§
From the * Department of Physiology, Institute of Hyperexcitability, Jefferson Medical College, Philadelphia, Pennsylvania 19107; Department of Medicine, Vanderbilt University School of Medicine; and § Department of Pharmacology, Vanderbilt University School
of Medicine, Nashville, Tennessee 37232
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ABSTRACT |
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The voltage sensor of the sodium channel is mainly comprised of four positively charged S4 segments. Depolarization causes an outward movement of S4 segments, and this movement is coupled with opening
of the channel. A mutation that substitutes a cysteine for the outermost arginine in the S4 segment of the second
domain (D2:R1C) results in a channel with biophysical properties similar to those of wild-type channels. Chemical
modification of this cysteine with methanethiosulfonate-ethyltrimethylammonium (MTSET) causes a hyperpolarizing shift of both the peak current-voltage relationship and the kinetics of activation, whereas the time constant
of inactivation is not changed substantially. A conventional steady state inactivation protocol surprisingly produces
an increase of the peak current at 20 mV when the 300-ms prepulse is depolarized from
190 to
110 mV. Further depolarization reduces the current, as expected for steady state inactivation. Recovery from inactivation in
modified channels is also nonmonotonic at voltages more hyperpolarized than
100 mV. At
180 mV, for example, the amplitude of the recovering current is briefly almost twice as large as it was before the channels inactivated. These data can be explained readily if MTSET modification not only shifts the movement of D2/S4 to more
hyperpolarized potentials, but also makes the movement sluggish. This behavior allows inactivation to have faster
kinetics than activation, as in the HERG potassium channel. Because of the unique properties of the modified mutant, we were able to estimate the voltage dependence and kinetics of the movement of this single S4 segment.
The data suggest that movement of modified D2/S4 is a first-order process and that rate constants for outward
and inward movement are each exponential functions of membrane potential. Our results show that D2/S4 is intimately involved with activation but plays little role in either inactivation or recovery from inactivation.
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INTRODUCTION |
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Voltage-dependent ion channels have evolved to react
rapidly to small changes in membrane potential. In the
superfamily of channels selective for sodium, calcium,
or potassium ions, this sensitivity to voltage is conferred
principally by four positively charged transmembrane
segments, known as S4 segments. Each S4 segment has
two to eight basic residues, either arginine or lysine, that are typically separated from each other by two hydrophobic residues. In response to a change of membrane potential, the positive residues move with respect
to the membrane electric field, and this movement is
coupled to the gating process that opens and closes the
ion-selective pore of the channel protein (for reviews, see Catterall, 1986; Patlak, 1991
; Keynes, 1994
; Sigworth, 1994
). Depolarization is expected to drive the
basic residues of S4 segments outward.
Mutations of S4 residues usually cause changes in gating, but these changes rarely provide direct insight into either the movement of the S4 segments or the interaction between S4 movement and gating. Furthermore, voltage-dependent gating typically involves at least two distinguishable processes, activation and inactivation. A step depolarization causes channels to open (activate) and then spontaneously close (inactivate). The relationship between S4 movement and each of these processes is purely speculative at the moment.
The most direct evidence for S4 movement comes
from experiments that use the cysteine scanning methodology introduced by Falke et al. (1988) and Akabas
et al. (1992)
. In studies of S4 function, a cysteine is substituted for a selected S4 residue by site-directed mutagenesis, and the expressed channel is then exposed
to a hydrophilic cysteine reagent while monitoring its
biophysical properties (Yang and Horn, 1995
; Larsson
et al., 1996
; Yang et al., 1996
, 1997
). The consequence
of the reaction between the introduced cysteine residue and the reagent is a change in either activation or
inactivation of the channel. For some S4 residues, a
change of membrane potential causes a change in the
surface accessibility of the cysteine, which can be monitored as a change in the rate of its modification by a
fixed concentration of cysteine reagent in either the
extracellular or intracellular solution bathing the channel. The voltage dependence of the modification rate
is, therefore, an assay of S4 movement that either exposes or buries individual residues (Horn, 1998
).
We have used this technique to explore the voltage-dependent movement of the S4 segment of the fourth
homologous domain (D4) of sodium channels (Yang
and Horn, 1995; Yang et al., 1996
, 1997
). In these experiments, we monitored the reaction between specific residues in D4/S4 and hydrophilic reagents by pronounced changes in the kinetics of inactivation. By
contrast, modification of D4/S4 cysteines had relatively
small effects on activation. Although we interpreted
these results as evidence that cysteine modification causes a change in the kinetics and voltage dependence of D4/S4 movement, we had no data to support
or reject this idea. Similarly, mutations in other S4 segments of sodium channels affect gating by unknown
mechanisms (Stühmer et al., 1989
; Chen et al., 1996
; Kontis and Goldin, 1997
; Kontis et al., 1997
).
Here we report that chemical modification of a cysteine introduced into the S4 segment of the second domain (D2/S4) causes a hyperpolarizing shift of activation, but has little effect on inactivation. We further provide evidence that chemical modification of this cysteine causes a marked decrease in the kinetics of D2/ S4 movement. These results contrast strongly with those obtained for modification of D4/S4, showing the unique contributions played by these two S4 segments in sodium channel gating.
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MATERIALS AND METHODS |
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Mutagenesis
The mutation R669C (D2:R1C) was constructed in hSkM1 using a single-step polymerase chain reaction mutagenesis strategy. Primers were designed to create the desired mutation and incorporate natural restriction sites for StuI (nucleotide [nt] 2051) and BsteII (nt 2179) in the final product. Amplifications (20 cycles) were performed using 20 ng of hSkM1 cDNA as template and Taq DNA polymerase. Final products were purified by spin-column chromatography (QIAGEN Inc., Chatsworth, CA), digested with StuI and BsteII, and the resulting 128-bp fragment ligated into the corresponding sites in the plasmid pRc/CMV-hSkM1. The amplified region was sequenced entirely in the final construct to verify the mutation and exclude polymerase errors.
