From the * Department of Molecular and Cellular Physiology, and Howard Hughes Medical Institute, Stanford University, Stanford,
California 94305
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ABSTRACT |
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Charged residues in the S4 transmembrane segment of voltage-gated cation channels play a key role in opening channels in response to changes in voltage across the cell membrane. However, the molecular mechanism of channel activation is not well understood. To learn more about the role of the S4 in channel gating, we constructed chimeras in which S4 segments from several divergent potassium channels, Shab, Shal, Shaw, and Kv3.2, were inserted into a Shaker potassium channel background. These S4 donor channels have distinctly different voltage-dependent gating properties and S4 amino acid sequences. None of the S4 chimeras have the gating behavior of their respective S4 donor channels. The conductance-voltage relations of all S4 chimeras are shifted to more positive voltages and the slopes are decreased. There is no consistent correlation between the nominal charge content of the S4 and the slope of the conductance-voltage relation, suggesting that the mutations introduced by the S4 chimeras may alter cooperative interactions in the gating process. We compared the gating behavior of the Shaw S4 chimera with its parent channels, Shaker and Shaw, in detail. The Shaw S4 substitution alters activation gating profoundly without introducing obvious changes in other channel functions. Analysis of the voltage-dependent gating kinetics suggests that the dominant effect of the Shaw S4 substitution is to alter a single cooperative transition late in the activation pathway, making it rate limiting. This interpretation is supported further by studies of channels assembled from tandem heterodimer constructs with both Shaker and Shaw S4 subunits. Activation gating in the heterodimer channels can be predicted from the properties of the homotetrameric channels only if it is assumed that the mutations alter a cooperative transition in the activation pathway rather than independent transitions.
Key words: Shaker; voltage-dependent gating; patch clamp ![]() |
INTRODUCTION |
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Voltage-dependent cation channels respond to changes
in the electric field across the cell membrane by undergoing conformational changes that open and close an
ion-permeable pore. These conformational changes involve the rearrangement of charges within the channel protein, making the rates of the conformational changes
voltage dependent. The movement of the charge within
the channel protein can be measured as gating current
(Schneider and Chandler, 1973; Armstrong and Bezanilla, 1974
; Keynes and Rojas, 1974
; Stühmer et al., 1991
;
Bezanilla et al., 1991
, 1994
; Schoppa et al., 1992
; Stefani et al., 1994
; Zagotta et al., 1994
a, 1994b). The elucidation of the molecular mechanism of the voltage-
dependent conformational change has been a major
goal of ion channel biophysics. This effort has been facilitated by the combination of powerful electrophysiological techniques and the ability to determine and alter the primary structure of channel proteins by recombinant DNA technology. For voltage-gated potassium
channels, the minimum requirement to make a functional channel is the association of four alpha subunits, each with a core region containing six putative transmembrane segments and a pore loop (MacKinnon,
1991
; Heginbotham and MacKinnon, 1992
; Kavanaugh
et al., 1992
; Liman et al., 1992
). A distinctive feature of
voltage-dependent cation channels is the fourth transmembrane segment (S4). The S4 is ~21 amino acids
long with positively charged basic amino acids, either
arginine or lysine, at every third position, separated by
hydrophobic and neutral residues (Noda et al., 1984
, 1986
; Salkoff et al., 1987
; Tanabe et al., 1987
, 1988
; Papazian et al., 1987
; Tempel et al., 1988
; Baumann et al.,
1988
; Ellis et al., 1988
; Kayano et al., 1988
). Because of
the high nominal charge density in this membrane
spanning segment and the fact that the motif is highly
conserved among voltage-gated cation channels, the S4
was initially proposed to be part of the voltage sensor
(Noda et al., 1986
; Catterall, 1988
; Durrell and Guy,
1992
).
If the S4 is part of the voltage sensor, the S4 and its
charges should move through the electric field in response to membrane depolarization. Evidence from
both skeletal muscle sodium channels and Shaker potassium channels indicates that a conformational change in
the S4 or in adjacent channel regions occurs in the channel protein upon activation (Yang and Horn,
1995; Yang et al., 1996
; Larsson et al., 1996
; Mannuzzu
et al., 1996
; Aggarwal and MacKinnon, 1996
; Seoh et
al., 1996
; Yusaf et al., 1996
). Another prediction, that
neutralization of S4 charges should decrease the
amount of charge moved during channel activation,
has also been confirmed in Shaker (Aggarwal and
MacKinnon, 1996
; Seoh et al., 1996
). These experiments provide strong evidence for a role for the S4 in
sensing voltage. However, the individual S4 charges do
not seem to contribute equally to the gating charge,
and properties of the charged residues other than the
charge are also important determinants of gating behavior (see also Perozo et al., 1994
). There is also evidence that a negatively charged residue in the S2 of
Shaker potassium channels may serve as a counter ion
for S4 charges and participate in the voltage-sensing
process (Papazian et al., 1995
; Seoh et al., 1996
; Tiwari-Woodruff et al., 1997
).
Mutation of nonbasic residues in the S4 through the
S5 segments can also cause significant changes in voltage-dependent gating in Shaker potassium channels
(Zagotta and Aldrich, 1990; Gautam and Tanouye,
1990
; Lichtinghagen et al., 1990
; McCormack et al., 1991
, 1993
; Lopez et al., 1991
; Schoppa et al., 1992
;
Logothetis et al., 1993
; Aggarwal and MacKinnon,
1996
) and in sodium channels (Auld et al., 1988
). In
experiments by Aggarwal and MacKinnon (1996)
, simultaneous substitution of several nonbasic S4 residues, with no change in the charged residues, alters the
voltage range over which gating charge moves but not
the total amount of gating charge moved. Mutation of
noncharged residues in this region could alter the coupling between voltage sensing and opening of the channel, or could alter movement of the charge
through the membrane electric field.
Evidence for cooperative transitions leading to potassium channel activation has come from studies of activation kinetics in Shaker (Sigworth, 1993; Zagotta et
al., 1994
b; Bezanilla et al., 1994
) and from studies of
steady state channel kinetics in heteromultimeric mammalian potassium channels (Tytgat and Hess, 1992
;
Hurst et al., 1992
). All of the several kinetic models that have been proposed to describe Shaker channel activation have in common multiple voltage-dependent transitions, with at least one transition involving the cooperative movement of subunits (Sigworth, 1993
; Bezanilla et
al., 1994
; Zagotta et al., 1994
a; McCormack et al., 1994
).
