Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Submitted 27 March 2003 ; accepted in final form 27 May 2003
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ABSTRACT |
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alcohol; anesthesia; gating; scanning mutagenesis; Shaw channels
Our earlier studies showed that the selective inhibition of Shaw2 K+ channels by 1-alkanols results from the apparent stabilization of the closed state (4); and from studies with chimeric channels, we found that this inhibition is determined by a 13 amino acid internal segment that constitutes the S4-S5 loop of the Shaw2 pore-forming subunit (14). Here, we applied alanine-scanning mutagenesis to investigate the contribution of the Shaw2 S5 and S6 segments. P410A in the COOH-terminal section of S6 caused the most remarkable change by converting the inhibition into a novel dramatic potentiation. P410 is the second proline in the highly conserved PVP motif that might help to control voltage-dependent activation of eukaryotic Shaker-related Kv channels (5, 6, 39). These results are discussed in terms of a working hypothesis that proposes allosteric interactions involving the S4-S5 loop and the COOH-terminal section of S6. Notably, the structural perturbation of the second proline of the S6 PVP motif can act as a switch that determines the overall response of the Shaw2 K+ channel to 1-alkanols.
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MATERIALS AND METHODS |
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Oocyte injection and electrophysiology. Xenopus laevis oocytes
were harvested according to a protocol approved by the IACUC of Thomas
Jefferson University. Wild-type and mutant cRNAs were injected into
defolliculated Xenopus oocytes (5-50 ng/cell) using a Nanoject
microinjector (Drummond, Broomall, PA). Currents were recorded 3-7 days
postinjection. The two-microelectrode voltage-clamp technique (TEV-200; Dagan,
Minneapolis, MN) was used to record whole oocyte currents. Microelectrodes
were filled with 3 M KCl (tip resistance was <1 M
). The bath
solution contained (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1
MgCl2, and 5 HEPES (pH 7.4, adjusted with NaOH). Current traces
(generally, 450-900-ms depolarizations) were low-pass filtered at 1 kHz (-3
dB) and digitized at 500 µs/point. The voltage offset recorded at the end
of an experiment was generally small (-0.75 ± 2.2, n = 22) and
was not subtracted from the command voltage. Correction was applied when
offset appeared to be greater than one standard deviation. The leak current
was subtracted offline, assuming Ohmic leak or using a P/4 procedure. In whole
oocyte experiments, 1-alkanols were applied externally as previously described
(4,
14). Whole oocyte currents
were recorded at 23°C using a temperature-controlled microscope stage
(PDMI-2, Medical Systems, Greenvale, NY).
Patch-clamp recording was conducted as described before
(17) using an Axopatch 200B
(Axon Instruments, Foster City, CA). Patch pipettes were constructed from
Corning glass 7052 (Warner Instrument, Hamden, CT). Typically, for macropatch
recording, the tip resistance of the recording pipettes in the bath solution
(see below) was 0.5-1 M. The pipette solution (external) was as
described above. For inside-out patches, the bath solution (internal)
contained (in mM) 98 KCl, 1 EGTA, 0.5 MgCl2, and 10 HEPES (pH 7.2,
adjusted with KOH). For single-channel recording, the tip resistance was 15-20
M
. Single-channel records were low-pass filtered at 2 kHz (-3 dB,
8-pole Bessel filter; Frequency Devices, Haverhill, MA) and digitized at 10
kHz. All patch-clamp experiments were recorded at room temperature (22
± 1°C).
Data acquisition and analysis. Voltage-clamp protocols and data
acquisition were controlled by a Pentium-class computer interfaced to a 12-bit
analog-to-digital converter (Digidata 1200B using pCLAMP 8.0; Axon
Instruments). Data analysis and curve fitting were conducted using Clamp-fit
(pCLAMP 8.0; Axon Instruments), Sigmaplot (Jandel, San Rafael, CA), or Origin
7.0 (Origin Lab). Equilibrium dose-inhibition experiments were analyzed as
described before (4). With the
pCLAMP 9.0 analysis software (Axon Instruments), a standard halfway threshold
method was used to idealize the single-channel records. The resulting event
list was then used to construct amplitude and open time histograms. To
estimate the mean unitary current, the amplitude histograms were described
assuming a Gaussian distribution. A Simplex maximum likelihood method was
applied to fit the logarithmically transformed distributions of open times
(300 µs) with an exponential function to estimate the mean open time.
Unless indicated otherwise, all pooled values are expressed as means ±
SE.
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RESULTS |
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Alanine substitutions in the distal section of S6 in Shaw2 (from T404A to
S414A) affected the kinetics of current activation in a few instances
(Fig. 2A). Families of
currents from mutants that exhibited the most significant changes are shown in
Fig. 2A. Whereas
activation of I405A is slower and clearly biphasic, V409A activation appeared
very fast and unresolved. The current-voltage relationships were, however,
similar for all alanine mutants (Fig.