Electrophysiology and Data Acquisition
Standard whole cell recording methods were used as previously
described (Yang and Horn, 1995). Supercharging reduced the
expected charging time constant for the cells to <10 µs. Series
resistance errors were <3 mV. Data were filtered at 5-10 kHz and
acquired using pCLAMP (Axon Instruments, Burlingame, CA).
Patch electrodes contained (mM): 105 CsF, 35 NaCl, 10 EGTA,
10 Cs-HEPES, pH 7.4. The bath contained (mM): 150 NaCl, 2 KCl, 1.5 CaCl2, 1 MgCl2, 10 Cs-HEPES, pH 7.4. Corrections were
made for liquid junction potentials. Most experiments were done
at room temperature (20-22°C). In a few experiments, the temperature was set at 11.1°C by use of feedback-regulated Peltier devices (Dagan Corp., Minneapolis, MN).
Methanethiosulfonate-ethyltrimethylammonium (MTSET),1 -ethylamine (MTSEA), and -ethylsulfonate (MTSES) were obtained
from Toronto Research Chemicals (North York, Ontario, Canada).
MTSET covalently attaches ethyltrimethylammonium to the reduced cysteine sulfhydryl via a disulfide bond, MTSEA attaches
ethylamine, and MTSES attaches ethylsulfonate. Aqueous stocks
of these reagents were kept at 4°C, and diluted in the bath solution immediately before use. The reagent solutions were presented to the cells with a macropipette placed in apposition to
the cell (Yang et al., 1997). In a few experiments, MTSET was introduced into the patch pipette solution to expose it to the cytoplasmic face of the channel (Yang et al., 1996
).
For modification of D2:R1C by MTS reagents, we used the following voltage protocol. In the presence of a fixed concentration of cysteine reagent (typically 50 µM MTSET), a single 9.7-s depolarization to a selected voltage from 150 mV was followed by a
10-s return to
150 mV. The depolarized voltage was either
60
or
20 mV. These pulses were followed by a 200-ms prepulse to
110 mV and a 20-ms test depolarization to
55 mV to measure
the amplitude of the currents. This voltage protocol was repeated
over a period of 10 min or until the channels were completely
modified. We estimated the first-order modification rate by fitting the time course of the change in peak amplitude of the current to a single exponential relaxation.
Data Analysis and Modeling
Whole cell data were displayed and analyzed by a combination of
pCLAMP programs, ORIGIN (MicroCal Software, Inc., Northampton, MA), and our own FORTRAN programs. Data from at least
three cells for each measurement are presented as mean ± SEM.
We fit data from individual cells to theoretical functions of
choice, and the reported values are the means and standard errors of the estimated parameters from these fits. Boltzmann functions (steady state inactivation and peak conductance-voltage
[G-V] relationship) were fit by use of a variable metric algorithm.
The midpoint (V0.5) and slope (q, the valence in units of the
number of elementary charges; Yang et al., 1997) were estimated
from these fits. The shift (
V) of the fitted G-V curve caused by
MTSET modification was used to estimate the stabilization of the
open state (French et al., 1996
). Specifically, the change in free
energy (
G °) is given by
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(1) |
where q is the slope of the G-V curve, NAvog = 6.022 × 1023
charges/mol and e0 = 1.602 × 1019 coulomb.
After modification by MTSET, the effect of 300-ms prepulses on
peak current at 20 mV was fit by a product of two Boltzmann functions (see Fig. 3 B), one corresponding to steady state inactivation at relatively depolarized potentials, and the other corresponding to the probability of D2/S4 being in an outward position. Because of the nonzero asymptote at hyperpolarized potentials, the
Boltzmann function describing D2/S4 position also has a nonzero
asymptote. The slope, midpoint, and limits of each Boltzmann function were estimated simultaneously by least squares minimization
using a variable metric algorithm. For accuracy and speed, the analytic derivatives of the fitted function with respect to each parameter were used in the estimation. A variable metric algorithm was
also used to fit the kinetics of recovery from inactivation in Fig. 4.
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RESULTS |
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Voltage-dependent Modification of D2:R1C by MTSET
Substitution of the outermost arginine of the S4 segment of domain 2 (D2) by cysteine has a rather modest
effect on the gating of the skeletal muscle sodium channel hSkM1, when expressed transiently in tsA201 cells.
Whole cell currents of this mutant, D2:R1C, are compared with those of the wild-type (WT) channel in Fig.
1. Families of currents in response to a series of depolarizations are shown in Fig. 1, A and B, and the corresponding peak current-voltage (I-V) and conductance-
voltage relationships are shown in Fig. 1, C and D. Boltzmann fits to the G-V data (theory curves in Fig. 1 D)
show that the cysteine substitution produces a 5.7-mV
depolarizing shift and a decrease in slope equivalent to
a reduction of 1.4 elementary charges (e0). These results are consistent with a role of this WT arginine of
D2/S4 in sensing the membrane potential. By contrast
with a cysteine substitution for the outermost arginine of the S4 segment of domain 4 (Chahine et al., 1994),
the D2:R1C mutation has little effect on the inactivation kinetics (Fig. 1, A and B).
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Modification of D2:R1C by the hydrophilic cysteine reagent MTSET (Fig. 2 A) mainly affects activation. MTSET causes a hyperpolarizing shift of both the peak current-voltage relationship (Fig. 2 B) and the kinetics of activation (Fig. 2 C), without affecting the time constant of inactivation (Fig. 2 D). Note: although the kinetics of inactivation appear slower after MTSET modification (Fig. 2 A vs. Fig. 1 B), this is primarily a consequence of the fact that currents at more hyperpolarized voltages are larger after MTSET modification, due to the shift in activation gating (Fig. 2 B), and to the fact that inactivation is slower at more negative voltages.
