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First published online October 17, 2008
Journal of Experimental Biology 211, 3442-3453 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.022608
Non-linear intramolecular interactions and voltage sensitivity of a KV1 family potassium channel from Polyorchis penicillatus (Eschscholtz 1829)
1 Department of Biological Sciences, University of Alberta, Edmonton, Alberta,
Canada T6G 2E9
2 Department of Biological Sciences, University of Calgary, Calgary, Alberta,
Canada T2N 1N4
3 Malaspina University College, 900 Fifth Street, Nanaimo, British Columbia,
Canada V9R 5S5
* Author for correspondence (e-mail: wgallin{at}ualberta.ca)
Accepted 2 September 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: site-directed mutagenesis, ion channel gating, electrophysiology
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Connor and Stevens, 1971b;
Connor and Stevens, 1971c;
Guan et al., 2007;
Kasten et al., 2007).
Understanding how the amino acid sequence of channel proteins affects their
voltage sensitivity is important for understanding how genetic variation can
cause changes in action potential form and excitability patterns, and
ultimately how different excitability properties have evolved.
The S4 transmembrane helical segment of voltage-gated ion channels is the
voltage sensor (Guy et al.,
1991; Noda et al.,
1984). This segment contains a basic amino acid at every third
position along the length of the helix in the form Arg-Xaa-Xaa or Lys-Xaa-Xaa.
Since this charged helix traverses the plasma membrane, changes in membrane
potential exert a force on the helix, causing it to move and change the
conformation of the channel. Each charged motif does not contribute equally to
voltage sensing (Bezanilla et al.,
1994; Papazian et al.,
1995; Seoh et al.,
1996). Furthermore, the potential field of the membrane is not
distributed completely or evenly over the S4 segment but is focused by aqueous
`canals' on both sides of the channel protein near the S4 transmembrane helix
(Ahern and Horn, 2005;
Baker et al., 1998;
Chanda et al., 2005;
Islas and Sigworth, 2001;
Starace and Bezanilla, 2004;
Yang and Horn, 1995).
Residues external to the S4 transmembrane helix also contribute to voltage
sensitivity of channel activation (Monks
et al., 1999; Papazian et al.,
1995; Perozo et al.,
1999; Tiwari-Woodruff et al.,
2000). In the Drosophila melanogaster Shaker channel, the
closed conformation is stabilized by salt bridges formed between Lys374 (K374)
in S4 and Glu293 (E293) and Asp316 (D316) in S2 and S3, respectively
(Durell et al., 2004;
Li-Smerin et al., 2000;
Papazian et al., 1995;
Tiwari-Woodruff et al., 1997).
The open conformation is stabilized by the interaction of Arg368 (R368) and
R371 in S4 with E283 in the S2 transmembrane helix, whereas E293 in S2 and
D316 in S3 interact with R377 (Papazian et
al., 1995; Silverman et al.,
2003). A neutralization mutant of E293 destabilizes the closed
conformation, shifting the voltage sensitivity of Drosophila Shaker
in a hyperpolarized (leftward) direction whereas neutralization mutants of
E283 and D316 shift the voltage sensitivity of these channels in a
depolarizing (rightward) direction
(Papazian et al., 1995).
The physical factors that affect the voltage of half activation
(V50) of a channel can be partitioned into two parameters,
the gating charge that moves in response to a change in membrane potential,
and the relative difference of internal free energy of the protein in the open
and closed states. At a membrane potential of 0 mV, the channel partitions
into open and closed states as a function of the internal energy difference
between the two states. When a membrane potential is applied, an additional
energy term is added, the energy difference between the gating charge in the
open-state versus the closed state conformation, proportional to the
product of the gating charge and the voltage of the potential field. This
additional energy term shifts the equilibrium between the open and closed
states. Conversely, by experimentally determining the V50
and the gating charge of the channel, it is possible to calculate the Gibb's
free energy (
G0) for channel opening. By evaluating
the differences in G0 of various mutations it is
then possible to define the way in which intra-molecular interactions
determine G0, gating charge, and thus the voltage
sensitivity. Using the formalism in this study, the probability that the
channel is closed increases as the G0
increases.
|
; Jegla et al.,
1995). In comparison with most other KV1 channels
(including jShak2), jShak1 has one less positively charged motif in the S4
region, with a charged residue being absent at the position equivalent to R362
in Drosophila Shaker (Fig.
