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Introduction
Voltage-gated K+ (Kv) channels show enormous
molecular diversity with approximately 40 pore-forming principal
subunits identified that constitute 12 subfamilies
(Kv1-Kv12)[1]. The heteromultimeric assembly of different subunits provides a
base for further diversity and leads to a huge number of
functionally diverse Kv channels with distinct biophysical,
pharm-acological, and regulation
properties[2]. Increasing evidence shows that the dysfunction of Kv channels is associated with
epilepsy, cardiac arrhythmias, skeletal muscle disorders,
neurodegenerative diseases, and other
diseases[3_5]. Thus, the pharmacological modulation of Kv channels was proposed
as a therapeutic strategy in the treatment of
disorders[4,6].
High-throughput screening (HTS) technologies have
been widely employed to search for drug candidates in
pharmaceutical industries. In contrast to those successfully
applied in screening enzyme inhibitors or ligands for the
G-protein coupled receptor, HTS technologies for ion channel
drugs remain a challenge. New methods, such as
fluorescence-based assays, radioactive efflux assays, and
radiolabeled-ligand binding assays, although representing
"industry standards", still rely on conventional patch-clamping
recording for assessing the functional interaction of a
compound with an ion channel[7,8]. Recently, a variety of
prototypes of high-throughput electrophysiology has emerged;
however, the throughput is generally
low[8,9]. Virtual screening complements bioactivity screening. Nowadays, both
ligand- and target-based virtual screening are used as
reliable methods in the discovery of drug candidates in
pharmaceutical industries[10,11]. Based on the crystal structure of
the KcsA channel from Streptomyces
lividans[12], we constructed a 3-D model of a eukaryotic Kv channel and
developed a computational virtual screening approach to search
large databases for novel Kv channel
blockers[13,14]. As a result, we found nearly a dozen hit compounds in both the
China Natural Products Database (CNPD) and the MDL
Available Chemicals Directory (ACD) that potently inhibited
voltage-activated K+ currents in rat hippocampal neurons.
Several hit compounds have been further characterized
with respect to their potency, efficacy and specificity. Among
them, OMBSA is the most potent hit compound found in the
ACD database[14] (Figure 1). However, it remains unclear
whether the hit compound inhibits voltage-activated
K+ currents through the blocking of Kv channels, modification of
gating, or shift of voltage
dependence[15]. In the present study, the inhibitory effects of OMBSA on voltage-activated
K+ currents in rat hippocampal neurons and the relevant
mechanisms were further investigated by using a whole-cell
voltage-clamp recording technique.
Materials and methods
Materials OMBSA (molecular weight 500.1, purity
>99.5%) was synthesized in our laboratory. Other chemicals were
purchased from Sigma-Aldrich China Inc (China).
Preparation of dissociated hippocampal neurons
Sprague-Dawley rats (5_9 d old) were provided by the Shanghai
Experimental Animal Center, Chinese Academy of Sciences
(Shanghai, China). Dissociated hippocampal neurons were
prepared as described previously[16]. Briefly, hippocampal
slices (500 μm) were cut in oxygenated ice-cold dissociation
solution containing (in mmol/L): 82
Na2SO4, 30
K2SO4, 5 MgCl2, 10 HEPES, and 10 glucose (pH 7.3 with NaOH). The
slices were incubated in dissociation solution containing
protease XXIII (3 g/L) at 32 oC for 8 min, and then placed in
dissociation solution containing trypsin inhibitor type II-S
(1 g/L) and bovine serum albumin (1 g/L) at 24_25
oC in an oxygen atmosphere. Before recording, the CA1 regions of
several slices were dissected and triturated using a series of
fire-polished Pasteur pipettes with decreasing tip diameters.
Dissociated neurons were placed in a recording dish and
superfused with an external solution containing the
following (in mmol/L): 135 NaCl, 5 KCl, 1
CaCl2, 2 MgCl2, 10 HEPES, 10 glucose, and 0.001 tetrodotoxin (pH 7.4 with NaOH).