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Some S4 residues in D4 are externally exposed only
upon depolarization (Yang and Horn, 1995; Yang et al.,
1996
). To examine the possibility that D2:R1C is also
accessible only when depolarized, we determined the
rate of modification by extracellular 50 µM MTSET at
three voltages,
150,
60, and
20 mV. To do this, we exploited the fact that the current at the foot of the activation curve increases dramatically after modification
(Fig. 2 B). We therefore monitored the modification by
measuring the peak current induced by a brief test
pulse to
55 mV every 20 s, while exposing the cell to
MTSET. If the membrane potential is maintained at
150 mV between these test pulses, there is very little
modification of the channels (data not shown). A 9.7-s
depolarization to
20 mV between the test pulses (see
MATERIALS AND METHODS for details) exposes this cysteine residue to MTSET, causing a progressive increase
in the amplitude of the current at
55 mV (Fig. 2 E; trace 0 represents the current before exposure to
MTSET). As expected for an outward movement of
D2/S4 when depolarized, the rate of modification by
MTSET increases with depolarization (Fig. 2 F ).
Certain D4/S4 residues are translocated completely
from an internally accessible to an externally accessible
position upon depolarization (Yang et al., 1996). To
test whether this also occurs for D2:R1C, we introduced
400 µM MTSET into the patch pipette solution and,
while maintaining the cells at a
140-mV holding potential, looked for shifts in gating equivalent to those
observed for external application. Over a period of 20 min, no such effects were observed (data not shown).
Furthermore, these cells remained susceptible to modification by extracellular 50 µM MTSET, showing that at
hyperpolarized voltages D2:R1C is inaccessible on either side of the channel, whereas at depolarized voltages it is exposed externally. This is exactly equivalent
to experiments involving the outermost basic residue of
D4/S4 (Yang et al., 1996
).
The above results can be interpreted as follows. Modification of D2:R1C by MTSET alters the conformation of D2/S4 to favor an open state of the channel. This could be explained if the probability of being in an outward position of D2/S4 is enhanced by the attachment of the SET adduct. In other words, the voltage-dependent probability of D2/S4 being in its outward conformation is shifted to more hyperpolarized voltages by MTSET modification. This shows, not remarkably, that D2/S4 plays a role as one of the four voltage sensors underlying activation. We were surprised, however, to see pronounced consequences of MTSET on what appeared to be inactivation at more hyperpolarized potentials.
Fig. 3 A shows the effects of a series of 300-ms
prepulses on the currents elicited by a test pulse to 20
mV after modification by MTSET. This relatively standard procedure, used to quantify steady state inactivation, produced a nonmonotonic effect of prepulse potential on the peak current of the test pulse. Depolarization of the prepulse from
190 to ~
110 mV
caused an anomalous increase in current; further depolarization reduced the current strongly, as expected for
steady state inactivation. Other characteristics of the currents are also affected by the prepulses, notably the kinetics of activation and inactivation (Fig. 3 A). The rates of these processes are both increased monotonically by
prepulse depolarization, an effect not observed for unmodified channels (see below). Fig. 3 B plots the normalized peak current during the test pulse as a function of prepulse voltage, showing a typical steady state
inactivation curve for D2:R1C before modification (
),
and the pronounced alteration after MTSET modification (
).
Sluggish Movement of D2/S4 Induced by MTSET Modification of D2:R1C
What could be responsible for the unusual effects of
prepulse potential on the currents during the test
pulse? We decided to explore the following hypothesis.
Suppose that cysteine modification causes not only a
hyperpolarizing shift in the steady state conformation of D2/S4, but also a drastic reduction in the rate of its
movement in response to changes of membrane potential. This could account for the effects observed in Fig.
3 A as follows.2 A long-duration prepulse to 190 mV
would drive all the S4 segments of a channel into their
inward positions. A subsequent depolarization to
20
mV would elicit a current only after all S4 segments
moved outward. The sluggish response of modified
D2/S4 to the depolarization is expected to have three
consequences on the macroscopic current elicited by a
step to
20 mV. First, the kinetics of activation would
be slow because the movement of D2/S4 would limit
the rate of opening. Second, the kinetics of inactivation
at
20 mV would be slow, because of coupling between
the rate of channel opening and the rate of macroscopic inactivation (Aldrich et al., 1983
). Finally, the
amplitude of the current would be small because some closed channels would inactivate before they had a
chance to open. A more depolarized prepulse, say to
110 mV, would set the modified D2/S4 in an outward
conformation. A subsequent depolarization to
20 mV
would drive the other S4 segments outward, allowing the rapid and efficient opening of the channels, because
only three S4 segments have to move. More depolarized prepulses would have the usual effect of producing
inactivation, and thus reducing the current amplitude.
Note that this model assumes that outward movement
of D2/S4 has a greater effect on activation than on inactivation, consistent with previous mutagenic studies
(Chen et al., 1996
; Kontis and Goldin, 1997
; Kontis et
al., 1997
).
We fit the normalized data of Fig. 3 B with a product
of two Boltzmann functions, one accounting for the
probability of D2/S4 being in an inward versus an outward conformation, and the other for the probability of
a channel being noninactivated. This makes the apparently extreme assumption that the conformation of
D2/S4 after modification by MTSET is independent of
the process of inactivation. However, in the voltage
range of most relevance to the D2/S4 movement (more negative than 110 mV), there is little contamination from steady state inactivation. The fit of the double Boltzmann model to the data for modified channels (theory curve in Fig. 3 B) suggests that 50% of the
D2/S4 segments are in outward conformations at a
membrane potential of
128 ± 3 mV, and that this
outward movement has a voltage dependence equivalent to a translocation of 1.19 ± 0.07 e0 across the membrane electric field.