1). The recently cloned KV1 channel from the Portuguese
Man-o-War, Physalia physalis, also lacks the first acidic residue in
S2 (S231 in PpKv1) and has a shortened S4
(Bouchard et al., 2006),
suggesting that these two structural differences may reflect a conserved
variant of KV1 channel architecture in the class Hydrozoa. Previous
mutagenesis analysis in jShak1
(Grigoriev et al., 1997)
demonstrated that the addition of length and charge in the S4 voltage sensor,
to either side of the conserved K294 residue (K374 in Drosophila
Shaker) had complex effects on channel properties, but did not simply shift
the voltage sensitivity of jShak1 to the range observed for vertebrate
KV1 channels. The current study was designed to determine how interactions between N227 and the S4 helix of jShak1 affect voltage sensitivity by setting the gating charge and the internal energy difference between open and closed states.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Jegla et al.,
1995) was amplified using primers WJG1182 and WJG1173 (Table S1 in
supplementary material). The PCR fragment was cut using unique XhoI
and SpeI sites and was inserted into the pXT7 plasmid and sequenced
to confirm that no PCR-induced errors were present. Translational efficiency
of transcribed mRNA is enhanced by using pXT7 as the expression vector because
the inserted coding region is flanked by 5' UTR and 3' UTR
sequences from Xenopus laevis globin mRNA.
To produce a non-inactivating channel, the full-length jShak1 in
pXT7 was used as a template for PCR amplification with primers WJG 2037 and
WJG 1246 (Table S1 in supplementary material). The PCR fragment and the
wild-type expression plasmid were cut with XhoI and HpaI
(New England Bioloabs, Ipswich, MA, USA) at unique restriction sites. The PCR
product was then ligated into the wild-type plasmid, to create a 23
N-truncated channel without the N-type inactivation ball, as described
previously (Jegla et al.,
1995). By removing fast, N-type inactivation
(Hoshi et al., 1990) the
steady-state conductance of the channels can be more accurately measured from
tail currents. This channel was designated as `wild type' for all subsequent
experiments.
The triplet mutations of the S4 sensor, whereby either Arg-Ile-Phe (RIF) or
Gln-Ile-Phe (QIF) was inserted on the N-terminal side of K294, or Ile-Phe-Arg
(IFR) was inserted on the C-terminal side of K294
(Fig. 1C), were constructed in
this plasmid as described previously
(Grigoriev et al., 1997). The
S2 mutations at Asn227 (N227; Fig.
1B) were created by overlapping PCR mutagenesis. Primers used to
create the N227E mutation were WJG1062 and WJG1063 and construction of N227D
used primers WJG1064 and WJG1065. In both instances the flanking primers were
WJG1149 and WJG1181. The resulting PCR products were digested with
HpaI and ClaI and ligated into the wild-type or S4 mutant
plasmids to create single (S2) and double (S2/S4) mutations, respectively. The
sequence of all mutants was confirmed to be as designed by sequencing the
cloned plasmid with the two flanking primers WJG1149 and WJG1181.
Expression plasmids and oocyte preparation
Purified plasmids were prepared from 10 ml overnight cultures [Terrific
Broth (Ausubel et al., 1987)
with 100 µgml–1 ampicillin] using a Wizard Miniprep Kit
(Promega, Madison, Wisconsin, USA). Plasmids were linearized with
XbaI and gel purified using the QiaQuick Gel Extraction Kit (Qiagen,
Mississauga, ON, Canada). Capped mRNAs were prepared by in vitro
transcription using a mMessage mMachine (Ambion, Austin, TX, USA) T7
polymerase kit, and stored at –80°C.
Female X. laevis that were 2 years old were reversibly
anaesthetized in 0.17% MS-222 (Sigma, St Louis, MO, USA) and a single lateral
incision made in the abdomen 1 cm from the midline, 1 cm caudal to the pelvic
girdle. A single ovary was removed and manually separated into 0.5 cm clumps.
Ovarian pieces were rinsed three times in MBM [in mmoll–1:
NaCl88, KCl1, Ca(NO3)20.33, CaCl20.41,
MgSO40.82, NaHCO32.4, Hepes10 (Tris base to pH 7.5),
sodium pyruvate 2.5, supplemented with penicillin G0.1 g l–1
and gentamycin sulphate 0.05 g l–1
(Huang et al., 1993)]. The
tissue fragments were incubated on a rotating shaker in 2 mg
ml–1 collagenase 1A (Sigma, Oakville, ON, Canada) in MBM at
room temperature. Released oocytes were removed from the collagenase solution
after 2 h, rinsed in MBM and 
200 eggs incubated per glass scintillation
vial, at 17°C overnight in fresh MBM. Eggs were de-folliculated by
immersion in a hypo-osmotic phosphate buffer [in mmoll–1:
K2PO4100 (pH 6.5 with HCl)] for 1 h. Following
treatment, eggs were left for 2 h in fresh MBM to recover prior to
manipulation. Mature stage V–VII oocytes were injected with 48.6 nl mRNA
(200–600 ng nl–1) and incubated in MBM at 17°C.
