1Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, Health Sciences Centre, Calgary, Alberta T2N 4N1; and 2Department of Biological Sciences, The University of Alberta, Edmonton, Alberta T6G 2E9 and Bamfield Marine Station, Bamfield, British Columbia V0R 1B0, Canada
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ABSTRACT |
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Grigoriev, Nikita G.,
J. David Spafford, and
Andrew N. Spencer.
Residues in a Jellyfish Shaker-Like Channel
Involved in Modulation by External Potassium.
J. Neurophysiol. 82: 1740-1747, 1999.
The jellyfish gene,
jShak2, coded for a potassium channel that showed
increased conductance and a decreased inactivation rate as
[K+]out was increased. The relative
modulatory effectiveness of K+, Rb+,
Cs+, and Na+ indicated that a
weak-field-strength site is present. Cysteine substituted mutants
(L369C and F370C) of an N-terminal truncated construct,
(jShak22-38) which only showed C-type inactivation, were used to establish the position and nature of this site(s). In
comparison with jShak2
2-38 and F370C, L369C showed a
greater relative increase in peak current when
[K+]out was increased from 1 to 100 mM
because the affinity of this site was reduced at low
[K+]out. Increasing
[K+]out had little effect on the rate of
inactivation of L369C; however, the appearance of a second, hyperbolic
component to the inactivation curve for F370C indicated that this
mutation had increased the affinity of the low-affinity site by
bringing the backbone oxygens closer together. Methanethiosulphonate
reagents were used to form positively (MTSET), negatively (MTSES), and
neutrally (MTSM) charged side groups on the cysteine-substituted
residues at the purported K+ binding site(s) in the channel
mouth and conductance and inactivation kinetic measurements made. The
reduced affinity of the site produced by the mutation L369C was
probably due to the increased hydrophobicity of cysteine, which changed
the relative positions of carbonyl oxygens since MTSES modification did
not form a high-field-strength site as might be expected if the
cysteine residues project into the pore. Addition of the side chain
-CH2-S-S-CH3, which is similar to the side
chain of methionine, a conserved residue in many potassium channels,
resulted in an increased peak current and reduced inactivation rate,
hence a higher affinity binding site. Modification of cysteine substituted mutants occurred more readily from the inactivated state
confirming that side chains probably rotate into the pore from a buried
position when no K ions are in the pore. In conclusion we were able to
show that, as for certain potassium channels in higher taxonomic
groups, the site(s) responsible for modulation by
[K+]out is situated just outside the
selectivity filter and is represented by the residues L369
and F370 in the jellyfish Shaker channel,
jShak2.
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INTRODUCTION |
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Various voltage-gated potassium channels are
modulated by the external potassium ion concentration,
[K+]out
(Baukrowitz and Yellen 1995; Lopez-Barneo et al.
1993
; Pardo et al. 1992
; Safronov and
Vogel 1996
;Tseng and Tseng-Crank 1992
). Recently
we were able to demonstrate that a Shaker-like,
high-threshold potassium channel (
-subunit), encoded by a gene
jShak2 cloned from the jellyfish Polyorchis
penicillatus, when heterologously expressed in Xenopus
oocytes, also demonstrates strong modulation of an A-like current by
[K+]out (Grigoriev
et al. 1999a
,b
). Increasing
[K+]out increases the
potassium conductance of jShak2 and simultaneously decreases
the inactivation rate. Both these effects are associated with C-type
inactivation (Grigoriev et al. 1999a
,b
). A possible physiological role for such modulation is to compensate for the effect
of potassium ions accumulating in extracellular space during repetitive
firing and leading to a change in the potassium equilibrium potential.
We were interested in examining the mechanism of this modulation by
determining the site(s) responsible in the channel vestibule. Because
this is an evolutionarily early channel, it is more likely to have
retained the ancestral mechanisms, without superimposed
specializations, than K+ channels from more
derived phyla.
It is well established that the mouth of the aqueous pore of potassium
channels as well as the selectivity filter are formed by the P-region
(Doyle et al. 1998; Heginbotham et al.
1994
; Lipkind et al. 1995
), which is
structurally conserved among most of the voltage-gated
K+ channels (Chandy and Gutman
1994
). C-type inactivation of Shaker channels
appears to be associated with specific residues in the P-region
(Baukrowitz and Yellen 1995
; Lopez-Barneo et al.