Our working hypothesis for the sluggish and shifted
voltage dependence of D2/S4 movement after modification produces a number of kinetic predictions, which
we explore here. One prediction, as discussed above, is
that for certain voltages the kinetics of D2/S4 movement will be slower than those of inactivation. An example is shown in Fig. 4 A. In this experiment, all channels were inactivated by a 100-ms depolarization to 20
mV from a holding potential of
180 mV. Subsequently, a variable-duration return to
180 mV induced recovery from inactivation (see Fig. 4 A, top, for
voltage protocol). A test pulse to
20 mV tracks the
process of recovery (Fig. 4 B,
). During recovery at
180 mV, the amplitude of the current was briefly almost twice as large as it was before the channels were
originally inactivated. This nonmonotonic recovery is
expected if the recovery from inactivation at
180 mV
is faster than the inward movement of D2/S4. For a recovery pulse of 2 ms, for example, D2/S4 has not had
enough time to move inward, whereas recovery from
inactivation is nearly complete. Longer duration recovery pulses cause the inward movement of D2/S4, which
reduces the peak current in the same fashion as described for the pulse protocols of Fig. 3. This nonmonotonic recovery behavior is less pronounced for
more depolarized recovery potentials, where there is
less inward movement of D2/S4. For example, at
100
mV, only ~20% of the modified D2/S4 segments are
expected to move inward (Fig. 3 B). Notice that recovery is preceded by a voltage-dependent delay of 200-
1,000 µs, as observed in previous studies of sodium channels (Kuo and Bean, 1994
; Ji et al., 1996
). As expected for our working hypothesis, recovery from inactivation is monotonic for unmodified D2:R1C channels
(Fig. 4 C).
We fit the recovery data for modified channels with a model in which D2/S4 movement has first-order kinetics, and recovery from inactivation is independent of the conformation of D2/S4. The ratio of peak currents of the second to the first depolarizations (Irec(t)/I1) is represented in this model as
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(2) |
where recovery has a single exponential relaxation with
rate rec after a voltage-dependent lag tlag, D2/S4 kinetics after modification are first order with rate
S4, pout is
the steady state probability of D2/S4 being in an outward conformation, and pin = 1
pout. Channels that
had recovered from inactivation, and therefore were
responsible for the currents during the test depolarization, were assumed to be in one of two kinetic states, corresponding to outward or inward conformations of
D2/S4. The relative amplitude of the current elicited
from these two conformations is designated in Eq. 2 as
Rout/in. Because of the postulated independence of recovery and D2/S4 movement, the fraction of recovered
current appears in Eq. 2 as a product of two probabilities.
The fit of the data in Fig. 4 B requires estimation of
four free parameters, rec,
S4, tlag, and Rout/in. The values of pout and pin were obtained from the fits of
prepulse inactivation in Fig. 3 B. Specifically, at
180
mV pout = 0.0761, and at
140 mV pout = 0.360. The
best-fit theory curves for this model are shown as the
solid lines for the nonmonotonic data at
180 and
140 mV (Fig. 4 B). The fit produces estimates of the
rate of recovery from inactivation (
rec) and the rate of
inward movement of D2/S4 (
S4). As expected,
rec >
S4 (Fig. 4 D). Furthermore,
rec is not affected strongly
by MTSET modification (compare Fig. 4 D,
and
),
further supporting the assumption that the conformation of D2/S4 has little influence on recovery from inactivation.
Our working hypothesis also predicts that activation
kinetics will depend on prepulse potential after D2:
R1C channels are modified by MTSET. To measure
these kinetics more accurately, we did a number of experiments at a reduced temperature of 11.1°C. We used
a holding potential of 150 mV, and applied a series of depolarizations from
90 to +65 mV, preceded by a
300-ms prepulse to either
180 mV (Fig. 5, A and B,
)
or
110 mV (
). For unmodified D2:R1C, the more
depolarized prepulse reduced the currents by a factor
of 0.703 with no effect on activation kinetics (Fig. 5 A).
After modification, the more depolarized prepulse increased the peak current by a factor of 1.70, consistent
with the results of Fig. 3, and also increased the rate of
activation (Fig. 5 B). This is the expected consequence
on activation kinetics, as discussed above, because from
the more hyperpolarized potential the outward D2/S4
movement is the rate-limiting step in activation in response to a depolarization.
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We showed in Fig. 3 A that, after modification,
prepulse voltage affects inactivation kinetics during a
depolarizing test pulse in experiments intended to
measure steady state inactivation. These kinetics were
measured at 11.1°C, and inactivation time constants at
20 mV are plotted in Fig. 5 C. The rate of inactivation during the test pulse increases with prepulse depolarization, an effect seen only after modification of D2:
R1C by MTSET (Fig. 5 C,
). Q10 values of ~2.1 are obtained for inactivation kinetics by comparing the data
in Figs. 2 D and 5 C. By contrast with inactivation kinetics, deactivation kinetics are not affected by MTSET
modification (Fig. 5 D). The latter result indicates that
D2/S4 of the mutant channel plays little role in deactivation, suggesting that one or more of the other three
S4 segments is responsible for the rapid closing of the
activation gate in response to a hyperpolarization.