Whole-cell voltage-clamp recording
Voltage-activated K+ currents were recorded in large, pyramidal-shaped
neurons using an Axopatch 200A amplifier (Axon Instruments, CA, USA) at 24_25
oC. Voltage protocols were controlled by pClamp 9.0 software
(Axon Instruments) via a DigiData-1322A interface (Axon Instruments). Recording
electrodes (with a tip resistance of 3_5 MΩ) were pulled
from borosilicate grass pipettes (Sutter Instruments,
USA) and filled with a pipette solution containing
the following composition (in mmol/L): 140 KCl, 2
MgCl2, 1 CaCl2, 10 HEPES, and 10 EGTA (pH 7.4 with KOH). Signals were
filtered at 2_10 kHz and sampled at frequencies of 10_40 kHz.
Series resistance was compensated by 75%_85%. Linear
leak and residual capacitance currents were subtracted online
using a P/4 protocol.
Drug application OMBSA was dissolved in DMSO to
prepare a stock solution of 10 mmol/L and stored at
_20°C. The stock solution was diluted to desired concentrations
with the external solution before use and applied to the
neuron using RSC-100 rapid solution changer with a 9-tube head
(BioLogic, France). DMSO (less than 0.1% in the final
dilution) had no observed effect on the voltage-activated
K+ currents. For intracellular dialysis, OMBSA in the
pipette solution was diffused into the recorded neuron
immediately after the patch membrane
ruptured[17].
Data analysis The peak amplitude of the fast transient
K+ channel (IA) was measured, whereas the amplitude of the
delayed rectifier channel (IK) was measured with a 300 ms
latency. The decay time constants (τ) of the currents were
obtained by fitting the decay time course with a
mono-exponential function. The concentrations of OMBSA to yield a
50% block of the K+ currents
(IC50) were obtained by fitting normalized concentration-inhibition relationships to the
following equation:
I/I0=1/(1+[{C}/IC50
]n),
where I0 and I are current amplitudes measured in the
control and in the presence of OMBSA, C is the
concentration of OMBSA in the external solution, and
n is the Hill coefficient. For analyzing the voltage-dependence of
steady-state activation or inactivation of the
K+ currents, normalized conductance or current was plotted against the
membrane potential and fitted to the Boltzmann equation:
Y=1/(1+exp[{V-V1/2
}/k]),
where Y is the normalized conductance or current,
V is the test potential,
V1/2 is the voltage at half-maximal
activation or inactivation, and k is the slope factor. The time course
of recovery of the K+ currents from inactivation was fitted
with a mono-exponential function:
I/Imax=A*(1-exp[
-Δt/t]),
where Imax is the maximal current amplitude,
I is the current after a recovery period of
Δt, τ is the time constant, and A is the amplitude coefficient. Data are presented as
mean±SEM. Statistical significance was assessed using a
Student's t-test or one-way ANOVA as appropriate, and
P<0.05 was considered significant. All analyses were performed
using GraphPad Prism 4 (GraphPad Software, Inc, USA) and
Excel 2000 software.
Results
Inhibition of voltage-activated K+ currents by OMBSA
in hippocampal neurons As shown in the left panel of Figure
2A, both IK and
IA were simultaneously recorded from a
dissociated pyramidal neuron by using the voltage protocols
and a subtraction procedure. Superfusion with OMBSA (30
μmol/L) remarkably reduced the amplitudes of both the
voltage-activated K+ currents elicited by all depolarizing steps
(middle panel of Figure 2A). The inhibitory effects had a
rapid onset and reached steady-state levels within 10 s. The
K+ currents recovered immediately upon washing out of the
compound (Figure 2B, 2C).