Because cooperativity can be a major determinant of
the voltage dependence of channel opening, assessment of the contribution of cooperative interactions to
the activation process is essential to a mechanistic understanding of potassium channel gating. Mutations that
change cooperativity can lead to large changes in the
slope of the conductance-voltage curve, giving the erroneous appearance of a change in the total gating
charge (Schoppa et al., 1992
; Sigworth, 1993
; Sigg et
al., 1994
; Zagotta et al., 1994
a).
In this study, we investigated further the role of the
S4 in channel activation by generating several Shaker
channel variants with chimeric S4 insertions. Much of
the previous work targeted specific amino acids for replacement based on presumed functions of the residues or their conservation among related channels. Our goal was to reveal properties of the S4 not evident
from this previous work. The strength of the chimeric
approach is that it does not make assumptions a priori
about the functional and structural importance of specific amino acids in the S4 sequence (see also Logothetis et al., 1993; Tang and Papazian, 1997
; Koopmann et al., 1997
).
While we generated several S4 chimeras, we focused
our study on the Shaw S4 chimera because its gating behavior differs most significantly from that of the Shaker
channel. Our analysis is based on a simplified model for
Shaker gating, which we use to differentiate changes in
the amount of charge moved during channel gating
from changes in cooperativity in the activation pathway. We also examine activation in channels assembled
from tandem heterodimer constructs with Shaker and
Shaw S4 subunits to investigate further whether the
mutations alter cooperative or independent transitions in the activation pathway. In the following paper, we
determine which of the amino acid substitutions in the
Shaw S4 chimera are responsible for differences in gating between Shaw S4 and Shaker and we present a
more detailed model that can account for changes in
both the kinetic and steady state properties of the mutant macroscopic ionic currents. The importance of
these residues in determining the properties of voltage-dependent activation was not previously recognized.
Preliminary results have been reported previously in
abstract form (Smith-Maxwell et al., 1993, 1994
; Ledwell et al., 1995
).
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MATERIALS AND METHODS |
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Chimera Construction
All S4 chimeras were generated in a mutant form of ShB (Papazian et al., 1987; Kamb et al., 1987
; Pongs et al., 1988
), designated ShB
6-46, in which fast N-type inactivation was removed by deletion of amino acids 6-46 (Hoshi et al., 1990
; Zagotta et al., 1990
). This allowed us to study activation in isolation from the fast (N-type) inactivation process. This Shaker construct still undergoes a relatively slow inactivation process (C-type inactivation). The time constant for C-type inactivation of ~1.5 s is sufficiently slow that it does not interfere with measurement of activation parameters (Hoshi et al., 1991
). ShB
6-46 cDNA was further modified by introduction of a unique "silent" StuI restriction enzyme
site 3' to the S4 coding region, at amino acid positions 380-382
(see Fig. 1). This modification, which does not alter the amino
acid sequence, was made using the polymerase chain reaction
method to generate a cassette that was inserted between two naturally occurring unique restriction enzyme sites within the
Shaker cDNA, StyI and NsiI. To generate the S4 chimeras, sense
and antisense oligonucleotides were made based on the S4 sequences of the cloned channels Shab, Shal, Shaw, and Kv3.2 (RKShIIIA) (Butler et al., 1989
; McCormack et al., 1990
). Sense and
antisense oligonucleotides were annealed and inserted into the
S4 of ShB
6-46 between the unique StyI site 5' to the S4 coding
region, at amino acids 357-359, and the silent StuI site 3' to the
S4 (see Fig. 1). An S4 chimera with the S4 of Shaker inserted into
Shaw was also constructed but did not express functional channels in oocytes. Verification of the inserted sequences was carried
out in all instances using the dideoxy termination sequencing
method (Sanger et al., 1977
).
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Terminology
Chimeric channels are designated as the species of S4 substituted
for the Shaker S4 sequence in ShB6-46. For instance, the Shal
S4 chimera was made by substituting the S4 sequence of Shal into
the Shaker channel in place of the Shaker S4 sequence. The specific amino acid replacements are shown in Fig. 1.
Dimer Construction
Tandem dimers were constructed by splicing together DNA coding for A and B protomers (Heginbotham and MacKinnon, 1992;
Ogielska et al., 1995
). The original protomers contain the sequence for Shaker potassium channels with the NH2-terminal deletion to remove N-type inactivation, ShH4
6-46. The amino acid
sequences of ShB and ShH4 are nearly identical, differing by
only two amino acids, both near the COOH-terminal end of the
protein (Schwarz et al., 1988
; Kamb et al., 1988
). The A protomer
contains DNA coding for a hydrophilic linker (NNNNNNAMV) inserted in frame between the coding regions of each protomer. Protomers were spliced together at the NcoI and KpnI restriction enzyme sites. Shaw S4 was spliced into the A protomer or B protomer
using RsrII and NsiI restriction enzyme sites. For the protomers Ak,
Bk, Aw, and Bw, the A and B designate the NH2- and COOH-terminal protomer, respectively, with each protomer containing either
the Shaker (k) or Shaw S4 (w) construct. All combinations of
dimers were constructed: AkBk, AwBk, AkBw, and AwBw.
Expression System
Channels were expressed in Xenopus oocytes by injection of
G(5')ppp(5')G capped cRNA from the different channel constructs. cRNA was transcribed in vitro from linearized plasmid
containing channel DNA constructs as described previously (Zagotta et al., 1989; Hoshi et al., 1990
). For Shaker and the S4 chimeras, cRNA was made from KpnI-linearized DNA with T7 RNA
polymerase. For Shaw and the S4 chimera in Shaw, cRNA was
made from SacI- or SalI-linearized DNA with T3 RNA polymerase. For the tandem dimers, cRNA was made from EcoRI-linearized DNA with T7 RNA polymerase. Recordings were typically
carried out 1-21 d after injection.
Electrophysiology and Data Analysis
Electrophysiological recordings of macroscopic ionic currents
were carried out from excised membrane patches in the inside-out configuration (Hamill et al., 1981). Currents were recorded
with either a List EPC-7 (Medical Systems Corp., Greenvale, NY)
or an Axopatch 1B (Axon Instruments Inc., Foster City, CA)
patch clamp amplifier and low pass filtered using an eight-pole
Bessel filter (Frequency Devices, Inc., Haverhill, MA). Macroscopic currents from Shaw and Shal S4 were filtered at 9 kHz. All
other data were filtered at 2 kHz unless otherwise noted. Two different systems were used to generate pulses and to digitize and
store data for later analysis. The first was a Digital Equipment
Corp. LSI 11/73-based minicomputer system (Indec Systems,
Sunnyvale, CA). The second was a Macintosh-based system with
hardware interface from Instrutech Corp. (Great Neck, NY) and
software from HEKA (Lambrecht, Germany). Patch pipettes were
constructed from VWR borosilicate glass and had initial resistances of 0.4-0.8 M
. No series resistance compensation was
used. The maximum error due to uncompensated series resistance for this series of experiments was ~4 mV; however, the error was typically <2 mV. Linear leak and capacitive currents were
subtracted with either a P/4 protocol or appropriately scaled averaged responses to repeated 20-mV depolarizing steps, both typically from a holding potential of
110 mV. The temperature was
held at 20 ± 0.2°C.