Electrophysiological methods
The N-truncated jShak1 wild-type channel had identical activation
properties to the full-length channel, but lacked fast N-type inactivation
(Jegla et al., 1995)
(Fig. 2A,B). The channels
expressed in pXT7 had higher rates of translation compared to mRNA prepared
from BlueScript (Grigoriev et al.,
1997; Jegla et al.,
1995), but had identical electrophysiological properties. The
mutants manifested variable C-type inactivation, with IFR-E having the most
inactivation at 150 ms, but all channels opened quickly, allowing for peak
currents to be obtained within 50 ms. Therefore, all electrophysiological
protocols were designed to provide current measurements prior to significant
C-type inactivation (Fig.
2C,D).
|
. Voltage-clamp recordings were obtained
using a GeneClamp 500B amplifier (Axon Div., Molecular Devices, Sunnyvale, CA,
USA) controlled by pClamp 9.0 software (Axon Div., Molecular Devices). Data
were acquired through a 1322A analogue/digital converter and analysed using
Clampfit 9.0 (Axon Div., Molecular Devices). Experiments were performed in
ND96 (in mmoll–1: NaCl96, KCl2, CaCl21.8,
MgCl21, Hepes5, pH 7.4). Measurements were performed with leak
subtraction (P/N=4) for all constructs.
Steady-state conductance was measured using a pulse protocol where the membrane is held at –90 mV for 10 ms, followed an activation test pulse from –140 mV to +90 mV in 2 mV steps for a duration of 50 ms. This was followed by a 20 ms tail-step to –120 mV for inward tails or –50, or –30 mV for outward tails, followed by a 200 ms return to a –90 mV holding potential (see Fig. S1 in supplementary material for examples of traces from a simplified –30 mV tail-step protocol). Because the deactivation kinetics in jShak1 are voltage dependent, with channels closing rapidly at hyperpolarized voltages, it was not always possible to use inward tail currents for steady-state conductance measurements, and outward tail currents were utilized. It is important to note that the S4 single and double mutations shifted the threshold for channel activation such that some channels (see Fig. S1 in supplementary material; N227E) were open at the –30 mV tail-step, and the –50 mV tail-step protocol was used for subsequent data analysis. Steady-state voltage-conductance curves were obtained by fitting the tail current to the sum of two exponential decays to increase detection of low probability opening events near the threshold voltage. The resultant fitted parameters were used to obtain calculated current values at 2 ms following the capacitative transient artefact (see Fig. S2 in supplementary material). These current values were normalized using a four-parameter Boltzmann curve (see below) to obtain a normalized measure of conductance.
The calculated current vs voltage data were fitted with a
four-parameter Boltzmann curve:
 | (1) |
Although it is possible to calculate a charge associated with the
transition between two states from the Boltzmann slope factor (b),
this derivation is only valid for a two-state equilibrium. The transition
between the open and the closed state of VKCs involves a large number of
intermediate states, each with their own voltage dependency
(Almers, 1978;
Sigg and Bezanilla, 1997). The
result of this complexity is that the charge calculated from the Boltzmann
slope factor consistently underestimates the gating charge, and that the
difference between the estimate and the true gating charge varies as a
complicated function of the intermediate states. Almers derived a robust
method for estimating gating charge, by determining the limiting slope of the
log(G/Gmax) vs V curve at very low
conductance (Almers, 1978).
This estimate of gating charge approaches the true gating charge
asymptotically. Sigg and Bezanilla demonstrated that the absolute value of the
gating charge determined by the limiting slope method is insensitive to the
nature of the state transitions in a number of realistic scenarios
(Sigg and Bezanilla, 1997). An
example of this analysis, including determination of the asymptotic value, as
implemented by Gonzalez et al. (Gonzalez
et al., 2000), is provided in Fig. S2 in supplementary material.
Briefly, slopes for successive windows of 15 points from the
log(G/Gmax) vs V curve are determined
and the values of the slopes reach a plateau at low values of
G/Gmax that represents the gating charge for the
channel. Graphs illustrating the asymptotic behaviour of the gating charge
(zlim) values for all 12 channels are provided in Fig. S3
in supplementary material. Although it is also possible to measure directly
the charge displacement associated with activation
(Perozo et al., 1993), this
method yields total charge movement, not just the charge movement that is
directly coupled to channel opening (Sigg
and Bezanilla, 1997).
Gibb's free energy values were calculated using the gating charge,
determined as described above, and the V50 values obtained
in Eqn 1:
 | (2) |
 | (3) |
Negative values of G0 indicate that the
equilibrium is shifted towards the open state, whereas positive values
indicate increased stabilization of the closed state compared to the open
state of the channel.
The significance of differences between electrophysiological parameters from different channels was assessed using a two-tailed t test.
Molecular modelling
The crystal structure of the RatKV1.2 potassium channel
(2A79.pdb) (Long et al., 2005)
was used to create a complete model of the RatKV1.2 channel, and
this model was used as the basis for homology modelling of jShak1 in the open
state. The crystal structure maps the coordinates for residues Cys32 to
Gly131, Thr219 to Ala243 and Met288 to Thr421. Two unresolved chains are
represented in the crystal structure by polyalanines. Chain C represents the
T1-S1 linker and S1 helix, and chain D represents the S3 helix. Chains C and D
are 52 and 21 residues long, respectively. The identity of the residues
constituting chains C and D was determined using the known structural elements
and secondary structure predictions from PredictProtein
(Rost et al., 2004), on the
complete primary sequence of RatKV1.2 (NCBI accession no. P63142).