1993
). Mutation of threonine449
dramatically alters the rate of C-type inactivation of
Shaker mutants and the channel's sensitivity to changes in
external potassium. The presence of charged residues such as lysine or
glutamate at this site increases the channel's sensitivity to
[K+]out, and the rate of
C-type inactivation. The lipophilic residues, tyrosine or valine at
position 449 render the channel insensitive to changes in
[K+]out and cause slow
inactivation (Lopez-Barneo et al. 1993
). Despite the
homologous position being occupied by phenylalanine, which is aromatic
and lipophilic, jShak2 demonstrated fast C-type inactivation and a high sensitivity to
[K+]out.
jShak2 also carries leucine369 in a
position occupied by methionine in most known voltage-sensitive channels. To elucidate the role of these residues in sensing
[K+]out, we tested two
jShak2 mutants: L369C and F370C.
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METHODS |
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Molecular biology
All jShak2 mutants were constructed using cassette,
PCR-based, site-directed mutagenesis as described previously
(Grigoriev et al. 1997). Mutants were verified by
sequencing in both directions using a Perkin-Elmer ABI 373A sequencer
and an ABI Prism Dye-Terminator Cycle Sequencing Kit. Construction of
the mutant jShak1
2-24 (jShak1T) was described
by Jegla et al. (1995)
. Capped mRNAs were prepared by
run-off transcription using a T7 mMessage mMachine kit (Ambion).
Whole cell, two-electrode recording from Xenopus oocytes
Xenopus oocytes were prepared and injected with mRNA
as previously described (Grigoriev et al. 1997). From 1 to 5 ng of mRNA were injected in each oocyte using a volume of 50 nl.
The amount of injected RNA was adjusted for each expressed channel type
to minimize the experimental artifacts introduced by a high level of
channel expression (Grigoriev et al. 1999a
). Whole cell
currents were recorded between 2 and 3 days after injection using a
two-microelectrode voltage clamp (CA-1, Dagan Corporation). Cells were
constantly microperfused either with a gravity-fed system or
computer-controlled pneumatic syringe pumps when a stable perfusion
volume (3 ml min
1) was required, such as with
the experiments using MTS reagents.
The potassium-free extracellular solution, [K+]out = 0, contained 100 mM NMG-Cl, 3 mM MgCl2, 10 mM HEPES-acid adjusted to pH 7.5 with NMG-Cl; while the solution, [K+]out = 100 mM contained KCl 95 mM, MgCl2 3 mM, 10 mM HEPES1/2K, pH 7.5. Intermediate concentrations of K+ were made by mixing these two solutions in the required proportion. The MTS reagents, MTSES, MTSET, and MTSM were obtained from Toronto Research Chemicals (Toronto, Ontario, Canada) and were dissolved in extracellular solution at a concentration of 500 µM and kept on ice. Reservoirs were loaded with the appropriate MTS reagent solution immediately before each modification experiment. Tetraethylammonium (TEA), tetramethylammonium (TMA), tetrapropylammonium (TPA), and tetrabutylammonium (TBA) chloride salts were obtained from Sigma-Aldrich. Experiments were carried out at 20°C using a temperature controller TC-10 (Dagan Corporation, Minneapolis, MN).
Data acquisition and experimental control
All data acquisition and experimental control was through a
Digidata 1200 (Axon Instruments, Foster City, CA) acquisition system
running pClamp 6.1 software. For most of the recordings currents were
elicited by depolarizing pulses from a holding potential 80 to +65
mV, with a duration of 100 ms. Analysis and fitting of experimental
data were done using the Clampfit program in pClamp 6.1 (Axon
Instruments) and SigmaPlot 4.00 (SPSS, Chicago, IL). Fitting of
[K+]out-effect curves
were done by using a combination of an equation similar to a
Michaelis-Menten relationship, and a linear relationship: X = [Xmax *
[K+]out/(KappX + [K+]out)] + [K+]out *
CX (Eq. 1) where
Xmax is the maximal parameter [either
conductance (g) or inactivation time constant (
) at
[K+]out = 100 mM],
KappX is the apparent constant of the hyperbolic
component, and CX is the slope of the linear
component. Conductance (g) was calculated for whole oocyte
recordings using intracellular potassium activity at 147 mM as reported
by Kusano et al. (1982)
. For g versus
[K+]out curves
conductances were normalized to g at
[K+]out = 100 mM. The
inactivation time constant (
) was determined by fitting inactivation
of the current traces with exponents. The
versus
[K+]out curves obtained
for the F370C mutant were optimally fitted with an equation having two
hyperbolic components:
= [
max1 *
[K+]out/(Kapp1
+[K+]out)] + [
max2 *
[K+]out/(Kapp2
+[K+]out)]
(Eq. 2). The g versus
[K+]out curve for the
MTSET modified L369C mutant was optimally fitted with a single
hyperbolic function: g = gmax1 *
[K+]out/(Kapp1g
+[K+]out) (Eq.
