From the Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, California 92697-4025
The role of the voltage sensor positive charges in the activation and deactivation gating of the rat
brain IIA sodium channel was investigated by mutating the second and fourth conserved positive charges in the S4
segments of all four homologous domains. Both charge-neutralizing (by glutamine substitution) and -conserving mutations were constructed in a cDNA encoding the sodium channel subunit that had fast inactivation removed
by the incorporation of the IFMQ3 mutation in the III-IV linker (West, J.W., D.E. Patton, T. Scheuer, Y. Wang,
A.L. Goldin, and W.A. Catterall. 1992. Proc. Natl. Acad. Sci. USA. 89:10910-10914.). A total of 16 single and 2 double mutants were constructed and analyzed with respect to voltage dependence and kinetics of activation and deactivation. The most significant effects were observed with substitutions of the fourth positive charge in each domain. Neutralization of the fourth positive charge in domain I or II produced the largest shifts in the voltage dependence of activation, both in the positive direction. This change was accompanied by positive shifts in the voltage dependence of activation and deactivation kinetics. Combining the two mutations resulted in an even
larger positive shift in half-maximal activation and a significantly reduced gating valence, together with larger positive shifts in the voltage dependence of activation and deactivation kinetics. In contrast, neutralization of the
fourth positive charge in domain III caused a negative shift in the voltage of half-maximal activation, while the
charge-conserving mutation resulted in a positive shift. Neutralization of the fourth charge in domain IV did not
shift the half-maximal voltage of activation, but the conservative substitution produced a positive shift. These data
support the idea that both charge and structure are determinants of function in S4 voltage sensors. Overall, the data supports a working model in which all four S4 segments contribute to voltage-dependent activation of the sodium channel.
The voltage-dependent activation of sodium channels
is essential for the generation of the upstroke phase of
the action potential in most excitable cells. In brain
neurons, the sodium channel consists of an and two
accessory
subunits (Catterall, 1984
), but it has been
shown in heterologous expression systems that the
subunit alone is sufficient to produce functional sodium channels (Noda et al., 1986
; Goldin et al., 1986
).
The amino acid sequence of the sodium channel
subunit has been determined. The channel consists of four
homologous domains that are 50-70% identical at the
amino acid level (Noda and Numa, 1987
). Hydropathy analysis suggests that each domain is made up of six
transmembrane segments, termed S1-S6, and two segments that extend partially through the membrane to
form part of the pore (Durell and Guy, 1992
). Voltage-gated calcium and potassium channels have similar structural features, with the exception that potassium
channel genes encode a single homologous domain
that is functional as a tetramer (Jan and Jan, 1989
). A
key feature that these voltage-gated channels share is
the amphiphilic S4 segment, containing multiple repeats of a motif consisting of a positively charged residue followed by two hydrophobic ones. The S4 positive
charges are the only positive residues predicted to be
within the membrane potential. Therefore, the S4 segments are believed to be the voltage-sensing apparatus
of the channel that was originally proposed by Hodgkin and Huxley (1952)
.
The voltage sensor hypothesis predicts that positive
charges within the sodium channel S4 segments, and
possibly other residues, determine the voltage-dependent properties of the channel. The involvement of S4
positive charges in voltage-dependent gating of sodium
channels was first shown by Stühmer et al. (1989). They
found that neutralization of S4 positive charges in domains I and II of the rat brain II sodium channel
shifted the voltage dependence of activation and decreased the apparent gating valence, consistent with
the hypothesis that the charged residues serve as voltage sensors. However, analysis of the role of the S4 residues in potassium channels has clearly demonstrated
that there is not a direct correlation between the individual charges and the gating valence, although there is
a relationship between the number of positive charges
and the gating charge (Papazian et al., 1991
; Liman et
al., 1991
; Logothetis et al., 1992
, 1993
; Tytgat et al., 1993
). In addition, substitutions of hydrophobic residues in either sodium or potassium channels can have
even larger effects on gating (Auld et al., 1990
; Lopez
et al., 1991
; McCormack et al., 1993
; Fleig et al., 1994
).
The best estimates for the size of the gating charges in
the Shaker potassium channel (Schoppa et al., 1992
) and the skeletal muscle sodium channel (Hirschberg et
al., 1995
) are that ~12 charges must move across the
field during activation, which is easier to explain if all
four S4 segments are involved in gating (Hirschberg et
al., 1995
). Recent experiments have shown that the domain IV S4 segment of the sodium channel (Yang and Horn, 1995
; Yang et al., 1996
) and the S4 segment of the
Shaker potassium channel (Mannuzzu et al., 1996
; Larsson et al., 1996
) actually move during gating, and that
some of the charged residues directly contribute to the
gating charge (Perozo et al., 1994
; Aggarwal and MacKinnon, 1996
).
Most of the studies examining the role of the S4
charges have been carried out on the voltage-gated potassium channel, which functionally contains four identical S4 segments. It is probable that sequence divergence of the four domains in the sodium channel resulted in differential roles of each of the S4 segments.