The corresponding regions of the primary sequence were threaded onto the
polyalanine backbones and the side chains were rebuilt using SwissPDBViewer
(Guex and Peitsch, 1997;
Peitsch, 1997;
Peitsch et al., 1995;
Schwede et al., 2003). The
missing inter-helical loop regions of the voltage sensor were rebuilt using
SwissPDB loop databases (Guex and Peitsch,
1997).
A homology model of jShak1 was constructed in SwissModel using the resolved
residues of the RatKV1.2 channel structure as a modelling template
(Fig. S4 in supplementary material). The jShak1 sequence was aligned with 140
voltage-gated potassium channel sequences using MUSCLE v3.6
(Edgar, 2004a;
Edgar, 2004b) and the
pair-wise alignment of jShak1 and RatKv1.2 was extracted. The
alignment of jShak1 and RatKV1.2 and corresponding secondary
structure predictions (Rost et al.,
2004) were used for the regions of jShak1 that were homologous to
regions of RatKV1.2. These regions were threaded onto the
corresponding regions of the RatKV1.2 crystal structure and the
side-chains were rebuilt using SwissPDBViewer
(Guex and Peitsch, 1997;
Schwede et al., 2003). The
inter-helix loops were rebuilt using SwissModel and SwissPDBViewer loop
databases (Guex and Peitsch,
1997; Schwede et al.,
2003). The resulting subunit model was energy minimized and
refined to remove steric clashes. Symmetry information from the
RatKV1.2 crystal structure was used to derive the coordinates of
the other three identical jShak1 subunits. The jShak1 channel model was
tetramerized, with loop and interface regions refined. Finally, the model was
energy minimized again to remove any residue clashes or irregularities at the
subunit interfaces.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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G0G0 for the 12
channels (r2=0.99).
The gating charges for the wild-type channel and the 11 mutant channels
ranged from 2.3 to 3.5 with a mean of 2.9
(Table 1). This is considerably
lower than the typical estimate for the Drosophila Shaker channel
(12). However, the limiting slope analyses of the gating charge for each
of these channels yielded a plateau value (Fig. S3 in supplementary material),
indicating that these are accurate measurements of the amount of charge
involved in the gating process.
|
S2 mutants
As has been observed for other KV channels, jShak1 can
be stabilized in the open state through formation of a salt bridge between an
acidic residue at position 227 in S2 and positively charged residues in S4.
Replacing the neutral asparagine at position 227 with an acidic residue,
aspartate, (N227D) had no significant effect on the V50 of
activation (21.8 mV for N227D vs 27.8 mV for wild type,
P=0.16), the Boltzmann slope factor (P=0.09) or gating
charge (P=0.76) compared with the wild-type channel
(Fig. 3A,
Table 1). The N227D mutation
did not alter the equilibrium between open and closed states, since
G0 is not significantly different from 0
(P=0.19; Table 1).
|
36 mV) of the V50 of
activation to –8.6±3.9 mV
(Fig. 3A,
Table 1;
P=5.7x10–7). This mutation had no effect on
the Boltzmann slope factor (P=0.19). Although the difference in the
gating charge is statistically insignificant (P=0.06), the low
P value suggests that there may be a small underlying difference that
could not be resolved with the available data. The N227E mutation
substantially favoured the open state compared to the wild-type channel
(G0N227E=–10 kJ
mol–1; P=1.8x10–7). Since
this change in the gating charge was an insignificant decrease, it appears
that this mutation acts primarily by stabilizing the open conformation, not by
changing the nature of the motion of the S4 helix and the potential field
during channel opening.
S4 mutants
Triplet Insertions
Although the effect of three-residue motifs inserted into S4 in the
full-length channel were reported in an earlier study
(Grigoriev et al., 1997), we
have now characterized these mutations in the N-truncated channel to provide
more accurate measures of V50, slope factor and gating
charge that can be compared to the values measured for other channel mutants
that were also constructed as N-truncated channels. Although the slope values
were similar to the previously published values, the V50
values differed noticeably from those previously reported for the
N-inactivating channel (Fig.
3B, Table 1). The
differences can be attributed to the errors arising from calculating
conductance from peak current using an estimated potassium ion reversal
potential (Grigoriev et al.,
1997) as compared with directly measuring conductance from tail
currents, as in this study.
