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Introduction
Large-conductance Ca2+- and voltage-gated potassium channels termed BK channels are widely distributed in many
tissues from pancreas to smooth muscle to
brain[1]. BK channels play a crucial role in the control of excitability and secretion.
Scorpion toxins such as Charybdotoxin (ChTX), Iberiotoxin (IbTX) and Slotoxin (SloTX) are among the most potent and
important tools for studying function and structure of ion channels. Most of the scorpion toxins have a well-conserved
three-dimensional structure stabilized by three or four disulfide
bridges[2]. They bond with high affinity and specificity to the
BK channels and causally to the voltage-gated
K+ channels
KV1.3[3,4]. The channels encoded with pore-forming Slo1
a and auxiliary b subunits usually have very different sensitivity to toxins in comparison to the channels encoded with Slo1
a subunits alone. Toxins have been used as tools to recognize the existence of
b subunits and to identify the stoichiometry of channels by their sensitivity to
toxins[5_9]. The reversibility of toxins such as ChTX and IbTX is usually very poor. This made
it difficult to study functions of BK currents, especially in current-clamp experiments. Based on the crystal structure of KcsA
channels, a docking model predicts that the residue Lys27 of ChTX inserts into the pore to occlude entranceway of ions and
the residue Phe266 is one of the binding sites by
p-p stacking with the aromatic residues Trp14 and Tyr36 of
peptides[4]. To understand the function of the residue
Phe266, we mutated the Phe266 to leucine (Leu) or alanine (Ala) to verify whether it is
a binding site between the ChTX peptides and the Slo1 channels. In the present work, we report that both the mutations
Slo1-F266L and Slo1-F266A have the equilibrium dissociate constant
Kd similar to the one of the wild-type mSlo1 channels, but
with a perfect reversible recovery. Our results oppose the idea that the residue Phe266 is a site associated tightly with the
ChTX peptide, whereas the reason for irreversibility of mSlo1 is still unknown.
Materials and methods
Site-directed mutagenesis The QuikChange protocol (Stratagene) was used to produce two point mutations
mSlo1-F266L and mSlo1-F266A. With the wild-type mSlo1 as a template and a pair of complementary mutagenesis primers, the
reactions were performed by polymerase chain reaction (PCR). The primers for the mutants mSlo1-F266L and mSlo1-F266A
are 5¡¯-CAGGGGACCCATGGGAAAATCTTCAAAACAA-CCAGGCACTTAC-3¡¯/5¡¯-GTAAGTGCCTGGTTGTTTTGAA-
GATTTTCCCATGGGTCCCCTG-3¡¯ and 5¡¯-CAGGGGACCC-ATGGGAAAATGCTCAAAACAACCAGGCACTTACG-3¡¯/5¡¯-
CGTAAGTGCCTGGTTGTTTTGAGCATTTTCCCATGG-GTCCCCTG-3¡¯, respectively. Then the enzyme DpnI was used to
cut the PCR reaction mixture to digest the template of the wild-type mSlo1. Finally, the PCR products were transformed into
competent bacterial cells to amplify the mutated plasmids of mSlo1. All mutant constructs were verified by sequence
analysis.
Expression in Xenopus oocytes After DNA was linearized with MluI, SP6 RNA polymerase (Roche) was used to
synthesize cRNA for oocyte injection. Methods of expression in Stage V_VI
Xenopus oocytes have been described
previously[5]. Oocytes were defolliculated by treatment with 2 mg/mL collagenase I (Sigma-Aldrich Corp, St Louis, MO, USA) in
zero-calcium ND-96 solution. Between 2 and 24 h after defolliculation, 1_2 ng of a (mSlo1) cRNA were injected into
Xenopus oocytes using a Drummond Nanoject II (Drum-mond Scientific Co, USA). After injection, oocytes were then incubated in
ND-96 solution at 18 ºC. Currents were recorded 2_7 d after cRNA injection. The ND-96 solution (pH 7.5) contained (in
mmol/L): 96 NaCl, 2 KCl, 1.8 CaCl2,
1 MgCl2, 2.5 Na pyruvate, and 10
H+-HEPES, supplemented with 100 IU/mL penicillin and 100 mg/mL streptomycin (only for
incubation).