2B). The most significant change was observed with I405A,
which appeared to operate at more depolarized voltages. In contrast to the
results from the alanine mutants in S5, S6 alanine mutants produced a very
different and striking pattern of responses to 15 mM 1-butanol
(Fig. 2C). Between
T404A and V409A, there was a progressive reduction in the inhibition by
1-butanol from 47% (wild-type, WT) to 20% (L407A and P408A produced
nonfunctional channels). P410A, however, exhibited a dramatic potentiation by
1-butanol (105%). A subsequent alanine mutation (V411A) reduced the inhibition
(26%), and the last two mutations (V413A and S414A) enhanced it modestly (64
and 53%, respectively). This striking pattern suggests that a single mutation
(P410A) acts as a switch that controls the overall response of the Shaw2
K+ channel to 1-butanol. No other Kv channels studied so far
(wild-type or mutant) exhibited this remarkable response to 1-alkanols or
general anesthetics (3,
4,
13). Ethanol also potentiated
the P410A currents, but, relative to 1-butanol, it is a less potent agonist.
At 180 mM, ethanol quickly and reversibly enhanced the P410A current by 94%
(not shown). Given the distinct polarities of ethanol and 1-butanol, this
finding is consistent with the presence of hydrophobic site of agonist action
for 1-alkanols in the P410A mutant.
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Dose dependence and electrophysiological properties of the potentiation
of P410A by 1-butanol. The dramatic potentiation of P410A by 1-butanol is
dose dependent and fully reversible (Fig.
3, A-C). Upon exposure of a whole oocyte to a
relatively high concentration of 1-butanol (100 mM), the P410A current was
quickly enhanced more than 10-fold and, upon washout, the current quickly
returned to the original control level
(Fig. 3B). A double
logarithmic dose-response curve shows that the potentiation induced by
1-butanol was apparent at concentrations as low as 1 mM (4% above the
control) and that higher concentrations potently enhanced the current up to a
level that was
1,100% above the control at 100 mM
(Fig. 3C), but there
was no evidence of saturation. In the presence of high 1-butanol
concentrations, the currents also exhibited a faster rising phase (see below)
and a slow decline that inactivated
30% of the peak current at the end of
a 900-ms depolarization to +70 mV (Fig.
3D and Fig.
4C). These kinetic changes were not caused by improper
voltage clamping of the oocytes (e.g., series resistance errors), because
similar changes were observed from oocytes expressing different levels of
current after 1-butanol application (between 12 and 23 µA; not shown).
Also, no comparable inactivation was observed when studying other Shaw2
mutants (e.g., F335A; Fig. 1)
that expressed currents of similar magnitude (10-15 µA).
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The potentiation of P410A by 1-butanol was further characterized to gain
insights into the mechanism of this response. First, we examined the
current-voltage relation of P410A in the absence and presence of 1-butanol (50
mM; Fig. 4, A and
B). Although there was an eightfold difference between
the peak currents under these conditions, both current-voltage relations
gradually increased with membrane depolarization in a similar fashion
(Fig. 4C). Due to the
characteristic low-voltage dependence of Shaw2 K+ channels, a
comparison of the conductance-voltage relations could not be reliably obtained
from whole oocyte currents recorded with the two-electrode voltage-clamp
method. The limited current-voltage relations observed here only activate a
very small fraction of the available channels, and depolarizations to voltages
>>+180 mV are necessary to approach maximum conductance
(33). Therefore, it is
difficult to test whether a leftward shift of the activation curve accounts
for the current potentiation by 1-butanol (see DISCUSSION).
Nevertheless, a closer examination of the current kinetics revealed evidence
of leftward-shifted voltage-dependent gating in the presence of 1-butanol.
Clearly, the activation time constants were reduced 23-fold in the
presence of 1-butanol, but their voltage dependence was not significantly
affected (parallel relationships in Fig.
4D). Assuming that the activation time constants depend
exponentially on membrane potential (solid lines,
Fig. 4D), the derived
apparent charges were 0.5 and 0.4 e0 in the absence and
presence of 1-butanol, respectively. This parallel shift in the voltage
dependence of the activation time constants is consistent with a negative
shift in voltage-dependent activation. The small apparent charges are also
consistent with the low voltage dependence of activation gating in Shaw2
channels (33).