The QIF mutation (Fig. 3B)
shifts the channel behaviour to favour the open state relative to the
wild-type channel (G0QIF=–10.9 kJ
mol–1, P=1.5x10–9). The
gating charge of this mutant is slightly greater than that for the wild-type
channel (3.3 vs 3.0, P=5.7x10–4), in
spite of the fact that the third arginine in S4 has been converted to an
uncharged glutamine residue. The IFR insertion mutant does not significantly
affect G0 (P=0.5), whereas the RIF
insertional mutant shifts the channel towards the closed state relative to the
wild-type channel (G0=7.1 kJ
mol–1; P=0.0035;
Fig. 3B,
Table 1), and both the
mutations have gating charges indistinguishable from that of the wild-type
channel (P=0.58 and P=0.66, respectively).
|
G0QIF-D=–11.7
kJ mol–1 compared with
G0QIF+G0N227D=–13
kJ mol–1; P=0.61;
Table 2). This similarity in
G0 for the double mutant compared to the sum
of the G0 values for the single mutants
indicates that the N227D mutation in S2 and the QIF mutation in S4 act
independently and therefore additively on the channel.
|
The double mutant QIF+N227E (QIF-E) did not shift the
V50 of activation, the gating charge or the
G0 significantly relative to the QIF mutant
alone, (Fig. 4B;
P=0.66, P=0.065, P=0.96, respectively). Gibb's free
energy calculations show that QIF-E favoured the open state compared with the
wild-type channel (P=2.7x10–6). However, the
sum of Gibb's free energies for the single mutants was more negative than that
observed in the double mutant (P=4.6x10–4),
indicating that the combined QIF and the N227E mutations antagonized each
other's independent abilities to stabilize the open state.
IFR mutations
The double mutant combining IFR+N227D (IFR-D) had no significant effect on
the V50 of activation relative to that of the wild type
(Fig. 4C; P=0.43).
This double mutant favours the open state of the channel to the same extent as
the wild-type channel (P=0.88). This effect was equivalent to the
summed effect of the single mutations (P=0.76).
The V50 of activation for the double mutant IFR+N227E (IFR-E) was shifted in the hyperpolarized direction by approximately 33.6 mV compared with the IFR mutant alone (Fig. 4D; P=1.9x10–4). Notably, the Gibb's free energy value for the IFR-E double mutation did not differ from the sum of the free-energy values for independent IFR and N227E mutations (P=0.86) indicating that these mutations in S2 and S4 acted independently in the double mutant.
RIF mutations
The double mutant RIF+N227D (RIF-D) failed to alter either the
V50 or gating charge compared with the single RIF mutant
(Fig. 4E,
Table 1; P=0.84 and
P=0.79, respectively). Combining the RIF and N227D mutations did not
modify the channel's equilibrium more than would be expected from the sum of
the independent single mutations (P=0.57;
Table 2), indicating that the
two mutations are acting independently.
In comparison, the RIF+N227E (RIF-E) mutant had a hyperpolarizing shift of
14.6 mV compared with the RIF mutant alone
(Table 1;
Fig. 4F;
P=8.6x10–4). This double mutation did not
significantly affect the gating charge (P=0.52). Double mutant cycle
analysis shows that the two mutations did not act independently
(G0RIF-E=4.2 kJ mol–1
compared with
G0RIF+G0N227E=–2.9
kJ mol–1; Table
2; P=0.03), since the two mutations interfered with each
other so as to move the equilibrium towards the closed state.
Homology modelling of jShak1 on the RatKV1.2 template
The open-state jShak1 homology model gives a continuous representation of
the intracellular domain and transmembrane domain of the channel, minus the N
terminus and C terminus. Loop regions missing from the original
RatKV1.2 crystal structure have been rebuilt from loop databases to
give a continuous model of the transmembrane domain, from the start of the
T1-S1 linker to the end of the S6 helix. Analysis of the Ramachandran plot for
the hybrid KV1.2 structure shows that 99% of residues were in
favourable regions of the plot, compared with 94% of residues in favourable
regions in the jShak1 model. PROCHECK and WHAT IF protein structure checks
were performed on the open-state homology model and the original
KV1.2 crystal structure. The results showed that models were of
comparable quality to the template crystal structure. The backbone
root-mean-squared deviation (RMSD) between the jShak1 open-state homology
model and the RatKV1.2 crystal structure was 0.13 Å.
It is important to note that the RatKV1.2 channel was
crystallized in the absence of an intact membrane (i.e. equivalent to a 0 mV
transmembrane potential), and so it represents the open conformation of the
channel since the V50 for RatKV1.2 has been
reported to be –17 mV (Scholle et
al., 2000).