3). All results are expressed as means ± SE.
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RESULTS |
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The jellyfish Shaker channel jShak2 does not
conduct in the absence of external potassium (Fig.
1A, inset)
(Grigoriev et al. 1999a,b
). Peak amplitude of
jShak2 current is increased (termed the "amplitude
effect") and the inactivation rate decreased as [K+]out is increased
(Fig. 1). This modulatory effect was shown to be associated with a
C-type inactivation mechanism (Grigoriev et al. 1999b
).
Other monovalent cations produced qualitatively similar effects on
jShak2 current, but with substantially lower efficiency than
potassium ions (Fig. 1A). Their effectiveness at modulating
both current amplitude and inactivation kinetics (Fig. 1B)
could be ranked as follows: K+ > Rb+ > Cs+ > Na+. This order corresponds to the IVth Eisenman
sequence for equilibrium ion exchange and may indicate the presence of
a comparatively weak-field-strength binding site for monovalent cations
(Eisenman 1961
; Hille 1992
). As for many
K+ channels, Cs+ blocked
jShak2 channels when applied to the cytoplasmic side of the
membrane (data not shown), which ruled out the possibility that the
sensor for external [K+] is associated with the
selectivity filter in the pore of jShak2.
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Inactivation of jShak2 channels is predominantly of the
C-type, whereas N-type inactivation is very weak and can be completely eliminated by N-terminal sequence truncation (Grigoriev et al. 1999b). To observe C-type inactivation uncontaminated by N-type inactivation, P-region mutants (Fig. 2)
were constructed in truncated jShak2
2-38. Both these
mutants, L369C and F370C, retained external potassium sensitivity (Fig.
3, A and B).
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For L369C the peak current increased dramatically in response to
elevation of the extracellular potassium concentration from 1 to 100 mM
(Fig. 3A), which could be accounted for by the lower affinity of this mutant to potassium ions at low concentrations. The
conductance versus
[K+]out curve for L369C
was less steep (Kappg increased 8-fold, from 2 to 16 mM) than for jShak22-38 (Fig. 3C). There was
only a moderate decrease in the inactivation rate for L369C (Fig. 3,
A and D) without any change in
Kapp
. The rate of recovery from C-type inactivation
for L369C was slightly less (
= 150 ms) than for Wild
jShak2
2-38 (
= 415 ms; Fig. 3E).
The potassium sensitivity of the amplitude effect in F370C remained
virtually unchanged relative to jShak22-38 (Fig.
3C). Unlike L369C, F370C showed a very different sensitivity
of inactivation to
[K+]out with two
components appearing, the first had a
K
app around 1 mM, and the
second component had a K
app
of 30 mM (Fig. 3D). It is likely that the appearance of a second hyperbolic component and the disappearance of the linear component represents an increased K+ affinity of
the low-affinity site, which, before mutation, is represented by the
linear portion of the curve. Recovery from inactivation for F370C was
slowed dramatically (>27-fold) relative to jShak2
2-38
(Fig. 3E).
We were able to use the substituted cysteine accessibility method
(SCAM) (Akabas et al. 1994) to evaluate the
accessibility of residues in positions 369 and 370. In addition, this
method allowed us to examine their state-dependent reactivity using
thiol-specific methanethiosulphonate reagents (MTS). Treatment of
cysteine with these reagents enabled us to attach functional side
groups through disulphide bonds. The reagent MTSET attached a group
carrying positive charge, whereas MTSES introduced a negative charge,
and MTSM added an uncharged group. Two of the reagents, MTSET and MTSES, did not affect the peak current of jShak2
2-38,
whereas MTSM treatment reduced current by 23% (Figs. 3F and
4A). We assume that the six
endogenous cysteine residues located on membrane-spanning and
cytoplasmic regions of jShak2
2-38 are inaccessible
from the outside because the hydrophilic reagents MTSES and MTSET had
no effect. However, the small inhibitory effect of MTSM could result from its lipophilicity (Fig. 3F).