Solutions Oocytes were bathed in the ND-96 solution. For all the experiments, currents were recorded in outside-out
patches. The intracellular recording solution was (in mmol/L): 160
MeSO3K, 10 H+-HEPES, and 2
MgCl2, adjusted to pH 7.0 with
MeSO3H. Pipettes were filled with a solution containing (in mmol/L): 160
MeSO3K, 10 H+-HEPES, and 5 HEDTA with
added Ca2+ to make 10 mol/L free
Ca2+, as defined by the EGTAETC program (E McCleskey, Vollum Institute), with pH
adjusted to 7.0. The solutions for 20 mmol/L Tetraethylammonium chloride (TEA) and 100 nmol/L Charybdotoxin (ChTX)
were made by adding the 20 mmol/L TEA and the 100 nmol/L ChTX into the intracellular solution, respectively. All of the
chemicals were obtained from Sigma.
Electrophysiology Recording pipettes were used to have a resistance of 2_6
MW while filled with internal solution. An outside-out patch was obtained by excising from oocytes. Currents were recorded with the EPC-9 patch-clamp amplifier and
PULSE software (HEKA Electronics, Germany). Data were typically collected with a sampling frequency of 20 kHz.
Macroscopic records were filtered at 2.9 kHz during digitiza-tion.
During experiments, the control, drug and recovery solutions were locally perfused onto the patches via a perfusing
pipette with seven solution channels. All experiments were performed at room temperature (22_25 ºC).
Data analysis Data were analyzed with IGOR (Wave-metrics, Lake Oswego, OR, USA), Clampfit (Axon Instruments, Inc
USA), and SigmaPlot (SPSS Inc USA) softwares. Unless stated otherwise, the data are presented as mean±SEM, significance
was tested by Student¡¯s t-test, and differences in the mean values were considered significant at
P£0.05.
The onset and recovery (offset) from blockade by ChTX were fit with the first-order blocking reaction, in which the time
constants of onset and offset were given by
ton=1/[f× (drug)+b] and
toff=1/b, where f is the forward drug blocking rate in
M-1s-1, b is the drug dissociation rate in
s-1.
During application of drug (for
t0<t£t
1),
I(t)=(I0-I
SS)×exp(-t/ton)+
ISS
(1)
During recovery (for
t>t1),
I(t)=I0-(I
0-Ir)×exp(-(t-
t1)/toff) (2)
Where I0 is the mean control current amplitude,
ISS is
I0×b/(f×[drug]+
b) and indicated a steady-state level of current during
blockade by a given drug concentration, Ir is the empirically determined current that is unblocked at the end of the drug
application period, t=0 at the time of the drug application, and
t1 is the time of drug washout. The equilibrium dissociation
constant Kd was defined by
Kd=b/[f×(drug)]
[10].
Results
Large-conductance Ca2+- and voltage-gated
K+ channels (BK channels) encoded by mammalian mSlo1 genes are
abundantly distributed in the nervous system. It regulates excitability in response to intracellular
Ca2+ and membrane potentials. BK channels likely share similar pore structural determinants and sensitivities to toxins with voltage-dependent
K+ channels (KV
channels)[4]. Some peptidyl scorpion toxins such as Charybdotoxin (ChTX) not only block
KV1.3 as well as a mutation F425H of shaker channels, but also block the BK currents encoded by both the Slo1
a subunits and the b subunits but with a higher
EC50[3,11,12]. In Figure 1, a conserved residue Phe266 (mSlo1) labeled with the symbol
¨< (the upper panel) is supposed to interact with the residues that are highlighted in the lower panel with the same
symbol[4,13].