Fig. 5A shows the alignment
of the S3-linker-S4 portion of the two structures. The RatKV1.2
linker is large and flexible and includes a helical region, as predicted from
the mutagenesis analysis by Mathur and others
(Mathur et al., 1997). The
jShak1 linker is short, nearly the length of a fully extended polypeptide, and
thus appears to anchor the C-terminal end of S3 to the N-terminal end of S4
during channel transitions between the open and closed state. The strong
similarities in the structures of the S4 helices and the L4-5 linkers in the
open states of these channels is illustrated in
Fig. 5B. When the membrane is
depolarized, translocation of the S4 helix is coupled to lateral movement of
the L4-5 segment that pulls the intracellular ends of the S5 and S6 helices
apart, opening the activation gate, allowing K+ flow through the
pore. Because of this essential function, it is expected that the three
mutations that insert a tripeptide into the S4 helix will distort the
conformation of the channel by displacing the N terminus of the S4 helix in an
outward (extracellular) direction, rather than disrupting the conformation of
L4-5. The length, shape and conformation of the S3-S4 linker has been shown to
affect voltage sensitivity (Gonzalez et
al., 2001; Mathur et al.,
1997), suggesting that in jShak1 increasing the length of the S4
helix might affect voltage sensitivity by creating a voltage sensor with
greater internal tension that alters the equilibrium between the open and
closed state, independent of charge content.
|
The alignment of the S2 helices (Fig. 6A) illustrates the positional equivalence of RatKV1.2 E226 and jShak1 N227 and of RatKV1.2 E236 and jShak1 E237, as shown in the alignment in Fig. 1B. The alignment of the S4 helices shows that the alignment of the S4 helices of RatKV1.2 and jShak1 are consistent with the alignment shown in Fig. 1B, with the exception that the SML tripeptide at the N-terminal end of the jShak1 S4 is part of the helix, and not part of the connecting linker (Fig. 6B).
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|
DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Li-Smerin et al.,
2000; Monks et al.,
1999; Perozo,
2000). In double mutant cycle analysis the
G0 values for two, independent single
mutations are compared with the observed G0
value for the double mutant channel. If the difference between the double
mutant and the summed G0 for the single
mutants is zero
[G0double–(G0mut1+G0mut2)=0],
then the single mutations are acting independently in the double mutant
channel (Horovitz, 1996).
Conversely, if there is a difference between the double mutant and the sum of
the single mutations
[G0double–(G0mut1+G0mut2)
0]
then the residues at the two positions are energetically coupled, either
through direct or indirect interactions. Differences in
G0 provide a measure of the strength of
pair-wise interactions between sites of mutations
(Horovitz, 1996). In our
study, if the difference between G0double
and
G0mut1+G0mut2
was greater than zero then the interaction between the two residues tended to
stabilize the closed conformation whereas a difference less than zero
indicated that the interaction between the two mutated residues tended to
favour the open conformation.
jShak1 has a number of structural features that make it useful for probing
structural interactions involved in stabilizing the open and closed
conformations of VKCs. The S2 transmembrane helix of jShak1 contains a single
acidic residue (E237) that is conserved in other KV1 channels, but
lacks a second acidic residue that is present in most other KV1
channels, being an asparagine in jShak1 (N227)
(Fig. 1B). The S4 voltage
sensor is shorter by a single triplet motif than other KV1
channels, containing 6 basic residues corresponding to R365-R380 in
Drosophila Shaker (Fig.
1B). Modelling indicates that the length of S4 is similar to other
KV1 channels, but the Ser-Met-Val (SML) amino acid sequence
replaces the N-terminal basic motif, so although the voltage-sensing element
is shorter, the total S4 helix is similar in length to that in other channels.
Finally, the S3-S4 linker in the wild-type jShak1 channel is the shortest
reported in any KV channel, being composed of only three amino
acids. This constrains the intramolecular distance between the S3 and S4
helices, such that small modifications to amino acid length (aspartic acid
vs glutamic acid) and relative packing in this region (IFR
vs RIF) can be observed to directly influence the internal energetics
of the channel. All of the jShak1 mutants, including those with the tripeptide
insert, open in response to voltage, demonstrating that both the voltage
sensor and linker are able to move during activation. However, changes in the
length, shape and conformation of the S3-S4 linker have been shown to affect
voltage sensitivity in Drosophila Shaker
(Mathur et al., 1997).
We determined a relatively small (2.9e) gating charge for jShak1 and
all of its mutants. This is much smaller than the total gating charge
determined for the Drosophila Shaker channel (12–13 e),
but other channels, e.g. Drosophila Shab (7.5 e)
(Islas and Sigworth, 1999) and
human herg (6 e) (Zhang et al.,
2004) show that a wide range of gating charge values can support
physiologically relevant voltage sensitivity. The number of gating charges is
a function of the total displacement of the charged residues in the
transmembrane potential field, so a lower gating charge could arise from (1)
fewer charges in the voltage sensor, (2) less displacement of the voltage
sensor charges through the potential field or (3) a wider, more diffuse
potential field such that the charges traverse a smaller proportion of the
potential field. It is noteworthy that a synthetic mutation that shortens the
loop connecting S3 and S4 in the Drosophila Shaker channel causes a
decrease in gating charge from 12 e to approximately 5 e
(Gonzalez et al., 2000); as
noted above, the jShak1 channel has a very short loop connecting S3
and S4.
