ICK gating modifier toxin specific for Kv4 channels. The Kd of
HpTx2 for two similar channels, Kv4.3 and Kv4.1, was 2.4 mM and 7.1 mM,
respectively. HpTx2 inhibition of Kv4.3 is highly voltage dependent, shifting
the current-voltage relationship to more depolarized potentials; consistent
with the classic behavior of gating modifier toxins. However, modification of
Kv4.1 gating is much less voltage dependent. Site directed mutagenesis of
the S3b interaction site of Kv4.3 and Kv4.1 shows that the same two conserved
bulky hydrophobic amino acids are required for HpTx2 interaction with each
channel. While the interior of the binding site is conserved between Kv4.3
and Kv4.1, three amino acids adjacent to the binding site are not conserved.
Swapping these amino acids between Kv4.3 and Kv4.1 swaps the phenotypic
response to toxin, while having minimal effect on gating properties of the chan-
nels. We modeled the activation gating of Kv4.3 and Kv4.1 and incorporated
the effects of HpTx2 into the kinetic parameters of activation. The model is
similar for both channels; it has four voltage-dependent transitions between
the closed states followed by voltage-independent transition to an open state.
Voltage-dependent transitions in Kv4.3 are more strongly affected by toxin.
In Kv4.1, the voltage-independent transition from the closed pre-open to
open state is most affected by HpTx2. Therefore, a higher proportion of toxin-
bound Kv4.1 channels are in the closed pre-open state, compared to toxin-
bound Kv4.3 channels. This decreases the voltage dependence of toxin-bound
Kv4.1 opening. The model closely recapitulates our experimental data. These
data show that amino acids near the HpTx2 binding site play a role in the
kinetics of Kv4 channel activation gating.
3399-Pos Board B446
A Potassium Channel Blocking Toxin Isolated From The Venom Of The
Scorpion Centruroides suffusus suffusus
Orsolya Szilagyi1, Monika Herceg1, Gerardo Corzo2, Ferenc Papp3,
Zoltan Varga1, Omar Barraza2, Pavel G. Espino2, Ricardo C. Rodrı¨¿1⁄2 guez
de la Vega2, Rezso Gaspar1, Lourival D. Possani2, Gyorgy Panyi1.
1University of Debrecen, Dept. of Biophysics and Cell Biology, Research
Center for Molecular Medicine, Debrecen, Hungary, 2Departamento de
Medicina Molecular y Bioprocesos, Instituto de Biotecnologı´a, Universidad
Nacional Auto´noma de Me´xico, CuernavacaMorelos, Mexico, 3University of
Debrecen, Cell Biology and Signaling Research Group of the Hungarian
Academy of Sciences, Department of Biophysics and Cell Biology, Research
Center for Molecular Medicine, Debrecen, Hungary.
Voltage-gated potassium channels are widespread among various cell types
playing roles in numerous cellular functions by controlling the membrane po-
tential. Pharmacological manipulation of these channels carries the potential of
selective interference with such well-defined cellular functions if molecules
that bind to them with high affinity and specificity are available. Scorpion
venoms have long been known for containing small peptide toxins bearing
these properties. We are particularly interested in peptides blocking T cell
Kv1.3 channels whose normal operation is essential for T cell mediated im-
mune responses.
We screened HPLC purified peptide fractions from Centruroides suffusus suf-
fusus scorpion venom for Kv1.3 channel blocking potency. Screening resulted
in one biologically active peptide component, which was purified to homoge-
neity and named Css20. The toxin’s amino acid sequence was completed by
Edman degradation and mass spectrometry analysis. It contains 38 amino
acid residues with a molecular weight of 4,000.3 Da, tightly folded by three di-
sulfide bridges.
We have found that Css20 preferentially blocked the currents of the voltage-
gated Kþ channels Kv1.2 and Kv1.3. Its blocking potency was tested on six
other potassium channels and a cardiac sodium channel, but the toxin proved in-
effective at 10 nM concentration. Dose-response curves of Css20 yielded an
IC50 of 1.3 and 7.2 nM for Kv1.2 and Kv1.3 channels, respectively. Interest-
ingly, despite the similar affinities for the two channels the association and dis-
sociation rates of the toxin were much slower for Kv1.2, implying that different
interactions may be involved in binding to the two channel types; an implication
further supported by in silico docking analyses. Based on the primary structure
of Css20, the systematic nomenclature proposed for this toxin is a-KTx 2.13.
3400-Pos Board B447
Differential Effects of Isoflurane on Mutant Cardiac IKs Channels
Ikuomi Mikuni, Carlos Torres, Martin Bienengraeber, Wai-Meng Kwok.
Medical College of Wisconsin, Milwaukee, WI, USA.
The slow delayed-rectifier potassium (IKs) channel mediates repolarization of
the cardiac action potential, and its dysfunction can lead to the long QT syn-
drome (LQTS). The IKs channel consists of two subunits, the pore-forming
a-subunit, KCNQ1, and the auxiliary b-subunit, KCNE1. Volatile anesthetics
have significant inhibitory effects on the IKs current, but their underlying mo-
lecular mechanism remains to be determined. We have previously shown that
F340 was a critical residue involved in this interaction, similar to the observation
reported for other putative IKs antagonists. In order to further elucidate this
mechanism, we investigated the effects of a volatile anesthetic, isoflurane, on
mutant constructs of the IKs channel. Two KCNQ1 mutants, A341C and
A344C, were constructed by site-directed mutagenesis. These two residues
were chosen due to their vicinity to F340, and to their roles in inherited
LQTS. Whole-cell current was recorded from transiently transfected HEK-
293 cells in the absence and presence of isoflurane (0.525 0.01 mM). Isoflur-
ane inhibited wild-type- (WT) KCNQ1 by 62.1 5 1.9% (mean5SEM, n¼9).
This inhibition was significantly attenuated when WT-KCNQ1 was cotrans-
fected with WT-KCNE1 (40.7 54.5%; n¼9). In contrast, isoflurane inhibited
the mutant A344C current by 25.5 5 2.0% (n¼13), but this inhibition was
not affected by the cotransfection with WT-KCNE1 (26.3 5 3.2%, n¼13).
The A341C mutant alone did not express any current. However, when A341C
was cotransfected with WT-KCNE1, an IKs-like current was elicited that was
inhibited by isoflurane by 63.15 2.9% (n ¼11). These results show that in ad-
dition to F340, the A341 and A344 residues also contribute to the anesthetic
binding environment. Furthermore, the differential impact of KCNE1 on the iso-
flurane effects on WT-KCNQ1 and the mutant constructs suggests a complex
role for the b-subunit in modulating the pharmacology of the IKs channel.
658a Wednesday, March 4, 2009


HpTx2 Interaction With Kv4.3 and Kv4.1 Reveals Differences in Gating Modification 

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HpTx2 Interaction With Kv4.3 and Kv4.1 Reveals Differences in Gating Modification

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