Such specialization would be consistent with the original formulation of Hodgkin and Huxley (1952), in
which potassium channels contain four identical gates,
whereas sodium channels contain three activation gates
and one inactivation gate. It has previously been proposed that the S4 region of domain IV plays a unique
role in coupling sodium channel activation to inactivation (Chahine et al., 1994
; O'Leary et al., 1995
). More
recently, Chen et al. (1996)
examined the importance
of the S4 charges in the all four domains of the human
heart sodium channel, confirming that the S4 region of
domain IV is uniquely involved in sodium channel inactivation.
To investigate the role played by the four S4 segments in the voltage dependence of activation and deactivation, we constructed charge-neutralizing and -conserving substitutions of the second and fourth positive charges in the S4 segment of each domain of the rat brain IIA (RBIIA)1 channel. We show that the positively charged residues in all four S4 segments contribute unequally to the voltage-dependent properties of the channel, consistent with the hypothesis that the different domains have specialized roles in gating.
Sodium Channel Mutations and In Vitro Transcription
The parent construct used in all experiments was the cDNA encoding the subunit of the RBIIA voltage-gated sodium channel that had the IFMQ3 mutation in the III-IV linker, which removes fast inactivation (West et al., 1992
). This mutation was included because activation is not complete before inactivation begins at
many voltages (Aldrich et al., 1983
). Therefore, if inactivation is
intact, the measured peak macroscopic current is less than the
theoretical peak current as determined by activation alone. All of
the mutations were made by site-directed mutagenesis using M13
subclones of the RBIIA channel, as previously described (Kontis and Goldin, 1993
). The mutated fragments were ligated into the full length cDNA in a plasmid vector containing a T7 RNA polymerase promoter and Xenopus
globin 5
and 3
untranslated sequences. Capped, full length transcripts were generated in vitro
using a T7 RNA transcription kit (Ambion Inc., Austin, TX), purified by phenol-chloroform extraction and ethanol precipitation, and dissolved in 10 mM Tris-HCl, pH 7.0, at ~1 µg/µl. Sodium channel
1 subunit RNA was prepared by in vitro transcription from a plasmid containing the cDNA flanked by
globin
sequences.
Expression of Channels in Xenopus Oocytes
Oocytes were removed from Xenopus laevis females and treated
for 90-120 min with 2 mg/ml collagenase in OR-2 buffer (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.5) to disperse the follicle cells. The oocytes were washed in the same
buffer to remove residual collagenase, and then washed in ND96
(96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM
HEPES, pH 7.5). The activation of sodium channels expressed in
oocytes is very slow at negative potentials in the absence of the 1
subunit, and does not reach complete saturation at potentials
near the threshold of activation (Patton et al., 1994
). To facilitate
measurement of peak currents and activation kinetics over a wide
voltage range, oocytes were co-injected with RNA encoding sodium channel
and
1 subunits at an approximate molar ratio of
1
:10
1. The dilution of
subunit RNA was determined empirically to achieve current amplitudes in the range of 0.5-5 µA. An
excess of
1 RNA was used in these experiments to assure that
functional differences could not be attributed to limiting
amounts of this subunit. The oocytes were injected in the center
of the vegetal pole. Injected oocytes were incubated in ND96
containing 550 mg/liter sodium pyruvate, 0.5 mM theophylline,
and 50 µg/ml gentamicin for 2 d at 20°C before electrophysiological recording.
Electrophysiological Recording
Voltage clamping of Xenopus oocytes was performed using the
DAGAN CA-1 high performance oocyte clamp, DigiData 1200 Interface (DAGAN Corp., Minneapolis, MN) and pCLAMP software (version 6.0.3; Axon Instruments, Inc., Burlingame, CA).
Oocytes were perforated with a 25-gauge needle in the center of
the vegetal pole and were mounted on the voltage clamp stage
fitted with a perfusion cannula connected to a syringe pump
filled with internal solution. The internal solution was (mM): 88 K+, 10 EGTA-CsOH, 10 HEPES-CsOH, and 10 Na+, pH 7.5. The
external solution consisted of (mM): 120 Na+-methyl ethane sulfonate (MES), 10 HEPES-CsOH, 1.8 Ca++-MES, pH 7.5. Care was
taken to exclude chloride ions in making these solutions to avoid
potential interference from native oocyte chloride channel currents. Agarose bridges coupling the headstage to the bath were
filled with 0.5% low melting point agarose in 1 M NaOH-MES
(pH 7.5) and were fitted with 100-µm platinum wires to improve
frequency response. The headstage manifold wells were filled
with 1 M NaCl. The temperature in these experiments was regulated at 15°C by a Peltier device coupled to a feedback controller (HCC-100A; DAGAN Corp.). Voltage was monitored through a
0.2-0.5 M microelectrode filled with 3 M KCl, which was inserted through the animal pole of the oocyte.