The energy required for channel opening is a function of the product of the
V50 and the number of charges that move during the opening
transition. Thus, the mutations that are described here could have caused
changes in the energy required for opening through two distinct effects;
changing the amount of charge translocated through the membrane potential
field, and changing the internal energy difference between the open and closed
state. The gating charge values were changed little by the various mutations
and there was little correlation between the values of the gating charges and
the V50 values; however, the V50
values were highly correlated with the G0 values.
Thus, the difference between the internal energies of the open and closed
states of the channels is the primary factor that is affecting
V50 in this set of mutations. The mutations are not
appreciably affecting the underlying mechanics of the gating process.
Effects of single mutations in S2
Substitution of N227 with aspartate did not have a significant effect on
the channel, whereas substitution of N227 with glutamate shifted the channel
equilibrium strongly toward the open state. This indicates that simply
converting the neutral asparagines to negatively charged aspartate at this
position does not lead to formation of a strong salt bridge, but that the
increased length of an additional methylene group in the side chain (a length
of 1.5 ångströms) in the glutamate residue, does lead to
significant stabilization. However, the energy difference between the
wild-type and the N227E mutant is only –10.5 kJ mol–1,
approximately half of what would be expected from a fully formed salt bridge
(Kumar and Nussinov, 1999).
The most straightforward interpretation of this result is that the S2 and S4
helices in jShak1 are strongly constrained from moving towards each other,
thus physically constraining the extent of interactions between the
side-chains of adjacent helices. The additional length in the N227E side-chain
allows the negatively charged carboxylate group to be closer to positively
charged S4 residues in the open state, thus yielding a more stable open state
than the shorter side-chain of the N227D mutation, but still not at the energy
minimum for a fully formed salt bridge.
Effects of S4 triplet insertion mutations
Insertion of RIF upstream of K294 produced a channel in which the energy
difference between the closed state and the open state was +15.1 kJ
mol–1, which is +7.1 kJ mol–1 more than for
the wild-type channel. We hypothesize that this difference can be attributed
to increased resistance to translocation of the longer S4 helix in the RIF
mutant, because of constraint by the short S3-S4 linker.
Insertion of QIF upstream of K294 produced a channel in which the energy
difference between the closed state and the open state was –2.9 kJ
mol–1, which is 10.9 kJ mol–1 less than for
the wild-type channel. Since the effect of a longer S4 helix should be the
same in QIF and RIF, the implication is that the glutamine residue in QIF is
causing an approximately 18 kJ mol–1 destabilization of the
closed state relative to the open state. This energy difference is
quantitatively similar to the typical stabilizing energy of a salt bridge
(Kumar and Nussinov, 1999).
Since the homologous residue in Drosophila Shaker is known to
interact with charged residues in S2 and S3 in the closed state
(Papazian et al., 1995), this
energy difference appears to be due to the absence of a salt bridge formed
between the inserted glutamine residue and one or both of residues E237 and
D260 (see Fig. 1) in the closed
state of the QIF mutant. Thus, stabilization of the closed state due to the
increased length of S4 is more than offset by the loss of charge interactions
that also stabilize the closed state.
|
Interactions between mutations in S2 and S4
There is no significant difference in G0 among
the leftward-shifted channels, QIF, QIF-D and QIF-E. This suggests that the
wild-type, N227E or N227D S2 helices had comparable interactions with the S4
helix. This seems reasonable since the arginine that normally interacts with
the amino acid at position 227 has been replaced by glutamine, preventing a
salt bridge forming between these two residues in the open state. However, the
sum of the single G0 for QIF and N227E
mutations predicted a greater stabilization of the open state
(G0QIF+G0N227E=–21
kJ mol–1) than was observed in the double mutant channel
(G0QIF-E=–10.9 kJ
mol–1) indicating an interfering interaction between the two
mutations, whereas a simple additive interaction was observed for the QIF-D
double mutant. Since the difference between the QIF-D and QIF-E double mutants
is that the side-chain of the acidic residue at position 227 has one more
methylene group in the glutamate than in the aspartate, this may indicate that
the S2 and S4 helices are packed so close together that the steric
interference caused by the additional methylene group causes more
destabilization than is gained by any polar interaction between the glutamate
carboxyl group at position 227 and the glutamine side chain. This is in
contrast to the effect of the N227E mutation on the wild-type channel,
suggesting that the addition of the QIF motif to the S4 helix has caused it to
be packed much closer to the S2 helix in the open state than is the case for
the wild-type S4 helix.
In the IFR double mutants, the presence of acidic S2 residues also favoured
the open conformation of the channel. The IFR-E mutation, with the longer
glutamate side-chain shifted the half activation voltage leftward by 29
mV as compared to the IFR-D mutation, which had an insignificant effect when
compared with the wild type. Double mutant cycle analysis showed that both the
N227D and the N227E mutation act independently with the IFR mutation. This
means that insertions of triplet motifs at different locations in the S4 helix
are having different effects on the way that S4 is interacting with S2.