The blocking behaviors of those toxins commonly show poor reversibility, therefore making it difficult to study functions
of BK channels, especially in current-clamp
experi-ments[5_7]. For mSlo1 channels (Figure 2A), an approximate 20%
irreversible component remains after a 3 min recovery period, compared to the unblocking currents. Fits of the onset and offset time
give ton=2.6 s and toff=64.5 s on graph, with a mean
ton=2.8±0.2 s and toff=73±14 s. The mean
Kd calculated from the fitted time
constants gives 3.9±0.5 nmol/L (n=3). To gain insight into the molecular determinants of peptide-channel complex such as
ChTX-mSlo1, Yao et al (2005) has reported that the aromatic residues Phe266 and Tyr294 of mSlo1 may stabilize binding of
ChTX by p-p stacking with the aromatic residues Trp14 and Tyr36 of
peptides[13]. In Figure 2B, we show that the mutation
mSlo1-F266L has ton=2.4±0.5 s,
toff=77.6±8.9 s and a mean
Kd=3.1±0.5 nmol/L (n=8). In Figure 2C, we find that the mutation
mSlo1-F266A has ton=2.6±1.2 s,
toff=73.6±20 s and mean
Kd=4.2±1.2 nmol/L (n=4). However, both the mSlo1-F266L and the
mSlo1-F266A mutations show a nearly completed recovery from inhibition by 100 nmol/L ChTX in most of cases (eg
n=8/9 for mSlo1-F266L).
There are three irreversible data of wild-type mSlo1 channels and eight reversible data of F266L channels shown as
labeled in Figure 3, respectively. Applying the recovery saline for 400 to 800 s, we only found a 70%_80% recovery arising
from mSlo1 and nearly a 100% recovery from the mSlo1-F266L. The irreversible recovery occurs sometimes in many blocking
experiments of the BK-type channels by applying toxins such as ChTX or IbTX, even though it was never brought to an
important place before[5,9]. Another interesting phenomenon is that the successive recovery level after the first application of
toxins is always
"reversible"[9]. The repeated recovery experiments (Figure 4) show that there is an irreversible component
arising from blocked mSlo1 channels that are only apparent after the first application of ChTX (Figure 4A). In contrast, the
irreversible component is never observed in both the mSlo1-F266L and mSlo1-F266A cases (Figure 4B, 4C).
Discussion
To date, the scorpion toxins have been used as a tool for exploring the structure and function of the ion-channel proteins.
It is important to know how it can associate with channel proteins and what function it exerts on the channels. Simulation of
peptide-channel complex interaction reveals that the residues Phe266 and Tyr294 in mSlo1 channels may bind to the aromatic
residues Trp14 and Tyr36 of the scorpion toxin peptides by
p-p stacking[4,13]. The mutant Y294V proves insensitive to both
TEA and CTX (n=8, data not shown), which means that Y294 is the binding site of CTX. However, the permanently lost
component in BK channels by ChTX or IbTX has never been paid enough attention before. We often selected the results of
the subsequent application of toxins so that we could examine the nature of the peptide-channel complex. In this study, we
were attempting to gain an insight into the interaction mechanism of ChTX-mSlo1 complex by mutations of the residue
Phe266.
Based on experiment results from both the mutants mSlo1-F266L and mSlo1-F266A in this study, we did not find any
significant difference on the equilibrium dissociation constant Kd, as well as time constants
ton and toff. Therefore, the results
in this study suggest that the residue Phe266 does not clearly show its ability for stabilizing binding to the peptide ChTX by
p-p stacking as the predication given by simulation of peptide-channel
complex[4]. The only difference we found in this study
is that the mutants eliminate irreversible components. It is unclear why the mSlo1 currents continue to contain an irreversible
component during the first application of toxin. A hypothesis is that the BK channels encoded with mSlo1 might have a
rundown in currents during a long recovering period. Consequently, we performed experiments to test the stability of mSlo1
currents and found that the steady-state currents of mSlo1
(n=4/4 patches) only showed less than the 10% rundown in -20
min (Unpublished data). Finally, we cannot completely exclude the possibility that the wild-type mSlo1 may need an extra
long time to recover its permanent part, or that the residue Phe266 itself might have a role in removing the irreversible
component. However, more precisely designed experiments should be undertaken to verify this idea in the future.
Yao J et al
Acta Pharmacologica Sinica ISSN 1671-4083
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