First-Order Kinetics for Modified D2/S4
Movement of D4/S4 is first order and voltage dependent in the unmodified cysteine mutant D4:R1C (Yang
and Horn, 1995). The data of Fig. 4 are also consistent
with this idea for MTSET-modified D2/S4. Here we
provide even stronger evidence that the movement of
modified D2/S4 is a first-order process. A stringent requirement for such a process is that the kinetics at any
voltage must relax as a single exponential with a time
constant independent of initial condition. We estimated the kinetics of D2/S4 movement at room temperature by working in a voltage range more negative
than that of inactivation (i.e., between
190 and
110
mV; Fig. 3 B). Fig. 6 A shows the normalized peak current at
20 mV for D2:R1C-SET in response to variable
duration prepulses to either
150 mV (Fig. 6 A,
and
; see inset) or
135 mV (
and
). In each case, the initial position of D2/S4 was set by a 500-ms conditioning pulse to either a highly negative voltage (Fig. 6 A,
D2/S4 inward, open symbols) or to
110 mV (D2/S4
outward, closed symbols). The data, plotted against a
logarithmic time axis, are well fit by single-exponential relaxations for all voltage protocols. Although the time
constants are smaller at
150 than at
135 mV (7.7 ± 0.1 vs. 9.4 ± 0.1 ms), they are independent of the initial
conditions at each voltage (see legend to Fig. 6 for details), consistent with a first-order process. These results
indicate that the rate of inward D2/S4 movement is the
same as the rate of outward movement at a given membrane potential.
|
We combined the time constants measured at 190,
150,
135, and
110 mV (Fig. 6 B,
) with steady
state estimates of the probability of D2/S4 being in an
outward position (Fig. 3 B), to obtain estimates of the
voltage-dependent rate constants for outward (
) and
inward (
) movement of this modified S4 segment. These two rate constants are plotted in Fig. 6 C, where
it is also shown that each is an exponential function of
membrane potential (solid lines). The time constants
for D2/S4 movement can be predicted from the theoretical values of
and
over a wide voltage range (
S4 = 1/[
+
]), as shown by the solid line in Fig. 6 B. These time constants may be compared with the estimates of
S4 = 1/
S4 that we obtained from a very different measurement, recovery from inactivation (Fig. 4). These estimates (Fig. 6 B,
) show a good correspondence with
the results of experiments like those in Fig. 6 A, supporting both that D2/S4 movement is first order and
that it is independent of recovery from inactivation.
Our data are therefore consistent with a very simple model for the voltage-dependent movement of D2/S4 after modification. The process is not only first order, but the rate constants are also exponential functions of membrane potential. The time constants in Fig. 6 B are also larger than those observed for both deactivation (Fig. 5 D) and recovery from inactivation (Fig. 4 D), consistent with our hypothesis that modification of D2: R1C by MTSET makes the D2/S4 movement sluggish.
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DISCUSSION |
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The S4 segments of voltage-gated ion channels are now
known to be the principal voltage sensors for gating.
This has been demonstrated primarily by two classes of
experiments, the effects of S4 mutations on both ionic
and gating currents, and the voltage-dependent accessibilities of specific residues using cysteine scanning
methods. The cysteine scanning studies provide evidence that S4 segments move when the membrane potential changes over an appropriate range (Yang and
Horn, 1995; Larsson et al., 1996
; Yang et al., 1996
, 1997
). Corroborative evidence using fluorescently tagged
S4 segments also supports the idea of voltage-dependent S4 movement (Mannuzzu et al., 1996
). Furthermore, the kinetics of S4 movement occur on the same
time scale as gating kinetics (Yang and Horn, 1995
; Mannuzzu et al., 1996
). It is important to note, however, that "movement" in all of these studies is defined
operationally. Some relative movement certainly occurs
between S4 segments and the membrane electric field,
the lipid bilayer, and/or other proteinaceous regions
of the channel. There has been some speculation how
this relative movement translocates charge (Armstrong,
1981
; Catterall, 1986
; Guy and Conti, 1990
; Sigworth,
1994
; Aggarwal and MacKinnon, 1996
; Larsson et al.,
1996
; Seoh et al., 1996
; Yang et al., 1996
). However, the
molecular details remain a mystery.
How does modification of S4 segments, either by mutagenesis or by reaction with cysteine reagents, alter gating, and what do such experiments tell us about S4 function? Two obvious consequences of S4 modification could be a change in the equilibrium conformation of the transmembrane segment with respect to the electric field and a change in the kinetics of S4 movement. Our data with D2:R1C suggest that the SET adduct affects both. It energetically biases the S4 segment to be in an outward position, by comparison with an unmodified cysteine. This is seen most clearly from the hyperpolarizing shift of the peak current-voltage relationship caused by MTSET modification (Fig. 2 B). The 14.4-mV shift is equivalent to a 3.7-kJ/mol stabilization of the open state (Eq. 1). Note that this calculation underestimates the effect of the modification on the steady state conformation of D2/S4, because this is only one of four S4 segments contributing to the opening of the channel.
We have also claimed that the kinetics of D2/S4
movement are markedly slowed by modification of D2:
R1C, based on the results of Figs. 3-6. Before considering the molecular implications of this interpretation,
we must first consider the alternative hypothesis, that
the sluggish kinetics induced by modification occur
downstream from actual S4 movement. How do we
know, for instance, that the slow kinetics are due specifically to a retardation of D2/S4 movement, rather than
to a slower conformational change that occurs after S4
movement? In fact, we cannot exclude this possibility,
although it is likely that the MTSET-modified S4 segment is capable of moving and thereby translocating
charge. We previously showed that another S4 segment, D4/S4, is capable of translocating a SET adduct
from the extracellular to intracellular face of the protein, where it can be cleaved off by a reducing agent
(Yang et al., 1996). Therefore, we will assume for discussion that the slowed activation kinetics caused by
MTSET are due to slowed S4 movement.