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Modification of F370C with MTSET and MTSM suppressed the current almost completely, whereas MTSES did not produce a strong effect on current amplitude (Fig. 3F) or inactivation rate (data not shown). MTSES treatment of F370C did not produce significant changes in potassium sensitivity while the residual currents that remained after F370C modification with MTSM and MTSET reagents were too small for reliable quantification of their potassium sensitivity (data not shown).
In contrast, both MTSM and MTSES treatment of L396C increased conductance (Fig. 4B) and slowed inactivation (Fig. 4C), whereas MTSET had the opposite effect. All three reagents modified the cysteine-substituted mutants within several seconds after initiating perfusion with MTS reagents. However, rates of return to the unmodified state were very slow, which allowed us to test the potassium sensitivity of channels that remained modified after removal of MTS reagents from the external solution.
The potassium sensitivity of L369C mutants was dramatically altered as
a result of cysteine modification (Fig. 4, B and
C). Positively charged MTSET reduced the potassium
sensitivity of channel conductance, making Kappg five
times greater (17 to 90 mM) when compared with unmodified L369C (Fig.
4B). This was accompanied by a slight overall decrease in
the steepness of the
/[K+]out curve, which
is most noticeable in the linear component of these curves (Fig.
4C). Modification of L369C by negatively charged MTSES and
neutral MTSM increased the sensitivity of whole cell conductance to
[K+]out making the
Kappg value similar to that for
jShak2
2-38. The Kappg after modification
by MTSES was 1.45 ± 0.3 mM (mean ± SE, n = 5), and by MTSM it was 1.0 ± 0.3 mM (n = 5);
although this value for jShak2
2-38 was 2.1 ± 0.2 mM (n = 7). MTSES and MTSM treatment also slowed
inactivation (Fig. 4, A and C) and made
inactivation more sensitive to potassium (Fig.
4C). None of these modification agents
appreciably affected the rate of recovery from inactivation (data not shown).
We examined the sensitivity of L369C to various monovalent
cations to determine if modification by MTSES altered the relative effectiveness of these ions in modulating K+
current amplitude (Fig. 4E). Ions with atomic radii closer
to that of potassium showed the greatest effect on peak current
amplitude. Addition of a negatively charged group to L369C did not
alter the order of the permeability sequence, but modification did
alter the relative effect of these ions so that the sequence
K+ Rb+ > Cs+ > Na+
Li+ became
K+ > Rb+ > Cs+ > Na+
Li+. Thus this sequence shows a
trend toward an Eisenman III sequence, which implies a weaker
field-strength site than in the unmodified channel. This conclusion
appears to contradict an obvious explanation that the increase in the
apparent affinity of this mutant is a result of the stronger
electrostatic interaction of K+ with the negatively charged
group acquired by cysteine369 after modification with MTSES.
Liu et al. (1996) demonstrated that for
Shaker channels, residues in homologous positions to
methionine448 and threonine449 became
preferentially available for modification in their inactivated state.
Modification of both L369C and F370C was slow (from 3 to 5 M
1s
1) in the closed state (data not shown).
However, the rate of modification was increased when the probability of
channels being in an inactivated state was increased. This is seen in
Fig. 4D, which shows that the rate of modification of
L396C by MTSES using 10-ms depolarizing test pulses (5.5 ± 1.5 M
1s
1, n = 5) was
indistinguishable from the rate in the closed state. However, long
100-ms pulses caused far more rapid (119.6 ± 10 M
1s
1, n = 6)
modification. If MTSES interacted preferentially with channels in their
open state, then one would expect a 5-fold increase (estimated by
integration of current during the pulse) in the modification rate seen
with 100-ms pulses relative to 10-ms pulses. Instead, we recorded a
20-fold increase in the rate. We also observed a somewhat slower rate
of modification at high concentrations of
[K+]out (data not shown). The slow recovery
from inactivation of F370C (Fig. 3C) did not allow us to
use the same stimulus protocol as for L369C (>0.05 Hz), and when we
used a lower frequency of stimulation we could not discriminate between
the inactivated and open states. Nevertheless, we obtained
qualitatively similar results for modification of
cysteine370, indicating that this residue became
preferentially available in the open and/or inactivated states but not
the closed state.