For Shaker, Shab S4, and Kv3.2 S4, conductance-voltage
curves were constructed by calculation of the chord conductance
(Gchord) from maximum currents (Imax) during the test pulse at
several voltages, assuming a reversal potential (Vrev) of 80 mV:
Gchord = Imax/(V
Vrev). Conductance-voltage curves generated
in this way are minimally affected by the nonlinearity of the single channel conductance if, as with these channel species, activation occurs over a more negative voltage range. For all other
channel species, conductance-voltage curves were constructed
from isochronal measurements of tail currents recorded at a
fixed voltage following steps to voltages that activate the channels. Typically, isochronal measurements were made between 0.2 and 1 ms after the end of the test pulse. Conductance-voltage
curves were normalized to the maximum value for comparison
between patches and between channel species. Conductance- voltage curves were fit by a first or fourth power Boltzmann function of the form
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with n equal to 1 or 4, to determine the voltage-dependent parameters of activation. G/Gmax is the conductance normalized to the maximum value for each channel. For a first power Boltzmann function, V1/2 is the voltage at which the channels are open half maximally. For a fourth power Boltzmann function, V1/2 is the voltage at which each subunit is activated half of the time. V is the voltage of the test pulse, z is the equivalent charge, F is the Faraday constant, R is the gas constant, and T is the absolute temperature. The slope factor is equal to RT/zF.
Typically, a holding potential of 80 mV followed by a 1-s
prepulse to
100 mV was used before the test pulses. For Shaw, 10-ms prepulses to
100 mV were often used. In some experiments with Shaw, a holding potential of 0 mV was used to minimize the capacitive transient during large positive voltage steps.
This can be done because Shaw does not inactivate even during
long depolarizations to 0 mV and its activation kinetics are not
sensitive to holding potential (data not shown; see also Wei et al.,
1990
; Tsunoda and Salkoff, 1995
).
The activation time course of the macroscopic ionic currents
was fit using a single exponential function for times greater than
the delay: I(t) = A{1 exp[(
t + d)/
]}. I(t) is the fit to the current at time t, A is the scale factor,
is the time constant, and d is
the delay required to obtain an adequate fit of a single exponential function, which was required for some channel species. Tail
currents were fit with a single exponential function.
Xenopus oocytes have endogenous potassium and chloride
channels, which are known to activate at more positive membrane potentials (Dascal, 1987). The currents measured in this
study from channel species requiring large positive voltage steps
to activate, such as from Shaw or the Shaw S4 chimera, were
clearly identified as currents through the exogenously expressed
potassium channels for the following reasons. Our intracellular
solutions typically contained 11 mM EGTA with little or no
added Ca++, which is expected to prevent activation of the calcium-activated chloride channels in Xenopus oocytes (Dascal,
1987
). We never saw Shaw or Shaw S4-like currents in membrane
patches from uninjected oocytes when using the standard recording solutions. Shaw S4 properties were similar whether expression levels gave currents of several nA or only 50-100 pA. This
was true for Shaw as well. Also, Shaw S4 and Shaw currents clearly
reverse near the calculated reversal potential for K+, between
80 and
100 mV, and not near the 0-mV reversal potential for
Cl
ions. Finally, expression levels for Shaw S4 and Shaw channels could give rise to several nanoamperes of current in a single patch, which is orders of magnitude larger than endogenous currents from similar membrane patches in uninjected oocytes.
Single channel currents for Shaker, Shaw, and Shaw S4 were
recorded from inside-out membrane patches using the standard
solutions (see below), unless otherwise stated. Currents were digitized at 20 kHz and filtered at 2 kHz, unless otherwise stated. Single channel conductance was calculated from single channel currents recorded during steps to voltages between 50 and +80 mV
by fitting a linear function to the single channel current-voltage
relation. Single channel current-voltage relations for all three
channels are linear in this voltage range. Results from single channel analysis of ShB
6-46 are taken from Hoshi et al. (1994)
.
Kinetic simulations were carried out as outlined in Zagotta et
al. (1994b). Transitions between conformational states are assumed to obey time-homogeneous Markov processes. Voltage-
dependent rate constants are assumed to be exponentially dependent on voltage.
Solutions
The standard extracellular (pipette) solution used for these experiments contained (mM): 140 NaCl, 2 KCl, 6 MgCl2, 5 HEPES (NaOH), pH 7.1. The standard intracellular solution (bath) contained (mM): 140 KCl, 2 MgCl2, 11 EGTA, 1 CaCl2, 10 HEPES
(N-methylglucamine), pH 7.2. For Shal S4, Shaw S4, and Shaw,
the intracellular solution was modified by removing MgCl2 and
CaCl2, unless otherwise noted. This was done because intracellular Mg++ blocks these channels in a time- and voltage-dependent
manner. Block of macroscopic currents becomes evident at voltages above +80 mV for these channels (data not shown), voltages
at which the probability of opening for Shal S4, Shaw S4, and
Shaw has not reached a maximum value. Even though intracellular Mg++ blocks Shaker channels also, it was not necessary to
record Shaker ionic currents in Mg++-free solution since Shaker
is fully activated at voltages well below those at which Mg++
blocks the channel. Similar block by intracellular Mg++ has been
shown for the closely related Kv1.1 potassium channel (Ludewig
et al., 1993). For Shaker and Shaw S4 channels, both the time
course of activation and the position of the conductance-voltage curve along the voltage axis is unchanged by Mg++ at voltages
where there is little or no block (data not shown).
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RESULTS |
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Alterations of Potassium Channel Gating by S4 Chimeric Substitutions
Chimeras were generated by substituting 21 amino acids from the S4 of several divergent potassium channels
for the S4 of Shaker potassium channels (Fig. 1). The
substituted potassium channel sequences were derived
from Drosophila channel genes Shab, Shal, and Shaw,
and from a rat Shaw homolog, Kv3.2 (RKShIIIA). Comparison of S4 amino acid sequences between these
channels and Shaker reveals significant differences in
amino acid composition. The sequence identity with
Shaker S4 ranges from as little as 43% for Shab to as much
as 62% for Kv3.2. The nominal net charge in the S4 regions ranges from +3 in Shaw to +7 in Shaker, assuming
that arginine and lysine have a net charge of +1,
glutamate has a charge of 1, and histidine is uncharged.