How does the SET adduct slow S4 movement? One possibility is that the bulk of cysteine-SET, with a volume nearly twice that of the WT arginine, increases the height of the activation barrier for translocation due to steric hindrance. This is apparently not the case, however, because modification by the smaller cationic reagent MTSEA had a qualitatively similar effect as MTSET on prepulse inactivation, whereas the intermediate-sized anionic reagent MTSES did not (data not shown). Charge by itself cannot be responsible for the sluggish movement, however, because the WT arginine, like MTSET, is cationic. Our data suggest, therefore, that D2/S4 movement is sensitive to the structure of the residue at this position of the S4 segment. Note that if modification prevented S4 movement altogether, we would expect a very different consequence, assuming standard models for activation. If, for example, the adduct trapped the S4 segment in an inward position, the channel would presumably be incapable of opening. By contrast, if it were stuck outward, activation would be shifted in a hyperpolarizing direction and channel opening would be easier, albeit less voltage dependent. This prediction is consistent with our results. However, a frozen S4 segment is inconsistent with the peculiar effects of hyperpolarizing prepulses.
Contrast between Channels and S4 Segments
Whereas many potassium channels are believed to be
homotetramers with four identical S4 segments, the subunits of sodium channels and the
1 subunits of calcium channels are monomers, each of which contains
four different S4 segments. The difference in primary
structure among the S4 segments of sodium channels is
highlighted by the fact that the number of basic residues ranges from four to eight in the four domains.
Some differences in the function of the individual S4
segments of both sodium and calcium channels have
been revealed by systematic mutagenesis (Chen et al.,
1996
; Garcia et al., 1997
; Kontis and Goldin, 1997
; Kontis et al., 1997
).
One of the most surprising results of this study is that
activation is so strongly affected by MTSET modification, whereas both steady state inactivation (Fig. 3 B)
and the voltage-dependent kinetics of inactivation
(Figs. 2 D and 4) are relatively insensitive to this treatment. Although the earliest voltage-clamp study of sodium channel gating proposed a model in which activation and inactivation were independent processes
(Hodgkin and Huxley, 1952), a myriad of later experiments showed that these processes are strongly coupled
in the sense that inactivation gets much of its voltage
dependence from activation (for reviews, see Armstrong, 1981
; French and Horn, 1983
; Bezanilla, 1985
;
Patlak, 1991
; Keynes, 1994
). However, some aspects of
sodium channel inactivation are inherently voltage dependent (Swenson, 1983
; Vandenberg and Horn, 1984
;
Greeff and Forster, 1991
; Sheets and Hanck, 1995
).
Furthermore, activation gating can be shifted substantially by mutations in the S4 segments of domains 1-3
without affecting inactivation kinetics (Stühmer et al.,
1989
; Chen et al., 1996
; Kontis and Goldin, 1997
; Kontis et al., 1997
).
The coupling between activation and inactivation is
not completely oblivious to the conformation of D2/
S4, because if opening is slowed by a hyperpolarized
prepulse, macroscopic inactivation is also slowed (Figs.
3 A and 5 C). This is consistent with the fact, as demonstrated by single channel studies (Aldrich et al., 1983;
Horn and Vandenberg, 1984
), that open channels tend
to inactivate more rapidly than closed channels. However, conformational changes of D2/S4 that bypass the
open state have no apparent effect on either the voltage dependence or kinetics of inactivation (Figs. 3 B, 4,
and 6 B). Overall, the data support the idea that some voltage sensors, especially D4/S4, strongly affect inactivation, whereas others, like D2/S4, play a relatively minor role in the inactivation process. Our results indicate that, as in the Hodgkin-Huxley model (see also
Keynes, 1994
), certain aspects of activation gating are
poorly coupled to inactivation. This component of the
activation machinery, which apparently includes D2/
S4, is likely to contribute to the asymmetrical gating
charge that is not immobilized by inactivation (Armstrong and Bezanilla, 1977
). Mutagenic studies are beginning to elucidate which voltage sensors are dedicated primarily to activation and which serve double
duty for both gating mechanisms.
After modification, the activation kinetics are slowed
by hyperpolarized prepulses (Figs. 3 and 5). A similar
phenomenon is seen for potassium current (Cole and
Moore, 1960); however, the effect is typically much
smaller in sodium currents (Armstrong and Bezanilla,
1974
). A partial explanation of this phenomenon in potassium channels is that there is substantial slow gating charge movement at very hyperpolarized potentials
(Bezanilla et al., 1994
). Our results suggest that charge
movement in sodium channels in this voltage range is
typically fast. Modification of D2/S4 shifts its voltage
dependence and slows its kinetics, sufficient to introduce a substantial Cole-Moore shift into these sodium
channels.
Another contrast between our studies and those involving potassium channels is that we have been able to
describe our data for both D2/S4 and D4/S4 by a first-order kinetic process. Detailed studies of potassium
channels suggest, however, that S4 movement occurs in
at least two sequential steps upon a change of membrane potential (Schoppa et al., 1992; Bezanilla et al.,
1994
; Sigg et al., 1994
; Zagotta et al., 1994
); similar arguments have been advanced for sodium channels,
based largely on gating current measurements in squid
axon (Keynes, 1994
). The most likely explanation for
the discrepancies in these studies is that the charge
translocation in sodium channels is also sequential but
has a predominant rate-limiting step. As evidence of
this possibility, the rate-limiting kinetic process revealed by D2/S4 modification does not have the full voltage dependence expected for an S4 segment, only
translocating ~1.2 e0. In fact, the sequential nature of
charge movement in potassium channels can be rather
subtle in WT channels (e.g., Schoppa et al., 1992
; Seoh
et al., 1996
). In the case of sodium channels, the data
presented here and previously (Yang and Horn, 1995
)
show that S4 movement can be represented as a first-
order process, but the supporting evidence always involves mutated S4 segments, begging the question
whether WT S4 segments behave in this manner. In
spite of this ambiguity, the modification employed in
this paper allowed us the unique opportunity to examine the voltage-dependent movement of a single S4 segment and also the relationship between the D2/S4 conformation and the gating behavior of the channels.