Although the RIF insert in S4 shifted the equilibrium to favour the closed state, and the RIF-N227D double mutant was functionally indistinguishable from the RIF mutant alone, the RIF-N227E mutant, with the longer acidic residue side-chain, shifted the equilibrium towards the open state compared with the RIF single mutant alone. The RIF mutation effect was independent of the N227D mutation, but the RIF mutation had an interfering interaction with the N227E mutation, similar to, but smaller in magnitude than, the interfering interaction between the QIF mutations and the N227E mutation.
These results can be explained in terms of three factors that affect the relative stability of the open and closed states, and hence the voltage sensitivity, of the channel (Fig. 7). First, the insertion of three amino acids in the S4 helix tends to stabilize the closed state relative to the open state. Since the interaction of S4 with the S4/S5 linker is conserved and central to the mechanism of channel opening and the S3/S4 linker is so short that it restricts the relative motion of S3 and S4, we infer that the increased length of S4 is creating internal constraint on S4 that resists its motion in the transition from the open to the closed state, thus causing the rightward shift in voltage sensitivity.
The second factor that affects the relative stability of the open and the
closed state is the interaction between the amino acid residue at position 291
and the two acidic residues in S2 and S3 in the closed state
(Papazian et al., 1995). In
the case of the QIF insertion mutant, stabilization of the closed state is
offset by the fact that the neutral glutamine residue is in a location that is
normally occupied by a positively charged arginine residue. In the wild-type
channel this arginine residue interacts with the acidic residues in S2 and S3,
helping to stabilize the closed conformation
(Fig. 7A). However, in the QIF
mutant the substituted glutamine residue cannot form these charge interactions
(Fig. 7C), thus destabilizing
the closed state and causing a significant leftward shift in the
V50 value, in spite of the constraint on movement caused
by the short S3/S4 linker.
The third factor affecting the relative stability of the open and closed
conformations is the interaction between the residue at position 291 and the
residue at position 227 in the open state. In the case of the wild-type S4
(Fig. 7B), and the RIF and IFR
insertions in S4, mutation of N227 to aspartate and glutamate both decrease
the G0 of the channels, although to different
extents. In all three cases the N227E mutation has a larger effect, probably
as a result of the side chain being one methylene group longer than that of
aspartate, thus allowing the positive charge at position 291 and the negative
charge at position 227 to interact more strongly. In the case of the QIF
double mutations, the N227D mutation yields a small decrease in
G0 but the N227E mutation yields no change over the
QIF mutant alone. We hypothesize that this is because of a steric crowding
effect of the longer glutamate side-chain with the glutamine at position 291,
which is not offset by any electrostatic energy gain, since there is no
positive charge on the glutamine side chain
(Fig. 7D).
In the absence of high-resolution structures of the ion channel of interest, homology models provide a three-dimensional map of a protein. They are structural predictors, which are highly dependent on the accuracy of the underlying sequence alignment and, as such, require progressive refinement as new results become available. In the case of the jShak1 open-state homology model, secondary structure predictions and loop database searches were used to bridge the gaps in the RatKV1.2 template structure, allowing the development of a more complete homology model.
A homology model, like the crystal structure on which is based, represents a single, state-dependent snapshot of jShak1. Although the open-state jShak1 model described here is a true homology model, there is no crystal structure of a voltage-gated channel in a closed state on which to base a comparable model.
Thus, to investigate and understand the details of the molecular mechanics of the transition between closed to open states in responses to changes in membrane potential it is valuable to use the structural model as a heuristic framework for formulating hypotheses that can be tested by mutagenesis. In turn, electrophysiological characterization of well-designed mutant channels provides dynamic data that can inform subsequent rounds of modelling and hypotheses about the detailed molecular mechanisms of channel opening and closing.
The model of the S2 and S4 helices (Fig.
6C) illustrates a basis for the difference between the wild-type
and N227E mutant of jShak1. In the wild-type channel
(Fig. 6C) the N227 residue is
not in close proximity to any of the basic residues of S4, whereas in the
RatKV1.2 model (Fig.
6C) the glutamate residue (E226) at the position homologous to
E227 in jShak1 is very close to one of the arginine residues (R303) in the S4
helix; this interaction would be expected to stabilize the open configuration
of the channel, by virtue of a salt bridge, and thus yield a
V50 value considerably more hyperpolarized than that for a
channel without an acidic residue in that position in S2. In this homology
model, the channel is in the open conformation, and the residues that are
thought to stabilize the closed state by forming salt bridges are not in close
proximity. It is clear that the process of channel closing will require
rotation and translocation of all three helices to form the predicted
interaction network between residues E237, D260 and positive residues in S4 of
jShak1, as described for Drosophila Shaker
(Papazian et al., 1995).
LIST OF ABBREVIATIONS
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Acknowledgments |
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Footnotes |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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