Macroscopic ionic currents and normalized conductance-voltage curves from the S4 chimeras and the Shaker parent channel are shown in Figs. 2 and 3 (see also Table I). All S4 chimeras activate at more positive voltages and with a shallower conductance-voltage relation than Shaker. However, Shaw S4 requires much larger positive steps to activate than the other chimeras. This shift in the voltage range of activation indicates that the S4 substitutions tend to bias gating toward the closed states more than the wild-type S4 sequence of Shaker. Activation kinetics of Shab S4, Shal S4, and Kv3.2 S4 are relatively fast, similar to Shaker. In contrast, activation kinetics of the Shaw S4 chimera are much slower.
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Contribution of S4 Charges to Voltage Dependence of Activation
One of the questions addressed in this study is whether
or not a correlation exists between the charges in the
S4 of the chimeras and the slope of the conductance-
voltage relation. S4-charged residues have been shown
to contribute to the gating charge associated with channel activation (Aggarwal and MacKinnon, 1996; Seoh
et al., 1996
). Although total gating charge per channel
determines the slope of the conductance-voltage relation
at very low open probabilities (Almers, 1978
; Schoppa et
al., 1992
; Zagotta et al., 1994
a, 1994b; Sigg and Bezanilla, 1997
), the shape and slope of the relationship at
higher open probabilities is additionally influenced by
interactions between subunits (Zagotta et al., 1994
a; Sigworth, 1993
). Therefore, we were interested in
studying the role of the S4 charges in determining the
slope of the conductance-voltage relation in the voltage range of moderate probability of channel opening
(Wei et al., 1990
; Schoppa et al., 1992
; Logothetis et al.,
1993
; Zagotta et al., 1994
a).
For all S4 chimeras, the conductance-voltage relation is significantly more shallow than for Shaker, as
might be predicted by the smaller number of basic residues present in the mutants (Fig. 3, Table I). The most
shallow slope of the conductance-voltage relation appears in Shaw S4, consistent with the S4 of Shaw having the least amount of charge (+3) of the chimeras as well
as the fewest of those charges thought to contribute
most to channel activation (Aggarwal and MacKinnon,
1996; Seoh et al., 1996
; Larsson et al., 1996
). However,
an almost equivalent slope occurs with Kv3.2 S4, which
has the highest nominal charge density (+6) in the S4
of all the chimeras. Kv3.2 S4 includes the same five basic residues present in Shab S4 and Shal S4, which have
fewer charges. Kv3.2 S4 contains all five of the S4 basic
residues thought to contribute most to the gating
charge. The only charge substitution in Kv3.2 S4 is a
neutralization at the seventh charged residue in Shaker,
a position that seems to contribute little to the overall charge movement associated with gating (Aggarwal and
MacKinnon, 1996
; Larsson et al., 1996
). Clearly, the
similar steepness of the conductance-voltage curves for
Shaw S4 and Kv3.2 S4, despite the large difference in
charge content of the S4 segments, indicates that properties of the S4 other than the amount of charge are important determinants of the voltage dependence of
channel opening. Furthermore, these results suggest
that molecular mechanisms other than charge movement, such as subunit cooperativity, greatly influence the shape of the conductance-voltage curve.
Distinguishing between Changes in Independent and Cooperative Transitions
To investigate further the role of S4 residues and cooperative interactions between subunits in gating, we
wanted to use a prototypical model for channel gating
that would incorporate several key features of voltage-dependent Shaker channel activation and thus would
yield insights that are readily generalizable to models with more complexity. All of the several kinetic models
that have been proposed to describe Shaker channel
activation have in common multiple voltage-dependent
transitions with at least one transition involving the cooperative movement of subunits (Bezanilla et al., 1994;
Zagotta et al., 1994
a; McCormack et al., 1994
; Sigworth, 1993
). The six-state model in Fig. 4 is the simplest kinetic model for a homotetrameric channel with both
independent and concerted conformational changes.
It incorporates a single independent conformational change in each of four identical subunits followed by a
single concerted conformational change, and it can reproduce many of the key features of Shaker activation.
Parameters for the six-state model were found that predict ionic currents similar to Shaker (control) and to
those predicted by a more complete model for Shaker
activation that quantitatively fits data from macroscopic ionic currents, single channel recordings, and gating
currents (Zagotta et al., 1994
a). The six-state model fits
the steady state and kinetic properties of the macroscopic ionic currents of Shaker reasonably well using
very few free parameters and predicts a considerable
amount of delay in the activation time course. While the cooperative transition in this six-state model in Fig.
4 is voltage independent, the steady state and kinetic
properties of Shaker activation can just as easily be fit
by a cooperative transition that is voltage dependent
(see Fig. 8; see also Zagotta et al., 1994
a; Smith-Maxwell
et al., 1998
). The six-state model does not fit as well as
the more complete model because it does not have enough transitions to fully account for the large delay in
Shaker activation or enough gating charge. More complex models for Shaker increase the amount of charge
moved during channel activation and the amount of delay in the activation time course by adding more independent transitions in each of the four channel subunits and by adding one or more concerted transitions.
General conclusions from this six-state model are directly and easily transferable to other models in the literature that have been proposed for Shaker, despite
differences in the details of the kinetic schemes (see
Smith-Maxwell et al., 1998
).
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Steady state and kinetic behavior for the Shaker
(control) model and for several variations are presented in Fig. 4. Control simulations have steady state activation properties and gating kinetics similar to Shaker
(see Fig. 2 and Fig. 8, bottom right). The opening kinetics are sigmoid and have a voltage-dependent pattern
of delay similar to the pattern observed for Shaker currents and for the complete model for Shaker activation
described by Zagotta et al. (1994a, 1994b). Shaker channel opening is delayed because it undergoes multiple
voltage-dependent transitions between closed states before it opens. The relative delay compared with the
overall rate of activation can be determined by scaling
the currents at all voltages to the maximum current
level and then changing the scaling of the currents
along the time axis so that the slope at the half maximal current is the same for all traces. This manipulation of the current traces facilitates comparison of the
relative delay, or sigmoidicity, at all voltages and between different channel species, despite differences in
current magnitude and absolute rates of activation
(Zagotta et al., 1994
a, 1994b). For the control simulations, as with Shaker, there is little delay in channel
opening during small depolarizing voltage steps due to
the relatively slow rate of return from the open state.
With larger depolarizations, the relative delay increases
because the relative contribution of the slow first closing
rate to the overall rate of activation decreases. With even
larger positive voltage steps, the relative delay tends to
saturate because the dominant transitions in channel activation have similar rates over this voltage range (Zagotta et al., 1994
a, 1994b). In contrast, with an independent model such as the Hodgkin-Huxley model
(Hodgkin and Huxley, 1952
), the sigmoidicity of the activation time course is a constant function of voltage.