Clearly the translocation of S4 charges in potassium,
calcium, and sodium channels involves a delicately orchestrated procession of steps that probably includes
hydration and dehydration of charged residues as they
traverse the core of the protein. The local environment at each end of the S4 segment undoubtedly plays a role
in the energetics of S4 movement. Our data with D4/
S4, for example, suggest the presence of a negatively
charged vestibule at the outer mouth of the S4 channel
(Yang et al., 1997).
HERG-like Behavior of a Sodium Channel
A class of cardiac arrhythmias has been linked to the
potassium channel gene HERG (Curran et al., 1995).
The currents of the HERG channel are inwardly rectifying, although the channel has the structure of a typical
outward rectifier (Sanguinetti et al., 1995
; Trudeau et
al., 1995
; Schönherr and Heinemann, 1996
; Smith et
al., 1996
; Wang et al., 1997
). The bizarre biophysical
properties of HERG are neatly explained by the fact
that inactivation is more rapid than activation upon depolarization, and recovery from inactivation is more
rapid than deactivation upon hyperpolarization (Sanguinetti et al., 1995
; Trudeau et al., 1995
; Schönherr
and Heinemann, 1996
; Smith et al., 1996
; Wang et al.,
1997
). Our results with modified D2:R1C sodium channels are similar to those obtained with HERG, although
the kinetic discrepancies between activation and inactivation are not as severe in the sodium channel. Specifically, prepulse inactivation and recovery from inactivation are nonmonotonic in both cases. As in HERG, the
biophysical abnormality of modified D2:R1C sodium
channels is due primarily to unusually slow activation kinetics. The data in both cases show that a delicate balance between the kinetics of these two processes is crucial for normal function.
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FOOTNOTES |
---|
Address correspondence to Richard Horn, Department of Physiology, Institute of Hyperexcitability, Jefferson Medical College, 1020 Locust Street, Philadelphia, Pennsylvania 19107. Fax: 215-503-2073; E-mail: hornr{at}jeflin.tju.edu
Received for publication 28 October 1997 and accepted in revised form 11 December 1997.
1 Abbreviations used in this paper: D2:R1C, R669C mutant of hSkM1; D2: R1C-SET, MTSET-modified D2:R1C; G-V, conductance-voltage; I-V, current-voltage; MTS, methanethiosulfonate; SET, S-ethyltrimethylammonium; WT, wild type.We thank Dorothy VanDeCarr for assistance in making the mutation and Carol Deutsch for insightful comments on the manuscript.
Supported by National Institutes of Health grants AR-41691 (R. Horn) and NS-32387 (A.L. George).
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Aggarwal, S.K., and R. MacKinnon. 1996. Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron 16: 1169-1177 [Medline]. |
2. | Akabas, M.H., D.A. Stauffer, M. Xu, and A. Karlin. 1992. Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science 258: 307-310 [Medline]. |
3. | Aldrich, R.W., D.P. Corey, and C.F. Stevens. 1983. A reinterpretation of mammalian sodium channel gating based on single channel recording. Nature 306: 436-441 [Medline]. |
4. |
Armstrong, C.M., and
F. Bezanilla.
1974.
Charge movement associated with the opening and closing of the activation gates of the
Na channels.
J. Gen. Physiol.
63:
533-552
|
5. | Armstrong, C.M., and F. Bezanilla. 1977. Inactivation of the sodium channel. II. Gating current experiments. J. Gen. Physiol. 70: 567-590 [Abstract]. |
6. |
Armstrong, C.M..
1981.
Sodium channels and gating currents.
Physiol. Rev.
61:
644-683
|
7. | Bezanilla, F.. 1985. Gating of sodium and potassium channels. J. Membr. Biol. 88: 97-111 [Medline]. |
8. | Bezanilla, F., E. Perozo, and E. Stefani. 1994. Gating of Shaker K+ channels. II. The components of gating currents and a model of channel activation. Biophys. J. 66: 1011-1021 [Abstract]. |
9. | Catterall, W.A.. 1986. Molecular properties of voltage-sensitive sodium channels. Annu. Rev. Biochem. 55: 953-985 [Medline]. |
10. | Chahine, M., A.L. George Jr., M. Zhou, S. Ji, W. Sun, R.L. Barchi, and R. Horn. 1994. Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation. Neuron 12: 281-294 [Medline]. |
11. | Chen, L.-Q., V. Santarelli, R. Horn, and R.G. Kallen. 1996. A unique role for the S4 segment of domain 4 in the inactivation of sodium channels. J. Gen. Physiol. 108: 549-556 [Abstract]. |
12. | Cole, K.S., and J.W. Moore. 1960. Potassium ion current in the squid giant axon: dynamic characteristic. Biophys. J 1: 1-14 [Medline]. |
13. | Curran, M.E., I. Splawski, K.W. Timothy, G.M. Vincent, E.D. Green, and M.T. Keating. 1995. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80: 795-803 [Medline]. |
14. |
Falke, J.J.,
A.F. Dernburg,
D.A. Sternberg,
N. Zalkin,
D.L. Milligan, and
D.E. Koshland.
1988.
Structure of a bacterial sensory receptor. A site-directed sulfhydryl study.