In addition to causing a reduction in the amplitude of
the IK, superfusion with OMBSA (30
mmol/L) markedly accelerated the decay of the current (Figure 3A). The
t for IK was drastically reduced from 271.3±6.2 ms to 23.6±3.9 ms
(n=6, P<0.01; Figure 3B). In contrast, OMBSA exerted a negligible
effect on the decay time course of
IA (Figure 3C). The value of τ
for IA was reduced from 20.9±1.1 ms to 16.5±2.3 ms
(n=6), but the change was statistically non-significant
(P>0.05; Figure 3D). The results suggest that the compound
differentially modulated the inactivation of
K+ currents.
Analyses of the concentration-inhibition relationships
of OMBSA have revealed that the compound preferentially
inhibited IK to
IA. As shown in Figure 4A, the threshold
concentration for the inhibition of
IK was between 0.1 and 1 μmol/L, and
IK was reduced by approximately 90% at 100
μmol/L. In contrast, the threshold concentration for the
inhibition of IA was between 1 and 10
μmol/L, and the amplitude of IA was reduced by approximately 70% at the maximal
concentration tested (300 μmol/L). The
IC50 value for the inhibition of
IK was 2.1±1.1 μmol/L (Hill coefficient
=0.94±0.16, n=6), and that for the inhibition of
IA was 27.8±1.5 μmol/L (Hill coefficient
=1.3±0.3, n=6).
Lack of effects of intracellular dialysis of OMBSA on
voltage-activated K+ currents In order to determine the
action site of the compound on the Kv channels, the effects of
intracellular dialysis of OMBSA on the
K+ currents were investigated. The concentration for intracellular dialysis was
100 μmol/L, which inhibited IK and
IA by 90% and 65%, respectively, when applied externally (Figure 4A).
Throughout the 10 min recording period, the relative amplitudes of
IK and IA in the neurons dialyzed with OMBSA were almost
identical to those in the respective control groups (Figure
4B,4C). At the end of the 10 min recording, the relative
amplitude of IK in the control and in the neurons dialyzed with
OMBSA were 89.5%±0.1% and 86.3%±0.1%, respectively
(n=6 for each, P>0.05); the relative amplitude of
IA in the control and in the neurons dialyzed with OMBSA were
82.7%±0.1% and 92.3%±0.1%, respectively
(n=7 for each, P>0.05). The results suggest that OMBSA acts at an extracellular site of
the Kv channels.
Voltage-independent inhibition of voltage-activated
K+ currents by OMBSA The compound was 13-fold more
potent in the inhibition of IK than
of IA. In order to cause comparable effect on the 2 types of
K+ currents, concentrations of 3 and 30
μmol/L were used for studying IK and
IA, respectively. The current-voltage
(I/V) curves of IK
in the control and in the presence of OMBSA (3
μmol/L) revealed that the compound did not change the threshold for the
activation of IK (Figure 5A). OMBSA apparently caused a
linear downward shift of the curve and reduced its amplitude
over the entire range of activation.
The relative amplitudes of IK elicited by depolarizing steps
at 0, +20, +40 and +60 mV were 47.7%±8.0%, 49.9%±10.8%,
46.6%±5.1%, and 47.7%±7.6%, respectively
(n=7, P>0.05, ANOVA; Figure 5B). Similar results were observed for the
I/V curve of IA in the presence of OMBSA (30
μmol/L; Figure 5C). The relative amplitudes of
IA elicited by depolarizing in steps at 0, +20, +40
and +60 mV were 51.4%±5.3%, 52.7%±3.0%,
52.6%±4.0% and 51.5%±4.0%, respectively
(n=6, P>0.05, ANOVA; Figure 5D). These results
demonstrated that the inhibition of
IK and IA by OMBSA was
voltage independent.