Since the channels under study are homotetramers,
identical substitutions within each subunit could alter
four independent transitions in the kinetic scheme. Alternatively, if the mutations change a cooperative transition, a single transition in the kinetic scheme can be
affected. To illustrate the predicted effects of such mutations, three distinct types of modifications to the control parameters are considered. In the first, the total
charge associated with channel gating is decreased by
decreasing the gating charge associated with either the
forward (z) or the backward (z
) rate constants in
each of the four subunits. In the second, the forward
rate constant of the independent transitions in all four
subunits (
0) is decreased without changing its voltage dependence. In the third, cooperativity is altered by decreasing the forward rate constant only in the concerted transition leading to channel opening (
) (see
Zagotta et al., 1994
a).
These different types of modifications of the gating transitions predict distinct changes in channel activation. Decreasing the amount of charge associated with each of the four voltage sensors decreases the slope of the conductance-voltage curve and predicts a shift to more negative voltages, regardless of whether charge associated with the forward or backward rate constants is decreased. This result was not observed with any of the chimeras, despite the fact that all chimeras decreased the number of basic residues in the S4 region. The predicted channel kinetics and sigmoidicity are similar to the properties of the control channel, though the voltage range is negatively shifted.
Decreasing the rate at which each individual subunit
changes conformation, 0, without changing its voltage
dependence, causes a positive shift in the conductance-voltage curve along the voltage axis but does not
change the slope of the curve. The slowing of the independent transitions slows activation kinetics but has little effect on the sigmoidicity of the kinetics when compared with the control. This voltage-dependent pattern
of sigmoidicity occurs, as with the control, because of
the cooperativity of the final transition (see also Zagotta et al., 1994
a).
Altering subunit cooperativity by decreasing the forward rate constant for the final opening transition, ,
causes the time course of activation to become less sigmoid. As
is decreased more and more, activation is
slowed and the cooperative transition becomes rate
limiting. When the transition is slowed sufficiently, the
activation time course becomes single exponential, as
shown in Fig. 4. Decreasing
also gives rise to a positive
shift in the conductance-voltage curve and a decrease
in the apparent voltage dependence. While the cooperative transition in our six-state model is voltage independent, making the transition voltage dependent does
not change the predictions (see Smith-Maxwell et al.,
1998
). Thus, we can decrease the slope of the conductance-voltage relation either by changing the cooperative interactions in a channel or by decreasing the
amount of charge moved per channel (see also Zagotta
et al., 1994
a). The amount of charge per channel can be determined independently from gating current measurements in combination with fluctuation analysis or
toxin binding to determine the number of channels
present (Schoppa et al., 1992
; Aggarwal and MacKinnon, 1996
). However, in terms of the model, we can distinguish between changes in cooperativity and changes in
the amount of charge moved independently in each
subunit, without charge per channel measurements, because they differ in their predictions for the position
of the conductance-voltage curve along the voltage
axis and in the effect on sigmoidal delay.
Interpretation of mutant data is not completely straightforward, however, since there is no reason to expect that a mutation, even a single amino acid substitution, will change only a single parameter of activation or a single transition. Even if a mutation were to change the energy of a single state, more than one transition will likely be affected, depending on how the states are connected together. The positive shift in the voltage range of activation for the S4 chimeras and the decrease in slope suggest that the substituted S4 segments may alter cooperativity in the Shaker channel in addition to any changes due to charge content of the S4. We tested this idea further in the Shaw S4 chimeric channel.
Comparison of the Shaw S4 Chimera to Shaw and Shaker
The most dramatic changes in channel function were introduced into Shaker by substitution with the S4 of Shaw. Before proceeding with the determination of the types of conformational changes that are altered, we compared Shaw S4 to both of its parent channels, Shaker and Shaw, to determine which gating properties resemble those of the parent channels. Current traces and conductance-voltage relations are shown in Fig. 5 and analysis of the gating kinetics is shown in Fig. 6.
|
|
The steady state voltage dependence of Shaw S4 activation is much more shallow than that of Shaker and is
shifted to more positive voltages, in the direction of the
conductance-voltage curve of the S4 donor Shaw, but
not to such extreme voltages. Fits of a first power Boltzmann function to conductance-voltage curves from
Shaw S4 and Shaker channels show that activation of Shaw S4 is shifted ~+120 mV, and the slope is decreased 2.7-fold. The S4 donor channel, Shaw, requires
even more positive voltages to fully activate than Shaw
S4 and has an even shallower voltage dependence (see
also Wei et al., 1990; Tsunoda and Salkoff, 1995
). We
cannot quantify the voltage dependence of Shaw activation by fitting with a Boltzmann function because the
probability of opening does not saturate within the limits
of our measurements (<+200 mV). The conductance-
voltage relation for Shaw is normalized to the value of the
conductance at +180 mV in Fig. 5 to facilitate comparison with Shaw S4 and Shaker. The probability of opening for Shaw increases with a very shallow voltage dependence over a wide voltage range. Shaw begins to open at
voltages more negative than Shaw S4, around
60 mV,
and does not reach a maximum open probability until
voltages well above the voltage where Shaw S4 open probability saturates. In contrast, the probability of opening for the Shaw S4 chimera, which begins to increase at voltages greater than 0 mV and saturates around +180 mV,
has a steeper voltage dependence than Shaw.
In Fig. 6 (left), currents recorded in response to positive voltage steps are shown for each channel species.
Superimposed on the current traces are fits by a single
exponential function as outlined in MATERIALS AND
METHODS. Note the different time scales, which were selected to enable comparison of the relative amounts of
delay at the beginning of the activation time course for
each of the three channel species. It is clear from these fits that Shaw S4 and Shaw currents follow a single exponential time course to activate with little or no delay.
This can be seen more clearly for a wide range of voltages by scaling the currents to compare the delay in activation relative to the overall time course of activation
(Fig. 6, middle). The lack of delay in the activation time
course of Shaw and Shaw S4 suggests that the activation kinetics are dominated by a single rate-limiting transition over a wide range of voltages. In contrast, Shaker
requires the addition of 1-4-ms delays for adequate fit
to the activation time course. Previous work on Shaker
has indicated that this large sigmoidal delay is the result of multiple voltage-dependent transitions, each
moving relatively small amounts of charge of similar
magnitude (Sigworth, 1993; Hoshi et al., 1994
; Zagotta
et al., 1994
a; Bezanilla et al., 1994
). A kinetic model for
Shaw has not yet been developed.