Oocytes were maintained at 15°C and clamped at 100 mV for
at least 5 min to allow temperature equilibration and full recovery from inactivation. Current-voltage data were acquired using
a sampling rate of 50 kHz with a filter frequency of 10 kHz. Depolarizations from a holding potential of
100 mV ranged from
90 to +55 mV in 5-mV steps, and lasted 57.5 ms. In the case of
some mutants, the voltage range of depolarizations was modified
to compensate for shifts in v1/2. Instantaneous tail currents were
acquired with the same sampling and filter frequencies using a
two-pulse protocol. The first step was a depolarization to +20 mV
for 1-3 ms to achieve maximal activation. For mutants that had
large positive shifts in activation, this prepulse was to +60 mV.
The second step was a 33.7-ms depolarization ranging from
90
to +55 mV in 5-mV steps. All data were acquired using P/4 subtraction and capacitance compensation. The series resistance
ranged from 1 to 1.5 k
.
Data Analysis
Electrophysiological data were analyzed using pCLAMP software.
Maximum inward tail currents were measured after repolarization to 100 mV for each current-voltage data record. Nonlinear curve fitting was performed using Sigmaplot (version 4.0; Jandel Scientific, San Rafael, CA), which uses the Marquardt-Levenberg algorithm for nonlinear regression. The fraction of activated
channels was calculated by measuring the peak inward current after repolarization to
100 mV from depolarizations ranging
from
90 to +55 mV, and normalizing the values to the maximum inward current. Normalized current-voltage relationships
were fitted with a two-state Boltzmann function,
![]() |
(1) |
where I/Imax is the normalized current after repolarization, v1/2 is the half-maximal voltage of activation, and zm is the apparent gating valence.
Activation kinetics were measured by fitting the rising phase of current traces during step depolarizations to a single exponential equation,
![]() |
(2) |
where I is the current, A is the initial amplitude, (t k) is the interval over which the fit is applied,
m is the time constant, and C is the
baseline. The voltage dependence of the activation kinetics was
analyzed over a voltage range of
30 to +30 mV by fitting the data
to an equation describing a single voltage-dependent exponential:
![]() |
(3) |
where 0 is the time constant at 0 mV membrane potential (corrected by subtracting the baseline,
min), k is the constant of voltage dependence, and
min is the lower limit of the activation time
constant.
Deactivation kinetics were measured by fitting the instantaneous tail currents to the single exponential equation,
![]() |
(4) |
where d is the time constant for deactivation and I, A, (t
k), and
C have the same meanings as in Eq. 2. The voltage dependence of the deactivation kinetics was measured by fitting the data to a
voltage-dependent single exponential equation,
![]() |
(5) |
where 50 is the deactivation time constant at a membrane potential of
50 mV (corrected by subtracting the baseline,
min), k
is the constant of the voltage-dependent variation of deactivation
kinetics, and
min is the lower limit of the deactivation time constant. The average and standard deviations for each voltage-dependent parameter were determined by individually fitting the
data. Statistical significance was evaluated using Student's unpaired t test.
Voltage Dependence of Activation Is Shifted by Substitution of Some, but Not All, Positive Charges in Each S4 Domain
The voltage-gated sodium channel consists of four homologous domains that each contain six segments (S1-
S6) that completely traverse the cell membrane (Guy
and Seetharamulu, 1986). The S4 segments are highly
amphiphilic, each having from four to eight positively
charged residues. Our working model of the sodium
channel predicts that all four domains participate in
the voltage-dependent conformational changes that are
manifested in the electrophysiological behavior of the
channel. To examine the role of the voltage sensors of
the sodium channel in activation gating, point mutations were made in the S4 segment of each domain.
The sequences of the S4 segments are aligned in Fig. 1
to show the repeated motif of a positively charged residue followed by two hydrophobic residues. The numbering shown labels the positive charges starting from the extracellular end of each segment toward the intracellular end, so that the first positive charge in domain
I is called 1R1. In this labeling system, substitution of
the second positive charge of the domain I S4 segment
with a glutamine is denoted as 1R2Q. Single mutants
were constructed in which charge neutralizing (gluta-mine) and charge conserving (arginine or lysine) substitutions were made for the second and fourth residues of each S4 segment. Two double mutants were
also constructed, one that combines neutralizations of
the fourth positive charges in domains I and II (1K4Q
and 2K4Q), and the other combining neutralizations
of the fourth positive charges in domains III and IV
(3R4Q and 4R4Q).
To measure peak macroscopic currents without interference from inactivation, all mutations were made in
the RBIIA sodium channel containing the IFMQ3 mutation, which removes fast inactivation (West et al.,
1992). Fig. 2 A shows representative currents from IFMQ3, demonstrating that the channels activate and
remain open for the duration of the 50-ms depolarization. The tail currents visible upon repolarization (Fig.
2 A, arrow) are shown in greater detail in Fig. 2 B, and
reflect the peak conductance upon repolarization to
100 mV. The traces in Fig. 2 C represent tail currents
elicited after fully activating all channels with a 2-3-ms
depolarization to +20 mV, followed by 75-ms steps to
voltages ranging from
90 to +55 mV. The time
course of the current decay during these voltage steps
reflects sodium channel deactivation.