1-Butanol increases the open probability of the Shaw2-P410A channel. We examined the effect of 1-butanol on the P410A channels in cell-free patches to test the possible dependence on soluble cellular components (e.g., second messenger systems). Clearly, in an inside-out patch expressing P410A (at 0 mV), 15 mM 1-butanol exerted a potent reversible potentiation that resulted from a fourfold increase in open probability (0.2, 0.8, and 0.3, before and after 1-butanol and after washout, respectively; Fig. 5). Thus 1-alkanols might induce the potentiation by acting directly on the pore-forming subunit or tightly associated regulatory components. Furthermore, the relatively rapid onset and reversal of the macroscopic response is not consistent with an effect mediated by a second messenger system (Fig. 3B).
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Analysis of a separate sparse record of unitary P410A currents from an
inside-out patch (Fig. 6;
opening frequency 0.26 Hz at 0 mV) allowed the estimation of the mean unitary
amplitude (i) and mean open time (O). Most openings
in this record occurred in isolation (Fig.
6, A-C). The mean closed time was, however, not
measured because the number of channels in the patch could not be determined.
Under control conditions (n = 122 openings), i was 2.6 pA
and
O was 6.3 ms (
6-fold longer than the mean open time
of wild-type Shaw2 K+ channel; Ref.
4). Assuming a reversal
potential of -90 mV under the ionic conditions of this experiment, the
estimated single-channel conductance was
25 pS, which is similar to that
of the wild-type Shaw2 K+ channel
(4). 1-Butanol (15 mM) slightly
reduced i (Fig.
6D, top) and
O (2.3 pA and 4.9
ms, respectively; n = 254 openings), but the opening frequency was
approximately doubled (0.54 Hz; Fig.
6D, center). Upon washout, the estimated
single-channel parameters were close to control values (i = 2.3 pA;
O = 6.9 ms; n = 148 openings) and the opening
frequency returned to basal levels (0.31 Hz, corresponding to an open
probability of
0.001). The absence of a significant effect of 1-butanol
on open durations was confirmed by comparing the cumulative distributions of
open times (Fig. 6D,
bottom). Thus mainly an increase in the opening frequency without
significant changes in unitary conductance or the stability of the open state
could account for the potentiation of the P410A activity by 1-butanol.
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DISCUSSION |
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The allosteric modulation of the Shaw2 channel by 1-alkanols may
involve critical interdomain interactions in the activation gate.
Crystallographic and biophysical analyses of bacterial K+ channels
(MthK and KvAP) firmly suggest that the COOH-terminal segment of S6 is likely
to be the activation gate of Kv channels
(18-20,
40). Although the S6 PVP motif
is not present at the equivalent positions in these bacterial channels, it is
highly conserved among all eukaryotic Shaker-related Kv channels, and separate
experimental evidence strongly suggests that the prolines in this motif favor
a kinked -helix structure at the COOH-terminal end of S6
(5,
6,
15). Thus it is hypothesized
that four kinked S6 segments at the inner mouth of the pore constitute an
internal activation gate in tetrameric Kv channels of eukaryotes
(5,
6). Our data showed that a
single proline to alanine mutation at the second position in the PVP motif of
the Shaw2 K+ channel (P410A) acts as a switch that controls the
overall response of the channel to 1-alkanols (Figs.
2,
3,
4,
5,
6). This striking finding has
important implications that we discuss here as a working hypothesis based on
the recently published crystal structure of a bacterial Kv channel (KvAP) and
our own data (4,
14,
19). Tightly coupled
voltage-dependent gating might depend on an interaction between the S4-S5 loop
and the COOH-terminal segment of S6
(25,
35), which in eukaryotic
Shaker-related Kv channels is more effective when the latter segment is kinked
at the PVP motif (Fig.
7A). Upon membrane depolarization, the positively charged
voltage sensors move outward, which drags the S4-S5 loop to allow the
expansion of the S5 cuff around the pore, and consequently, a concerted
lateral displacement of the S6 COOH-terminal segments follows. This
displacement causes the opening of the channel's internal mouth. Importantly,
the S4-S5 loop is a region that critically influences the modulation of Shaw2
and Kv3.4 channels by 1-alkanols
(4,
14). It is therefore
conceivable that the 1-alkanol response of the Shaw2 K+ channel
depends on how 1-alkanol binding is coupled to a gating mechanism that, in
turn, depends on the relationship between the S4-S5 loop and the COOH-terminal
segment of S6. When the S6 segment is kinked at the PVP motif, there is
inhibition of Shaw2 channels by 1-alkanols
(Fig. 7A). In the
closed state, the S4-S5 loop and S6 form a protein-protein interface that may
constitute the binding cavity of 1-alkanols, which is rapidly occupied only
from the internal side (14).