Time constants obtained from fits to the activation time course similar to those shown in the Fig. 6 (left), from several patches with the Shaw S4 chimera and its parent channels, are plotted in Fig. 6 (right). Also plotted are time constants from single exponential fits to tail currents at negative membrane potentials. Tail currents measured during closing of all three channels are reasonably well fit by a single exponential function. Shaw S4 opens and closes much more slowly than either Shaw or Shaker. The kinetics of Shaw S4 are strongly voltage dependent, as are those of Shaker. The voltage range over which the kinetics of Shaw S4 change most dramatically is shifted to more positive voltages compared with Shaker and is shifted to the same extent as the probability of channel opening. In contrast to both Shaker and Shaw S4, the opening and closing kinetics of Shaw exhibit a very shallow voltage dependence over a wide range of voltages, encompassing 360 mV. Thus, while the activation time course for both Shaw and Shaw S4 is dominated by a single, rate-limiting transition, for Shaw S4 this transition is strongly voltage dependent while for Shaw it is nearly voltage independent. This difference in voltage dependence suggests that the rate-limiting conformational changes in the activation of Shaw and Shaw S4 may be different.
Differences between Shaw S4 and the parent channels can also be seen at the single channel level (Fig.
7). The first latencies of Shaw S4 are substantially
longer than the first latencies of Shaker and Shaw, consistent with altering transitions in the activation pathway (data not shown). However, once the channel
opens, the Shaw S4 single channel events much more
closely resemble the behavior of Shaker than Shaw in
their kinetics and single channel conductance (Fig. 7).
The single channel conductance for Shaw S4 of 9.5 pS (n = 18) is the same as the 9-10 pS conductance of
Shaker (Hoshi et al., 1994), and much smaller than the
23.3 pS (n = 3) single channel conductance of Shaw
(see also Tsunoda and Salkoff, 1995
).
|
The rate of inactivation of Shaw S4 ionic currents
(1.57 ± 0.28 s [SEM] at +50 mV, n = 5) closely resembles the 1.5-s C-type inactivation rate of Shaker (Hoshi
et al., 1991). In contrast, there is no detectable inactivation in the macroscopic ionic currents of Shaw during
positive voltage steps lasting 20-30 s (data not shown;
see also Wei et al., 1990
; Tsunoda and Salkoff, 1995
). These results are consistent with the Shaw S4 substitution primarily altering conformational changes in the
activation pathway and not introducing global changes
that perturb other channel functions.
Alteration of Cooperative Gating by Shaw S4 Substitution
The slower gating kinetics and decreased sigmoidicity
suggest that the Shaw S4 mutations alter activation gating fairly specifically, slowing a highly cooperative transition in the activation pathway sufficiently to make it
rate limiting. Since introducing the S4 of Shaw into all
four subunits of Shaker changes the activation time
course from sigmoid to single exponential, the rate-limiting transition in Shaw S4 cannot be due to the slowing
of independent transitions in each subunit. This suggests that the single rate-limiting transition in Shaw S4
is a highly cooperative transition in the activation pathway. The simplified model for Shaker gating is illustrative (Fig. 4). While slowing the independent transitions slows channel gating, the time course of channel opening maintains considerable sigmoidicity, a characteristic clearly different from the single exponential time
course of Shaw S4 activation. However, we can simulate
currents with properties similar to Shaw S4 simply by
slowing the forward rate of the final cooperative transition in the activation pathway (Fig. 4, bottom). Decreasing only the rate constant, , with no other changes,
leads to slowing of activation kinetics, a positive shift in
the voltage range of activation, a decrease in the slope
of the conductance-voltage curve, and activation kinetics with a single exponential time course over the entire
voltage range of activation. These are all characteristic
of Shaw S4 macroscopic currents.
Although we used the six-state model for our predictions, as discussed previously, it does not fully account
for the large delay in the activation time course of
Shaker or the large amount of charge associated with
activation gating. We used the six-state model to interpret the results of the S4 substitutions because it is the
simplest model possible that will describe several key features of activation gating in Shaker. Therefore, conclusions from this model are fairly general and are not
obscured by complexities introduced by models with
many more transitions. However, more complex models are necessary to fit quantitatively the wild-type and
mutant kinetics. For example, simply decreasing the
forward rate constant of the cooperative transition in
the six-state model predicts many of the functional
changes observed with Shaw S4, but it does not predict
the maximum probability of opening observed in the
single channel records. However, as seen in the next
paper (Smith-Maxwell et al., 1998), modifications to
the final closing step in the more complete model for
Shaker (Zagotta et al., 1994
a) can quantitatively account for Shaw S4 kinetics. Therefore, our conclusion
that a late cooperative step is altered in Shaw S4 is valid
both for a simple model that incorporates the key features of Shaker gating and a more complex model that
quantitatively fits Shaker activation.
Shaw S4 and Shaker Tandem Dimers
The change to slow, single exponential kinetics upon
substituting the S4 of Shaw for the S4 of Shaker indicates that a single transition in the activation pathway
has become rate limiting. Since we can assume from
studies with the Shaker channel (MacKinnon, 1991;
Heginbotham and MacKinnon, 1992
; Kavanaugh et al.,
1992
; Liman et al., 1992
) that the Shaw S4 chimera assembles into a tetramer with four identical subunits,
the rate-limiting transition in Shaw S4 activation most
likely arises from highly cooperative interactions between subunits. To test further the hypothesis that the
Shaw S4 mutation slows a cooperative transition, we constructed tandem heterodimers containing the slowly activating Shaw S4 subunit and the rapidly activating
Shaker subunit in either position of the dimer (AkBw
and AwBk) along with homodimers containing either two
Shaker subunits (AkBk) or two Shaw S4 subunits (AwBw)
(see Tytgat and Hess, 1992
; McCormack et al., 1992
;
Heginbotham and MacKinnon, 1992
; Ogielska et al.,
1995
). Macroscopic currents and normalized conductance-voltage curves from the dimer constructs are
shown in Fig. 8, along with predictions for the independent or cooperative movement of subunits. Values
from fits of Boltzmann functions to data from the
dimer constructs are given in Table II. Macroscopic
currents recorded from channels assembled from the
homodimer constructs, AwBw and AkBk, activate with
properties that are indistinguishable from the properties of channels assembled from Shaw S4 and Shaker
monomers, respectively (Figs. 2 and 8, Tables I and II),
demonstrating that the nine amino acid linker between
subunits in the tandem dimer constructs does not perturb channel activation.