The voltage dependence of activation for IFMQ3 and
all of the mutant channels is shown in Fig. 3, which depicts the normalized peak tail currents (as determined
in Fig. 2 B) plotted against the depolarization potential. Parameters of the Boltzmann fits to the data are
shown in Table I. The gating valences were determined from the slopes of the Boltzmann equations. Although
most of the mutations resulted in statistically significant
shifts in the v1/2 for activation, only a few caused pronounced changes. The largest shifts were observed
when the fourth positive charge was neutralized. Neutralization of the fourth charge in domain I (1K4Q) or
II (2K4Q) resulted in a significant positive shift in v1/2, and the combination of the two substitutions (1K4Q:
2K4Q) resulted in a positive shift that was greater than
the sum of the two individual changes. The substitution
in domain I also significantly decreased the gating valence, and the double substitution resulted in an even
greater reduction in valence.
Table I. Parameters of the Voltage Dependence of Activation |
In contrast, neutralization of the fourth positive charge in domain III (3R4Q) resulted in a negative shift in v1/2. However, the charge-conserving substitution of the same residue (3R4K) resulted in a positive shift, emphasizing the importance of both charge and structure at this position. No significant shift was observed for neutralization of the fourth charge in domain IV (4R4Q). In addition, none of the substitutions in either domain III or IV significantly affected the gating valence. Combining the two substitutions of the fourth charge in domains III and IV (3R4Q:4R4Q) resulted in a channel with properties similar to 3R4Q alone, supporting the idea that charges in the S4 segment of domain IV are not as involved in voltage-dependent activation.
Mutations of Positive Charges in the S4 Voltage Sensors Can Affect the Voltage Dependence of Activation Kinetics
The voltage dependence of sodium channel activation
is reflected in the behavior of activation kinetics over a
defined voltage range. Fig. 4 shows the time constants
obtained by fitting the current data obtained from
IFMQ3 with a single exponential equation as described
in MATERIALS AND METHODS. The value min is the minimum approached as v becomes more positive and the
constant k defines the voltage dependence. The value
of k can be used to calculate the voltage step required
for an e-fold change in
m for the condition when
min
goes to zero, since the relevant issue is the effect of the
charge, not the limiting
min. The term
0 represents
the value of
m when there is no potential across the
membrane after subtracting
min.
Table II. Parameters of Activation Kinetics |
Data for all of the mutants were similarly analyzed,
and the activation time constants are plotted on a log
scale against step potential in Fig. 5, with the parameters of the fits shown in Table II. Differences in 0 between each of the mutants and IFMQ3 can be expressed as a shift along the voltage axis, assuming that
there is no difference in k between the mutant and
IFMQ3 channels. Although this assumption is not completely correct for most of the mutants, the values for k
are similar enough between the mutants and IFMQ3 so
that the calculated shifts are generally valid. That is, the
value of the shift changed by <1.7 mV depending on
whether the mutant or IFMQ3 value for k was used for
all of the mutants except 1K4Q:2K4Q. For that double
mutant, the calculated voltage shift was 24.7 mV if the
mutant value for k was used.
As with the voltage dependence of activation, the
most significant differences were observed for neutralizations of the fourth positive charges in domains I and
II. Each of these neutralizations resulted in a positive
voltage shift in m, and the combination of the two resulted in a shift that was approximately the sum of the
individual changes. Therefore, neutralization of either of these charges shifts the voltage dependence of both
activation and activation kinetics in the positive direction.
Substitutions of the comparable charges in domains
III and IV resulted in less dramatic effects on the kinetics of activation. The 3R4Q mutation resulted in a positive voltage shift in m, in contrast to the negative shift
in the voltage dependence of activation that was observed for the same mutant (Table I). The 4R4Q mutation caused a negative voltage shift in
m, and this mutant did not demonstrate any significant shift in the
voltage dependence of activation. Combination of the
two mutations (3R4Q:4R4Q) resulted in a channel with
kinetics similar to those of IFMQ3, which is not surprising considering that each mutant shifts
m in the opposite direction.
Voltage Dependence of Deactivation Kinetics Is Altered by Some Positive Charge Mutations in the S4 Voltage Sensors
Upon returning to hyper-polarized membrane potentials, activated sodium channels rapidly deactivate. The
rate of deactivation slows in a voltage-dependent manner as the membrane potential becomes more positive.
Fig. 6 shows the time constants obtained by fitting the
instantaneous tail currents obtained from IFMQ3 with
a single exponential equation as described in MATERIALS AND METHODS. The term 50 is the time constant
at a membrane potential of
50 mV after subtracting
the baseline (
min), k is the exponential constant, and
min is the minimum time constant approached at increasingly negative potentials. As with activation, k can
be used to calculate the voltage step required for an
e-fold change in
d for the condition when
min goes to
zero, and differences in
50 can be expressed as shifts
along the voltage axis, with the same assumptions as described earlier for
m. The value of
50 mV was chosen
arbitrarily because the
d values become extremely
large and subject to greater error at 0 mV.