Effects of OMBSA on activation and steady-state
inactivation of voltage-activated K+ currents in hippocampal
neurons Superfusion of OMBSA (3 μmol/L) had no significant
effect on the steady-state activation of
IK, nor did it affect its steady-state inactivation and the time course of recovery
from inactivation (Figure 6A,6C,6E). OMBSA (30
μmol/L) had no effect on the voltage dependence of the steady-state
activation of IA, but resulted in a significant hyperpolarizing
shift (nearly 8 mV) of its steady-state inactivation curve, and
significantly slowed down its recovery from inactivation
(Figure 6B,6D,6F). The effects of OMBSA on the kinetic
parameters of IA and
IK are summarized in Table 1.
Discussion
Due to the lack of HTS techniques for ion channel
drugs[7,9], virtual screening approaches have been developed for
discovering new blockers of Kv
channels[13,18]. In a virtual
screening study[14], a comprehensive assessment of electrostatic and
hydrophobic interactions with the Kv channel, and
solvation free energy suggests OMBSA to be a hit compound
with the most potent Kv channel-blocking activities in the ACD
database.
In the present study, we showed that OMBSA potently
inhibited both IK and
IA in rat hippocampal neurons. The
compound is 500-fold more potent than tetraethylammonium
(TEA) in the inhibition of IK (the
IC50 value of TEA was 1.05±0.21
mmol/L[19]), and approximately 180-fold more potent than
4-aminopyridine in the inhibition of
IA (the IC50 value of
4-aminopyridine was 4.92±0.65 mmol/L, unpublished data).
Furthermore, several interesting clues were found that
seemed to be helpful in elucidating the interaction of the
compound with Kv channels: (1) the onset of the inhibition
and recovery from the inhibition were fast (Figure 2B,2C),
suggesting that OMBSA rapidly binds to, and dissociates
from the binding site on Kv channels; (2) the inhibition was
voltage independent (Figure 5), which was similar to the
blocking of IK by externally-applied TEA in hippocampal
neurons[19]; and (3) intracellular dialysis of OMBSA was
ineffective (Figure 4B,4C). The results demonstrate that the
compound is most likely to act as a blocker at the outer
mouth of the Kv channels, as predicted by the molecular docking
model in the virtual screening[13,14]. A similar mechanism has
been found for 14-benzoyltalatisamine, a hit compound found
in the CNPD database, which selectively blocks
IK through binding to its external pore entry with partial insertion into
the selectivity filter[19].
It is also evident that the mode of actions of OMBSA on
the 2 types of Kv channels differs: (1) OMBSA is 13-fold
more potent in blocking IK than
IA; (2) OMBSA may bind to
IK with 1:1 stoichiometry (Hill coefficient
=0.94±0.16), and to IA with 2:1 stoichiometry (Hill coefficient
=1.3±0.3); and (3) in addition to acting as a channel blocker
molecule, OMBSA differentially modulates the kinetics of
the Kv channels; it markedly accelerated the inactivation
of IK without significant effect on that
of IA. Moreover, the compound did not change the activation, steady-state
inactivation of IK, and its recovery from inactivation, but caused
a significant hyperpolarizing shift of the voltage dependence
of the steady-state inactivation of
IA, and slowed down its recovery from inactivation, which led to fewer fast
transient K+ channels available for activation.
In vitro studies have shown that loss of intracellular
K+ ions through enhanced
IK (mainly the Kv2.1 channel)
mediates apoptosis of cultured cortical neurons induced by a
variety of treatments, and the blocking of the Kv channel
reduces neuronal death[20_22]. A number of
in vivo studies also showed that transient forebrain ischemia resulted in a
progressive increase of IK and a transient upregulation of
IA in hippocampal CA1 pyramidal neurons that led to neuronal
injury and programmed cell death[23,24], while blocking
IK by TEA effectively promoted neuronal survival in the CA1
region[25,26]. Recently, the inhibition of an A-type transient
K+ current was found to protect cerebellar granule cells against
low K+/serum deprivation-induced
apoptosis[27,28]. Because OMBSA potently blocks both
IK and IA in hippocampal CA1
pyramidal neurons, it will be interesting to test whether the
compound possesses neuroprotective effects.
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