|
Macroscopic currents from heterodimer constructs AkBw and AwBk activate in a voltage range roughly midway between Shaker and Shaw S4, with a shallow voltage dependence much more like that of Shaw S4 than Shaker (Fig. 8). Heterodimer activation kinetics are slower than Shaker, though not nearly as slow as Shaw S4 (Fig. 8), and yet the time course of activation approximates a single exponential like Shaw S4 (Fig. 9). Currents from heterodimer channels scaled for analysis of sigmoidicity show little relative delay or voltage- dependent change in the delay. The voltage independence of the scaled activation time course in the heterodimers is similar to that of Shaw S4 and unlike the large voltage-dependent changes in relative delay found in Shaker. These results suggest that only two subunits in the channel tetramer need the Shaw S4 substitution to alter cooperativity in the activation process in such a way as to make a single transition rate limiting.
|
Conductance-voltage curves from channels formed
from heterodimers of both orientations were compared as a test for misassembly of subunits in the channel tetramer (Fig. 8). The curves from AkBw and AwBk
are very similar. However, small differences in the voltage range of activation occur in the direction expected for a slight preferential insertion of the NH2-terminal
subunit. Similar observations have been reported previously for concatenated channel subunits (McCormack
et al., 1992; Ogielska et al., 1995
). The close agreement
between results from the two heterodimer constructs indicates that only a small proportion of the functional
channels include improperly assembled subunits. We
tested further for misassembly of heterodimers by looking for kinetically distinct populations of channels that
might be proportionately more active at different voltages. We found no change in deactivation rates after activation of channels at different voltages, supporting
the idea that the heterodimer constructs tend to assemble into a homogeneous population of channels. Therefore, the relatively small percentage of channels we encountered with incorrect subunit assembly should not
affect our conclusions.
Independent and Cooperative Models: Predictions for Heterodimers
We constructed several models to determine whether
activation of the dimers is more consistent with the independent movement of Shaker and Shaw S4 channel
subunits or with cooperative interactions between them.
Predictions of a simple independent model, based on
the model of Hodgkin and Huxley (1952), are illustrated in Fig. 8 (top right). This model assumes that each
of the four channel subunits undergoes a single independent voltage-dependent conformational change
leading to channel opening. The normalized conductance-voltage curves of AkBk and Shaw S4 were each fit
with a fourth power Boltzmann function to determine
the voltage-dependent properties of activating a single
subunit in the tetrameric channel protein. The Boltzmann function used for these fits is given by:
![]() |
where V1/2 is the half activation voltage and z is the equivalent charge associated with activation of each subunit. For a channel with two Shaw S4 and two Shaker subunits, the prediction for steady state channel activation is given by:
![]() |
where Vw and Vk are the half activation voltages for each Shaw S4 and Shaker subunit, respectively, and zw and zk are the equivalent charge for each Shaw S4 and Shaker subunit. The model predicts that the conductance-voltage curve for channels with two Shaw S4 and two Shaker subunits will be much closer to the curve for Shaw S4 than the curve for Shaker because the channel will not open until all subunits are activated, including the less likely to activate Shaw S4 subunits. The dimers AkBw and AwBk activate at much more negative voltages than predicted by the independent model.
The large disparity between the heterodimer data
and the prediction of the independent model argues
strongly that the independent movement of the subunits cannot account for the dominant transition in
Shaw S4. This evidence for a cooperative transition in
gating agrees with earlier findings from studies of gating using concatenated dimers and tetramers (Tytgat
and Hess, 1992; Hurst et al., 1992
) and from detailed
analysis of Shaker gating kinetics (Sigworth, 1993
; Zagotta et al., 1994
a, 1994b; Bezanilla et al., 1994
).
To test this idea further, we used two different cooperative models to compare with the data from AkBw and
AwBk and to investigate the predictions of cooperative
subunit interactions. The first cooperative model makes
the simplest assumption, that channel opening involves
a single fully cooperative or concerted transition, wherein all four channel subunits activate simultaneously by undergoing a single voltage-dependent conformational change from the closed state into the open
state. Therefore, the energetic contribution to channel
opening by each of the four subunits will be additive. The free energy change for activation of each subunit,
G, was calculated from fits of a first power Boltzmann
function to the normalized conductance-voltage relation for homotetrameric channels with all Shaker subunits or all Shaw S4 subunits from the following:
![]() |
where K is the voltage-dependent equilibrium constant
for channel opening given by K = ezF(V V1/2 )/RT. The free
energy change for activation of each of the four subunits in each homotetramer was calculated from
![]() |
The total free energy change, Gtot, for opening a channel with two Shaw S4 and two Shaker subunits was calculated from
Gtot = 2(
Gw +
Gk), where
Gw and
Gk
represent the free energy change for activation of single
Shaw S4 and Shaker subunits, respectively. The predicted
conductance-voltage curve for a channel with two Shaw
S4 and two Shaker subunits was then reconstructed from
![]() |
and is represented by the dotted line in Fig. 8 (bottom
right). Even with this overly simple model, the predicted conductance-voltage relation for channels with
half Shaw S4 subunits and half Shaker subunits approximates the heterodimer data much more closely than the independent model. This model assumes the maximum possible cooperativity between subunits. The prediction of heteromultimeric conductance-voltage curves
at voltages more negative than the heteromultimer data
is consistent with the well known departure from first order gating in Shaker channels (Sigworth, 1993; Bezanilla et al., 1994
; Zagotta et al., 1994
a) and suggests that the
relative contribution of cooperative transitions to channel opening differs in Shaker and Shaw S4.
In a second model, we investigated the prediction of
activation schemes with both independent and cooperative transitions by using the simple six-state gating
model in Fig. 4. We assumed for Shaker that a single independent voltage-dependent transition in each of the
four subunits is followed by a concerted transition that
opens the channel. For Shaker, we used two variations
of the model, one in which the final concerted transition is voltage independent, as in Fig. 4, and one in
which a small amount of charge movement is incorporated into the final concerted transition. We considered a voltage-dependent variation because there is evidence in Shaker for a transition near the open state
that moves a small amount of charge (Koren et al., 1990;
Bezanilla et al., 1994
; Hoshi et al., 1994
; Zagotta et al.,
1994
a). The independent transitions have identical
properties in both variations of the Shaker model. As
with the six-state model in Fig. 4, we assumed that the rate constants of all voltage-dependent transitions have an
exponential dependence on voltage (see legend to Fig.
4). We calculated the probability of channel opening,
Popen, from the equilibrium constants for each transition:
![]() |
![]() |
Popen was normalized to the maximum Popen and plotted as a function of voltage.