Table III. Parameters of Deactivation Kinetics |
Data for all of the mutants were similarly analyzed,
and the deactivation time constants are plotted on a log
scale against step potential in Fig. 7. The values for 50,
voltage shift, k, and the voltage required for an e-fold
change are shown in Table III. As was the case for the
voltage dependence of activation and activation kinetics, neutralizations of the fourth positive charges had
the most significant effects. Neutralization of the fourth
charge in domains I or II resulted in a positive shift in
d, and neutralization of the fourth charge in domain
III (3R4Q) resulted in a large negative shift. However,
the charge-conserving mutation of the same residue
(3R4K) caused a shift in the positive direction of comparable magnitude. The opposite effects of the charge-neutralizing and -conserving substitutions at this position are comparable with the results observed for the
voltage dependence of activation. Substitution of the
fourth charge in domain IV (4R4Q) resulted in no significant changes, as was the case for the parameters examined previously. However, combining the mutations in domains III and IV (3R4Q:4R4Q) resulted in the largest negative shift in deactivation kinetics.
This study examines the roles in activation gating of the
four putative voltage sensors (S4 segments) in the sodium channel. Previous reports have demonstrated that
the S4 regions in the sodium channel are involved in
voltage-dependent gating (Stühmer et al., 1989; Chen
et al., 1996
), and our results are consistent with that interpretation. However, distinct differences were observed in the effects of comparable mutations in each of the
four domains, suggesting that there is a significant extent of differentiation between the domains.
It is clear from previous results with sodium (Chen et
al., 1996) and potassium (Papazian et al., 1991
; Liman
et al., 1991
; Logothetis et al., 1992
, 1993
; Tytgat et al.,
1993
) channels that each of the charges in the S4 regions are not equally important for activation gating. In
this study, elimination of charges at only five positions
altered the v1/2 for activation, and elimination of the
charges at only two positions (1K4Q and 2R2Q) decreased the gating valence. The changes in gating valence must be considered approximations because
these values were determined from the slope of the
Boltzmann fits. Limiting slope analysis is a more accurate means of determining zm, but it is necessary to
measure current at extremely low probability of opening to approximate limiting slope (Sigworth, 1995
).
The measurement of such small currents was too unreliable in these experiments to provide informative data,
although the general trends determined from limiting
slope analysis were comparable with those determined
from fits to the Boltzmann equation (data not shown).
Even if limiting slopes were used, it is not possible to
accurately determine the extent to which any one
charge functions as a voltage sensor for activation by
this type of analysis, for a number of reasons. First,
many of the mutations significantly shift the v1/2 for activation, indicative of a change in the relative stabilities
of the resting and activated states. This change could result from an interaction between the charged residue
and an amino acid of the opposite charge elsewhere in
the channel in one state, as has been shown to be the
case for the Shaker potassium channel (Papazian et al.,
1995). The net effect could be a reduction in the apparent gating valence. Second, the apparent overall
gating event may be largely determined by one charge
or domain that dominates the kinetic process. Therefore,
the conductance-voltage curve will reflect the movement
of that one gate, and neutralization of charges in other
domains may have no effect on the measured gating valence. Finally, if any one charge represents just 1 of the
12 charges that move across the field during activation
of the sodium channel (Hirschberg et al., 1995
), then
neutralization of that charge will remove ~8% of the
total gating charge. A decrease of 8% would not be detected by the analysis of conductance-voltage curves.
The most definitive approach to determining the role
of each charged residue in gating is to examine the effects of the mutations on the gating currents, as has
been done for the Shaker potassium channel (Perozo et
al., 1992
, 1994
; Sigg and Bezanilla, 1997
). These studies
are in progress.
Of the mutations that shifted the v1/2 for activation,
the most pronounced effects were observed when the
fourth charge in each of domains I, II, and III was neutralized. It might be expected that neutralization of a
single charge in a domain that contains fewer charges
would have larger effects. Domain I contains only four
positive charges, domains II and III each contain five
positive charges, and domain IV has eight positive
charges. The mutations in domains I and II shifted the
v1/2 in the positive direction, indicating that these two
mutations stabilized a resting state compared with an
activated state of the channel. Consistent with this interpretation, both mutations also resulted in positive shifts in the time constants for activation (m) and deactivation (
d). The double mutant combining these two
neutralizations resulted in shifts in the v1/2 and time
constants in the same direction, only larger, indicating
that the effects of the two neutralization mutations are
at least additive.