Introduction of the aromatic residue, phenylanine, in the
P-region of Shaker channels (mutant T449F) makes it
highly sensitive to TEA due to the formation of a bracelet of
pore-lining aromatic residues (contributed by each subunit) in the
channel mouth, which provides a structure that favors interaction with
flat, symmetrical molecules such as TEA (Heginbotham and
MacKinnon 1992). Administration of TEA to
jShak2
2-38 channels caused partial channel block and a decrease in the inactivation rate (Fig.
5A), which can be
explained by assuming that TEA in the channel mouth prevents potassium
ions from passing through the pore while simultaneously preventing transition of the channel to an inactivated state (Grissmer and Cahalan 1989
). As expected, the mutant (F370C), where
phenylalanine had been replaced by cysteine at a homologous position,
showed reduced sensitivity to TEA (Fig. 5B) when
compared with the Wild, N-terminal deleted mutant
(jShak2
2-38). Among all quaternary ammonium (QA)
ions, TEA had the most noticeable effect on peak current and
inactivation rate (Fig. 5C). The smallest ion, TMA, had
no effect on either peak current or inactivation, whereas the larger
ions, TPA and TBA, showed some blocking effect, but inactivation was
affected minimally (Fig. 5C). TPA and TBA had no effect
on current amplitude or inactivation rate at concentrations of 10 mM or
less (data not shown). The sensitivity of jShak2 to TEA
blockade (Kd = 1.2 mM) (Jegla et
al. 1995
) is lower than that reported for Shaker
T449F (Kd = 0.35 mM), indicating
that the relative positions of the aromatic rings in
jShak2 are less favorable for the formation of a
high-affinity receptor. Comparison of the blocking effects of other
quaternary ammonium ions (TMA, TPA, and TBA) with TEA showed that this
site is most specific for TEA (Fig. 5C). The smaller
ion, TMA, had no effect, whereas the larger molecules, TPA and TBA,
blocked at a lower efficiency. A similar profile of blocking by QA ions
was reported for Shaker T449F (Heginbotham and
MacKinnon 1992
), indicating that the relative positions of the
four phenylalanine residues in the channel mouth of this mutant is
similar to that for jShak2.
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DISCUSSION |
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This study indicates that residues L369 and
F370 participate in the formation of site(s)
responsible for the modulatory effect of [K+]out in an
evolutionarily early voltage-gated potassium channel. Occupancy of
these sites by potassium decreases the probability of C-type
inactivation occurring by a "foot in the door mechanism" (Grigoriev et al. 1999b; Labarca and
MacKinnon 1992
; Lopez-Barneo et al. 1993
).
According to Eisenman's theory (Eisenman 1961
), the
observed selectivity sequence for monovalent cations in
jShak2 indicates that a low-field-strength binding site
for K+ is present at or close to the selectivity filter. A
model of the pore suggested by Lipkind et al. (1995)
shows that potassium ions interact with four carbonyl oxygens of the
peptide backbone of the hairpin structures formed by four P-region
sequences lining the pore. A recent study using X-ray analysis of the
structure of the potassium channel pore from Streptomyces
lividans clearly demonstrated that main chain carbonyl oxygen
atoms line the selectivity filter (Doyle et al. 1998
).
The weak nature of the binding site is created by low energy
interactions between permeable ions and oxygen atoms. The narrowest
pore section formed by carbonyl oxygens occurs within the conserved GYG
sequence (Heginbotham et al. 1994
) and is the presumed
selectivity filter (Fig. 6). Exterior to
the selectivity filter, the vestibule becomes wider and the interaction of K+ with other potential binding sites becomes weaker.
The oxygen rings of the GDL sequence form a less narrow opening that
presumably reduces affinity for K+, but this site is able
to hold the larger impermeable ion, Cs+. The section of the
pore formed by L and F has even less probability of interacting with
potassium, or other monovalent cations and therefore may be a possible
candidate for the sites with lowest affinity. The mutation L369C
produced a lower affinity site than in the Wild protein. If we assume
that side chains project into the channel lumen, then this lowered
affinity could be due to reduced steric hindrance of K+ by
the shorter side groups of cysteine. Alternatively, the lower hydrophobicity of cysteine alters interactions between residues in the
hairpin structure (
and
angles of rotation of peptide backbone),
which, in turn, changes the position of wall-forming carbonyl oxygens
and decreases the strength of the potassium-oxygen interaction.