The concerted rearrangements that open the channel can only take place when
all S4-S5/S6 interfaces are free. Thus, if a 1-alkanol occupies one site [the
Hill coefficient for the inhibition at equilibrium is
1
(4,
14)], the channel remains
locked in the closed conformation. To explain the potentiation, we assumed
that the P410A mutation reduces or eliminates the S6 COOH-terminal kink
(Fig. 7B). In this
case, there is no S4-S5/S6 interface, but rearrangements similar to those
explained above still mediate channel activation and pore opening. The crystal
structure of KvAP suggests that S4 and the S4-S5 loop may be in contact with
membrane phospholipids (19).
Thus, through an interaction with the lipid-protein interface, 1-alkanols
could reposition S4 and the S4-S5 loop to facilitate voltage-dependent
activation. Accordingly, faster current activation at lower voltages results
from the repositioning of the voltage sensors (Figs.
3D and
4D). Mechanical models
based on the crystal structure of KvAP are, however, still speculative. To
develop a physical model of the allosteric modulation of Shaw2 K+
channels by 1-alkanols, it is necessary to understand the interactions between
the S4-S5 loop and the COOH-terminal tail of S6. Interestingly, mutating the
equivalent proline (P469A) in Kv3.4 (a mammalian Shaw homologue) impaired
voltage-dependent gating and also conferred activation by 15 mM 1-butanol
(
50%; not shown). The latter suggested that the potentiation by
1-alkanols may only be weakly dependent on the distinct structures of the
S4-S5 loops in Shaw2 and Kv3.4 (contrary to the inhibition, Ref.
14).
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Biophysical basis of the allosteric modulation of the Shaw2-P410A
mutant by 1-alkanols. The electrophysiological characterization of the
P410A channel provided further information about the mechanism underlying the
potentiation by 1-butanol (Figs.
3,
4,
5,
6). The main effect of
1-butanol was a rapid dose-dependent increase in peak current that was not
associated with significant changes in single-channel conductance or mean open
time. This rapid reversible response, which is independent of soluble cellular
components, suggested a direct interaction between 1-butanol and a channel
subunit or a closely associated regulatory protein. Namely, 1-butanol
increased the open probability of the P410A mutant by enhancing the opening
frequency. A more detailed analysis of closed times was, however, not possible
because the number of channels in the patch could not be estimated (see
RESULTS). Nevertheless, the simplest general interpretation of
these observations suggests that 1-butanol increases the open probability of
the channel through destabilizing nonconducting states connected to the main
open state of the channel. If the affected closed state is in the activation
pathway, the voltage-dependent activation curve is expected to exhibit a
leftward shift. The analysis of the current-voltage relations
(Fig. 4), however, revealed no
indication of such a shift. This result is not surprising because the examined
voltage range (up to +70 mV) only appears to activate a very small fraction of
the available channels at the foot of the activation curve (<0.01; Ref.
33). Indeed, by modeling
activation of Shaw2 K+ channels as a first-order transition (C
O; with a midpoint potential of +200 mV and an apparent gating charge
of 0.6 e0), we found that a shift of -90 mV cannot be
detected upon normalizing the current-voltage relations at the foot of the
activation curve (-100 to +100 mV) where the open probability is <0.05
(even though a drastic eightfold potentiation of the current is clearly
apparent). Interestingly, 1-butanol also induced faster current activation of
the P410 mutant, which was fully reversible and appeared to be concentration
dependent and voltage dependent (Fig.
3D and Fig.
4D). The presence of faster current activation in the
presence of 1-butanol is consistent with the presence of a leftward-shifted
voltage-dependent activation curve. Further electrophysiological studies are
necessary to investigate the properties of the apparent inactivation and the
exact relation between the kinetic changes and the potentiation of the
current.
The dose dependence of the current potentiation by 1-butanol
(Fig. 6C) is
consistent with an allosteric mechanism involving a low-affinity interaction
that enhances activation gating. At the examined membrane potentials (up to
+70 mV), P410A channels operate at a very low open probability (<0.01).
Analogously, in the presence of submaximal agonist concentrations that
activate 10% of the available channels, similar dose-response curves have
been observed for the potentiation of GABA and glycine receptor channels by
1-alkanols or general anesthetics
(8,
26,
27,
37). Characteristically, the
dose-response curves for the potentiation of ligand-gated ion channels and G
protein-coupled K+ channels by 1-alkanols do not exhibit evidence
of saturation, which is also consistent with the presence of a low-affinity
interaction (8,
22,
24,
26,
27,
37). Taken together, our
results shed more light on the electrophysiological, kinetic, and structural
basis of the allosteric modulation of activation gating in Shaw2 K+
channels by 1-alkanols. In particular, by demonstrating the dramatic effect of
a single mutation, this study underscores the significance of investigating
Shaw2 K+ channels as an archetypal system to learn more about the
intricate interactions that constitute the protein-based theories of alcohol
intoxication and general anesthesia.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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