On the other hand, neutralization of the fourth
charge in domain III (3R4Q) shifted the v1/2 for activation in the negative direction, suggesting that an activated state has been stabilized compared with a resting
state. The negative shift in d is consistent with this interpretation, but this mutant also exhibited a positive shift in
m, which is the opposite of what would be expected. It is clear that this residue in domain III is particularly sensitive to the structure of the amino acid, because the charge-conserving mutation (3R4K) demonstrated large positive shifts in v1/2 and
d, and a small
positive shift in
m. Therefore, substitutions at this position alter the conformation of the protein either in
both resting and activated states, or in a transitional
state between the two, accounting for the slowing down
of both activation and inactivation. The 3R4Q mutation significantly increased the time constants for activation (
0) and deactivation (
50), consistent with an
increase in the energy barrier for the transition. Neutralization of the charges in domain IV had the smallest
effects on activation, with only a minor shift in v1/2 for
4R2Q and no shift for 4R4Q.
Most of the mutations that affected the kinetics of activation and deactivation had comparable effects on
the two time constants, with the notable exception of
3R4Q as described above. However, it is not possible to
compare the absolute values of the time constants for
activation (m) and deactivation (
d) because there is
essentially no overlap in the voltage regions for which
the two were determined. The time constants for activation (
m) were determined at potentials equal to or
more positive than
30 mV (Fig. 5). In contrast, the
time constants for deactivation (
d) for most of the mutants were determined at potentials equal to or more
negative than
40 mV (Fig. 7). The maximum values
for
m were ~10 ms at
30 mV, and the maximum values for
d were ~2 ms at
40 mV. Because these voltages were in the regions in which each time constant
was most rapidly increasing, we do not know if this apparent difference represents a real feature of the channel kinetics.
Two previous studies have also examined the effects
of mutations that neutralize the S4 charges in the sodium channel. Stühmer et al. (1989) studied the effects
of mutations in domains I and II in the rat brain II
channel using macropatch recording of Xenopus oocytes. Chen et al. (1996)
investigated the effects of mutations of the first and third charges in all four domains
of the human heart 1 channel using two-electrode voltage clamping of oocytes. All three studies used different electrophysiological recording techniques, and Chen
et al. (1996)
examined a completely different channel
(the rat brain II and IIA channels are identical in the four S4 regions). In addition, both of the previous studies examined sodium channels with fast inactivation intact, whereas our experiments were carried out with
fast inactivation removed. Despite these differences, the
results for the wild-type channel are remarkably similar
in all three cases. For the voltage dependence of activation, Stühmer et al. (1989)
obtained v1/2 of
32.7 ± 7 mV, Chen et al. (1996)
obtained v1/2 of
31.9 ± 0.79 mV, and we obtained v1/2 of
29.1 ± 2.7 mV. With respect to gating valence, Stühmer et al. (1989)
based
their analysis on three identical gating charges, so their
values should be equivalent to 1/3 of the other two. Multiplying the values from Stühmer et al. (1989)
by
three, they obtained zm of 6.3 ± 0.6 e0, Chen et al.
(1996)
obtained zm of 5.6 ± 0.4 e0, and we obtained zm
of 5.1 ± 1.1 e0. It might have been expected that removal of fast inactivation would shift the v1/2 of activation in the negative direction (Cota and Armstrong, 1989
; Gonoi and Hille, 1987
). However, this is not the
case in all preparations (Nonner et al., 1980
; Oxford,
1981
; Stimers et al., 1985
; Wang and Strichartz, 1985
;
Wang et al., 1985
), and it was not previously observed
for either the rat brain II (Stühmer et al., 1989
) or IIA
(Patton and Goldin, 1991
) channel.
The results of the three studies are summarized in Table IV. In domain I, there appears to be a trend such that charges closer to the cytoplasmic side have more effect on the gating process. Neutralization of the charge at position 3 or 4 resulted in a significant depolarizing shift in v1/2 and a significant decrease in zm, whereas neutralization of the charge at position 1 or 2 did not have a significant effect on either parameter. In contrast, neutralization of each of the charges in domain II significantly shifted v1/2 in the positive direction, and neutralization of each of the first three charges significantly decreased zm. All of the mutations in domains I and II that had significant effects on v1/2 shifted it in the positive direction. On the other hand, neutralization of either charge 1 or 4 in domain III shifted v1/2 in the negative direction, indicating that the presence of either of these charges stabilize a resting state. Only the neutralization of charge 3 in domain III reduced zm. Neutralization of charges 2 or 3 in domain IV shifted v1/2 in the positive direction, and neutralization of charge 1 or 3 reduced zm. These results suggest that there is a fundamental difference in the roles of each of the four domains with respect to activation gating, but that all are involved to some extent.
Table IV. Summary of Charge-neutralizing Mutation Effects on Activation |
Hodgkin and Huxley (1952) originally proposed that
voltage-gated sodium channels contain three activation
(m) gates and one inactivation (h) gate. When sodium
channels were first cloned, it was tempting to speculate
that those gates would correspond to the four S4 regions (Noda et al., 1984
). However, it is clear that at
least some positive charges in the S4 regions of all four domains are involved in activation as voltage-sensing elements, and that each of the four domains has specialized to some extent. It has previously been suggested that
domain IV plays a unique role in coupling activation to
inactivation (Chahine et al., 1994
; O'Leary et al., 1995
;
Tang et al., 1996
; Chen et al., 1996
). The accompanying
paper examines the effects of the S4 charge mutations on
sodium channel inactivation (Kontis and Goldin, 1997
).