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MTSES modification of side chains that project into the pore should
provide a structure that forms a high-field-strength binding site
because of the introduction of strong negative charge. This appears not
to be the case because adding a strong binding site should alter both
the order of the modulatory effect produced by different monovalent
cations and produce a shift to a higher Eisenman sequence. However, the
Eisenman sequence remained at IV, and even showed a trend toward the
IIIrd sequence, indicating a weakening of the purported site. These
results enable us to rule out the possibility that the cysteine side
chains of L369C project into the pore during ion passage. Therefore
changes in the relative positions of carbonyl oxygens as a result of
conformational changes in the hairpin remains the most plausible
explanation for changes in sensitivity to
[K+]out produced by mutation L369C, as well
as changes evoked by its modification using MTS reagents. It is
interesting that modification of L396C with MTSM produces side chains
with the structure, -CH2-S-S-CH3, which
enhances the apparent affinity of the modulatory sites to potassium.
This side-chain construct resembles the side chain of methionine,
-CH2-CH2-S-CH3, which is conserved
in jShak1 and many other potassium channels. It is
possible that the presence of such side-chain structure is optimal for
formation of high affinity potassium sites by placing backbone oxygens
in positions that favor high-affinity binding. If it can be assumed
that the side chains of C369 and C370 are able
to interact with neighboring side chains, then the positioning of
backbone oxygens also explains the differences noted in the modification profiles of L369C and F370C (Fig. 3F). For
example, it is likely that the absence or presence of negatively
charged D368 in proximity to side chains of
C369 and C370 will affect and
angles of
rotation of the peptide backbone, which, in turn, will change the
relative positions of carbonyl oxygens. Pascual et al.
(1995)
also observed different profiles after modifying
cysteines in positions homologous to L369C and F370C in Kv2.1 channels
with MTS reagents carrying different functional groups. This hypothesis
presents a paradox because the side chains of C369 and
C370 should be buried to prevent interaction between them
and potassium ions, yet they are accessible for modification by
hydrophilic MTS reagents. This paradox can be resolved if we assume
that side chains can rotate in the pore when potassium is absent.
Guy and Durell (1994)
have suggested stabilization of
the pore lining by potassium ions. The buried position is stabilized
when K+ is in the pore, thereby preventing cysteines being
modified by MTS reagents. Disruption of potassium flux allows side
chains to rotate and exposes them to the water-filled pore, where they are available for MTS modification. This scenario also explains the
preferential modification of L369C in the inactivated state. A similar
explanation for state-dependent modification of residues in homologous
positions was suggested by Liu et al. (1996)
for fruit-fly Shaker channels.
The appearance of a hyperbolic component in place of the linear
component of versus [K+]out curves when
cysteine replaces phenylalanine at position 370 can be explained by
assuming that loss of the rigid phenylalanine side chain brings the
backbone oxygens closer together in this section of the pore producing
a higher affinity site for potassium.
Such a change in the architecture of the external channel vestibule probably allows for a more severe conformational change during C-type inactivation, which can explain the drastic decrease in the rate of recovery from inactivation when cysteine replaces phenylalanine at position 369.
In conclusion we have been able to show that the modulatory effect of external K+ is likely to be evolutionarily ancient and that the mechanism of K+ binding to a specific site(s) just exterior to the selectivity filter so as to interfere with C-type inactivation has been conserved. Selection for different amino acids at this homologous site, leucine369 and phenylnalanine370 in the case of jShak2, presumably depended on the requirement for operating in different external K+ regimes and on the variability of these regimes.
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ACKNOWLEDGMENTS |
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We thank Bamfield Marine Station for providing excellent facilities. We are grateful to T. Baukrowitz and P. Rubin for stimulating discussion and valuable advice. Toronto Research Chemicals (Toronto, Ontario, Canada) provided invaluable technical advice related to application of MTS reagents. We especially thank Dr. W. Gallin for providing technical guidance for the molecular biological aspects of this study, which were carried out in his laboratory.
Partial salary support for N. G. Grigoriev was provided by the Western Canadian University Marine Biological Society. J. D. Spafford was supported by an Alberta Heritage Foundation for Medical Research studentship. This work was supported by a Natural Sciences and Engineering Research Council Research grant to A. N. Spencer.
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FOOTNOTES |
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Address for reprint requests: A. N. Spencer, Bamfield Marine Station, Bamfield, British Columbia V0R 1B0, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 30 March 1999; accepted in final form 16 June 1999.
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REFERENCES |
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