J. Biol. Chem.
263:
14850-14858
|
15. | French, R.J., and R. Horn. 1983. Sodium channel gating: models, mimics, and modifiers. Annu. Rev. Biophys. Bioeng. 12: 319-356 [Medline]. |
16. | French, R.J., E. Prusak-Sochaczewski, G.W. Zamponi, S. Becker, A. Shavantha, Kularatna, and R. Horn. 1996. Interactions between a pore-blocking peptide and the voltage sensor of a sodium channel: an electrostatic approach to channel geometry. Neuron 16: 407-413 [Medline]. |
17. | Garcia, J., J. Nakai, K. Imoto, and K.G. Beam. 1997. Role of S4 segments and the leucine heptad motif in the activation of an L-type calcium channel. Biophys. J. 72: 2515-2523 [Abstract]. |
18. | Greeff, N.G., and I.C. Forster. 1991. The quantal gating charge of sodium channel inactivation. Eur. Biophys. J. 20: 165-176 [Medline]. |
19. | Guy, H.R., and F. Conti. 1990. Pursuing the structure and function of voltage-gated channels. Trends Neurosci 13: 201-206 [Medline]. |
20. | Hodgkin, A.L., and A.F. Huxley. 1952. A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. 117: 500-544 [Medline]. |
21. | Horn, R. 1998. Explorations of voltage-dependent conformational changes using cysteine scanning. In Methods in Enzymology. Ion Channels. Academic Press Inc., New York. In press. |
22. | Horn, R., and C.A. Vandenberg. 1984. Statistical properties of single sodium channels. J. Gen. Physiol. 84: 505-534 [Abstract]. |
23. | Ji, S., A.L. George Jr., R. Horn, and R.L. Barchi. 1996. Paramyotonia congenita mutations reveal different roles for segments S3 and S4 of domain D4 in hSkM1 sodium channel gating. J. Gen. Physiol 107: 183-194 [Abstract]. |
24. | Keynes, R.D.. 1994. The kinetics of voltage-gated ion channels. Q. Rev. Biophys 27: 339-434 [Medline]. |
25. |
Kontis, K.J., and
A.L. Goldin.
1997.
Sodium channel inactivation is
altered by substitution of voltage sensor positive charges.
J. Gen. Physiol
110:
403-413
|
26. |
Kontis, K.J.,
A. Rounaghi, and
A.L. Goldin.
1997.
Sodium channel
activation gating is affected by substitutions of voltage sensor positive charges in all four domains.
J. Gen. Physiol.
110:
391-401
|
27. | Kuo, C.-C., and B.P. Bean. 1994. Na+ channels must deactivate to recover from inactivation. Neuron 12: 819-829 [Medline]. |
28. | Larsson, H.P., O.S. Baker, D.S. Dhillon, and E.Y. Isacoff. 1996. Transmembrane movement of the Shaker K+ channel S4. Neuron 16: 387-397 [Medline]. |
29. | Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Direct physical measure of conformational rearrangement underlying potassium channel gating. Science 271: 213-216 [Abstract]. |
30. |
Patlak, J..
1991.
Molecular kinetics of voltage-dependent Na+ channels.
Physiol. Rev.
71:
1047-1080
|
31. | Sanguinetti, M.C., C. Jiang, M.E. Curran, and M.T. Keating. 1995. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81: 299-307 [Medline]. |
32. | Schönherr, R., and S.H. Heinemann. 1996. Molecular determinants for activation and inactivation of HERG, a human inward rectifier potassium channel. J. Physiol. 493: 635-642 [Abstract]. |
33. | Schoppa, N.E., K. McCormack, M.A. Tanouye, and F.J. Sigworth. 1992. The size of gating charge in wild-type and mutant Shaker potassium channels. Science 255: 1712-1715 [Medline]. |
34. | Seoh, S.A., D. Sigg, D.M. Papazian, and F. Bezanilla. 1996. Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. Neuron 16: 1159-1167 [Medline]. |
35. | Sheets, M.F., and D.A. Hanck. 1995. Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin. J. Gen. Physiol. 106: 617-640 [Abstract]. |
36. | Sigg, D., E. Stefani, and F. Bezanilla. 1994. Gating current noise produced by elementary transitions in Shaker potassium channels. Science 264: 578-582 [Medline]. |
37. | Sigworth, F.J.. 1994. Voltage gating of ion channels. Q. Rev. Biophys. 27: 1-40 [Medline]. |
38. | Smith, P.L., T. Baukrowitz, and G. Yellen. 1996. The inward rectification mechanism of the HERG cardiac potassium channel. Nature 379: 833-836 [Medline]. |
39. | Stühmer, W., F. Conti, H. Suzuki, X. Wang, M. Noda, N. Yahagi, H. Kubo, and S. Numa. 1989. Structural parts involved in activation and inactivation of the sodium channel. Nature 339: 597-603 [Medline]. |
40. | Swenson, R.P. Jr.. 1983. A slow component of gating current in crayfish giant axons resembles inactivation charge movement. Biophys. J. 41: 245-249 [Abstract]. |
41. | Trudeau, M.C., J.W. Warmke, B. Ganetzky, and G.A. Robertson. 1995. HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269: 92-95 [Medline]. |
42. | Vandenberg, C.A., and R. Horn. 1984. Inactivation viewed through single sodium channels. J. Gen. Physiol. 84: 535-564 [Abstract]. |
43. | Wang, S., L. Shuguang, M.J. Morales, H.C. Strauss, and R.L. Rasmusson. 1997. A quantitative analysis of the activation and inactivation kinetics of HERG expressed in Xenopus oocytes. J. Physiol. (Camb.) 502: 45-60 [Abstract]. |
44. | Yang, N., and R. Horn. 1995. Evidence for voltage-dependent S4 movement in sodium channels. Neuron 15: 213-218 [Medline]. |
45. | Yang, N., A.L. George Jr., and R. Horn. 1996. Molecular basis of charge movement in voltage-gated sodium channels. Neuron 16: 113-122 [Medline]. |
46. | Yang, N., A.L. George, and R. Horn. 1997. Probing the outer vestibule of a sodium channel voltage sensor. Biophys. J. 73: 2260-2268 [Abstract]. |
47. | Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1994. Shaker potassium channel gating III: evaluation of kinetic models for activation. J. Gen. Physiol. 103: 321-362 [Abstract]. |