Address correspondence to Dr. Alan L. Goldin, Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697-4025. Fax: 714-824-8598; E-mail: AGoldin{at}uci.edu
Received for publication 26 February 1997 and accepted in revised form 23 July 1997.
Dr. Kontis' present address is Hycor Biomedical Inc., Garden Grove, CA 92841.We thank Dr. Raymond Smith, Dr. Michael Pugsley, Ted Shih, Marianne Smith, and Dan Allen for helpful discussions during the course of this work, Dan Allen and Ian Jester for help with the experiments, and Mimi Reyes for excellent technical assistance.
This work was supported by grants from the National Institutes of Health (NS-26729) and the National Science Foundation (IBN9221984). A.L. Goldin is an Established Investigator of the American Heart Association.
RBIIA, rat brain IIA.
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. | Aldrich, R.W., D.P. Corey, and C.F. Stevens. 1983. A reinterpretation of mammalian sodium channel gating based on single channel recording. Nature (Lond.). 306: 436-441 [Medline]. |
3. | Auld, V.J., A.L. Goldin, D.S. Krafte, W.A. Catterall, H.A. Lester, N. Davidson, and R.J. Dunn. 1990. A neutral amino acid change in segment IIS4 dramatically alters the gating properties of the voltage-dependent sodium channel. Proc. Natl. Acad. Sci. USA. 87: 323-327 [Abstract]. |
4. | Catterall, W.A.. 1984. The molecular basis of neuronal excitability. Science (Wash. DC). 223: 653-661 [Medline]. |
5. | 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]. |
6. | 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]. |
7. | Cota, G., and C.M. Armstrong. 1989. Sodium channel gating in clonal pituitary cells. The inactivation step is not voltage dependent. J. Gen. Physiol. 94: 213-232 [Abstract]. |
8. | Durell, S.R., and H.R. Guy. 1992. Atomic scale structure and functional models of voltage-gated potassium channels. Biophys. J. 62: 238-250 [Abstract]. |
9. | Fleig, A., P.C. Ruben, and M.D. Rayner. 1994. Kinetic mode switch of rat brain IIA Na channels in Xenopus oocytes excised macropatches. Pflugers Arch. 427: 399-405 [Medline]. |
10. |
Goldin, A.L.,
T. Snutch,
H. Lubbert,
A. Dowsett,
J. Marshall,
V. Auld,
W. Downey,
L.C. Fritz,
H.A. Lester,
R. Dunn,
W.A. Catterall, and
N. Davidson.
1986.
Messenger RNA coding for only the ![]() |
11. | Gonoi, T., and B. Hille. 1987. Gating of Na channels: inactivation modifiers discriminate among models. J. Gen. Physiol. 89: 253-274 [Abstract]. |
12. | Guy, H.R., and P. Seetharamulu. 1986. Molecular model of the action potential sodium channel. Proc. Natl. Acad. Sci. USA. 83: 508-512 [Abstract]. |
13. | Hirschberg, B., A. Rovner, M. Lieberman, and J. Patlak. 1995. Transfer of twelve charges is needed to open skeletal muscle Na+ channels. J. Gen. Physiol. 106: 1053-1068 [Abstract]. |
14. | 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. (Cambr.). 117: 500-544 [Medline]. |
15. | Jan, L.Y., and Y.-N. Jan. 1989. Voltage-sensitive ion channels. Cell. 56: 13-25 [Medline]. |
16. | Kontis, K.J., and A.L. Goldin. 1993. Site-directed mutagenesis of the putative pore region of the rat IIA sodium channel. Mol. Pharmacol. 43: 635-644 [Abstract]. |
17. |
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
|
18. | 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]. |
19. | Liman, E.R., P. Hess, F. Weaver, and G. Koren. 1991. Voltage-sensing residues in the S4 region of a mammalian K+ channel. Nature. 353: 752-756 [Medline]. |
20. | Logothetis, D.E., S. Movahedi, C. Satler, K. Lindpaintner, and B. Nadal-Ginard. 1992. Incremental reductions of positive charge within the S4 region of a voltage-gated K+ channel result in corresponding decreases in gating charge. Neuron. 8: 531-540 [Medline]. |
21. | Logothetis, D.E., B.F. Kammen, K. Lindpaintner, D. Bisbas, and B. Nadal-Ginard. 1993. Gating charge differences between two voltage-gated K+ channels are due to the specific charge content of their respective S4 regions. Neuron. 10: 1121-1129 [Medline]. |
22. | Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1991. Hydrophobic substitution mutations in the S4 sequence alter voltage-dependent gating in Shaker K+ channels. Neuron. 7: 327-336 [Medline]. |
23. | Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Direct physical measure of conformational rearrangement underlying potassium channel gating. Science (Wash. DC). 271: 213-216 [Abstract]. |
24. | McCormack, K., L. Lin, and F.J. Sigworth. 1993. Substitution of a hydrophobic residue alters the conformational stability of Shaker K+ channels during gating and assembly. Biophys. J. 65: 1740-1748 [Abstract]. |
25. | Noda, M., S. Shimizu, T. Tanabe, T. Takai, T. Kayano, T. Ikeda, H. Takahashi, H. Nakayama, Y. Kanaoka, N. Minamino, et al . 1984. Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature (Lond.). 312: 121-127 [Medline]. |
26. | Noda, M., T. Ikeda, H. Suzuki, H. Takeshima, T. Takahashi, M. Kuno, and S. Numa. 1986. Expression of functional sodium channels from cloned cDNA. Nature (Lond.). 322: 826-828 [Medline]. |
27. | Noda, M., and S. Numa. 1987. Structure and function of sodium channel. J. Receptor Res. 7: 467-497 [Medline]. |
28. | Nonner, W., B.C. Spalding, and B. Hille. 1980. Low intracellular pH and chemical agents slow inactivation gating in sodium channels of muscle. Nature (Lond.). 284: 360-363 [Medline]. |
29. | O'Leary, M.E., L.-Q. Chen, R.G. Kallen, and R. Horn. 1995. A molecular link between activation and inactivation of sodium channels. J. Gen. Physiol. 106: 641-658 [Abstract]. |
30. | Oxford, G.S.. 1981. Some kinetic and steady-state properties of sodium channels after removal of inactivation. J. Gen. Physiol. 77: 1-22 [Abstract]. |
31. | Papazian, D.M., L.C. Timpe, Y.N. Jan, and L.Y. Jan. 1991. Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence. Nature (Lond.). 349: 305-310 [Medline]. |
32. | Papazian, D.M., X.M. Shao, S.-A. Seoh, A.F. Mock, Y. Huang, and D.H. Wainstock. 1995. Electrostatic interactions of S4 voltage sensor in Shaker K+ channel. Neuron. 14: 1293-1301 [Medline]. |
33. |
Patton, D.E.,
L.L. Isom,
W.A. Catterall, and
A.L. Goldin.
1994.
The
adult rat brain ![]() ![]() |
34. | Patton, D.E., and A.L. Goldin. 1991. A voltage-dependent gating transition induces use-dependent block by tetrodotoxin of rat IIA sodium channels expressed in Xenopus oocytes. Neuron. 7: 637-647 [Medline]. |
35. | Perozo, E., D.M. Papazian, E. Stefani, and F. Bezanilla. 1992. Gating currents in Shaker K+ channels. Implications for activation and inactivation models. Biophys. J. 62: 160-171 [Abstract]. |
36. | Perozo, E., L. Santacruz-Toloza, E. Stefani, F. Bezanilla, and D.M. Papazian. 1994. S4 mutations alter gating currents of Shaker K channels. Biophys. J. 66: 345-354 [Abstract]. |
37. | 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 (Wash. DC). 255: 1712-1715 [Medline]. |
38. |
Sigg, D., and
F. Bezanilla.
1997.
Total charge movement per channel. The relation between gating charge displacement and the
voltage sensitivity of activation.
J. Gen. Physiol.
109:
27-39
|
39. | Sigworth, F.J.. 1995. Charge movement in the sodium channels. J. Gen. Physiol. 106: 1047-1051 [Medline]. |
40. | Stimers, J.R., F. Bezanilla, and R.E. Taylor. 1985. Sodium channel activation in the squid giant axon. Steady state properties. J. Gen. Physiol. 85: 65-82 [Abstract]. |
41. | 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 (Lond.). 339: 597-603 [Medline]. |
42. | Tang, L., R.G. Kallen, and R. Horn. 1996. Role of an S4-S5 linker in sodium channel inactivation probed by mutagenesis and a peptide blocker. J. Gen. Physiol. 108: 89-104 [Abstract]. |
43. |
Tytgat, J.,
K. Nakazawa,
A. Gross, and
P. Hess.
1993.
Pursuing the
voltage sensor of a voltage-gated mammalian potassium channel.
J. Biol. Chem.
268:
23777-23779
|
44. | Wang, G.K., M.S. Brodwick, and D.C. Eaton. 1985. Removal of sodium channel inactivation in squid axon by the oxidant chloramine-T. J. Gen. Physiol. 86: 289-302 [Abstract]. |
45. | Wang, G.K., and G. Strichartz. 1985. Kinetic analysis of the action of Leiurus scorpion-toxin on ionic currents in myelizated nerve. J. Gen. Physiol. 86: 739-762 [Abstract]. |
46. | West, J.W., D.E. Patton, T. Scheuer, Y. Wang, A.L. Goldin, and W.A. Catterall. 1992. A cluster of hydrophobic amino acid residues required for fast Na+ channel inactivation. Proc. Natl. Acad. Sci. USA. 89: 10910-10914 [Abstract]. |
47. | 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]. |
48. | Yang, N., and R. Horn. 1995. Evidence for voltage-dependent S4 movement in sodium channels. Neuron. 15: 213-218 [Medline]. |