The solid and dashed lines through the data for
Shaker dimer AkBk in Fig. 8 (bottom right) show the fit of
both variations to the AkBk data, demonstrating that
both variations can reasonably approximate steady state
Shaker activation. Simulated currents from the Shaker
variation without charge movement in the final transition are shown as the control in Fig. 4. To simulate
Shaw S4, the final conformational transition leading to
channel opening in the Shaker model was slowed and
the voltage dependence was increased. The solid line
through the Shaw S4 data shows that this simple modification of the six-state Shaker model gives a good approximation for the effect of the introduced mutations on the conductance-voltage curve. Simulated currents
from this model have single exponential activation kinetics like Shaw S4 (data not shown; but see simulation
in Fig. 4, = 10).
Since modification of a single transition can account
for the steady state behavior of Shaw S4, we can assess
the role of energy additivity in the heterotetramers for
this single transition in a multi-state scheme that approximates the gating of the channels. Rate constants
taken from the final transition before opening in the
models for Shaw S4 and AkBk were used to calculate the energetic contribution of each Shaw S4 and Shaker
subunit to the transition. The free energy change, G,
contributed by each subunit to the concerted transition
was calculated from the rate constants in the models
for the homotetramers where
![]() |
The total free energy change, Gtot, for the concerted
transition with two Shaw S4 and two Shaker subunits
was calculated from the free energy change per subunit, as for the two-state concerted model above, from
Gtot = 2(
Gw +
Gk). The resultant equilibrium constant for the final step in the heterodimer model, K5,
was computed from K5 = e
Gtot/RT.
The predicted probability of opening, Popen, was calculated from the equation for the steady state probability of opening (Eq. 1), changing only the equilibrium constant for the concerted transition, K5. Popen was normalized to the maximum open probability. The curves for the predictions are shown in Fig. 8 (bottom right). The solid line through the heterodimer data is from the Shaker model with a voltage-dependent concerted transition and the dashed line is from the variation with the voltage-independent concerted transition. This multi-state model with both independent and cooperative transitions, in which only the cooperative transition is changed, improves on the fit of the two-state cooperative model to the heterodimer channel data, presumably because the multi-state model more accurately describes gating in these channels and takes into account the different contributions of cooperativity to gating in Shaker and Shaw S4.
To test further the validity of the predictions from the six-state model, we simulated ionic currents for the heterodimer channels. Rate constants taken from the final transition before opening in the models for Shaw S4 and AkBk were used to calculate the energetic contribution of each Shaw S4 and Shaker subunit to the rate constants in the final transition in the heterodimer model. Forward and backward rate constants were calculated separately. The activation energy contributed by each Shaker and Shaw S4 subunit, respectively, is given by:
![]() |
where kk and kw are the rate constants for Shaker and Shaw S4, respectively, A is the attempt frequency, and kB is the Boltzmann constant. The predicted rate constant for the final transition for channels with two Shaker and two Shaw S4 subunits was calculated from
![]() |
The calculated rate constants for the forward and backward transitions were then incorporated into the final transition of the six-state model, assuming all other transitions were unchanged, and the predicted currents for the heterodimer channels were simulated. Activation kinetics of simulated currents predicted by this analysis are similar to the kinetics of the heterodimer channels (data not shown) and the activation time course has a greatly reduced sigmoidicity, similar to that found with the heterodimer mutant channels (Fig. 9). Our results indicate that activation involves multiple voltage-dependent transitions with at least one cooperative transition, that the Shaw S4 substitution alters a cooperative transition, and that the contribution of the cooperative transition to channel opening differs in the Shaker and Shaw S4 channels.
Since macroscopic ionic currents from channels with
two or four Shaw S4 subunits can be simulated simply
by altering the final transition in the activation pathway, making it rate limiting, it is likely that the same
transition is affected in both the heterodimer and homomeric Shaw S4 channels. While the two cooperative models provide good estimates of steady state activation and sigmoidicity for the heterodimer constructs,
they do not fit the heterodimer results perfectly. The
small discrepancy between the cooperative predictions
and the data may be the result of not using the full model for Shaker activation (Zagotta et al., 1994a) or it
may be due to the slight differences in gating we observe when only two Shaw S4 subunits are present, as
opposed to when all four channel subunits have the
Shaw S4 substitution. Activation kinetics of Shaw S4 have
a much steeper voltage dependence than activation kinetics of Shaker. Exponential fits to the activation time
constants estimate the equivalent charge associated
with the final opening transition to be 0.42 electronic
charge for Shaker (Zagotta et al., 1994
b) and 0.78 electronic charge for Shaw S4 (Smith-Maxwell et al., 1998
).
However, for channels with two Shaw S4 and two Shaker subunits, similar fits of activation time constants
estimate the equivalent charge to be 0.45 electronic
charge, suggesting that while two Shaw S4 subunits are
sufficient to make the transition rate limiting, they are
not sufficient to change the voltage dependence of the
rate-limiting transition.
The fact that the cooperative models fit the heterodimer data much better than the independent model and that the cooperative models give qualitatively similar results despite differences in the specific details of the kinetic schemes, indicates that some form of cooperativity provides the best explanation for the steady state activation properties of heteromultimeric channels with both Shaker and Shaw S4 subunits. Cooperativity can be introduced into kinetic schemes in a number of different ways and there is as yet no consensus on the mechanism by which cooperativity participates in Shaker channel activation. Our intention was not to explore extensively all possible classes of models that could lead to this behavior, but rather to determine whether an independent scheme or some form of cooperative scheme provides a more likely explanation for our results. Along with the analysis of Shaw S4 kinetics presented earlier, the heterodimer results strongly suggest that a cooperative transition in the activation pathway is slowed by the Shaw S4 substitution.
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DISCUSSION |
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We used chimeric substitutions to explore further the
role of the S4 in voltage-dependent gating. Chimeric
substitutions have been used in the past to pinpoint regions of importance for gating in several voltage-
dependent cation channels (Tanabe et al., 1990, 1991
;
Stocker et al., 1991
; Chen et al., 1992
; Logothetis et al.,
1993
; Zhang et al., 1994
; Nakai et al., 1994
; Mathur et al., 1997
; Tang and Papazian, 1997
; Shieh et al., 1997
;
Koopmann et al., 1997
). We substituted into a Shaker
channel background S4 segments from several divergent potassium channels with different kinetic and
steady state gating properties (Butler et al., 1989
; Wei
et al., 1990
; Tsunoda and Salkoff, 1995
; McCormack et
al., 1990
). Activation of all S4 chimeras in our study differed from Shaker, however, the S4 substitutions did
not confer on Shaker the gating properties of the S4
donor channels.
Relationship Between Net S4 Charge and Conductance-Voltage Relations
The total charge associated with channel opening has
been estimated for Shaker, using several different methods, to be between 12 and 14 electronic charges (Schoppa
et al., 1992; Zagotta et al., 1994
b; Aggarwal and MacKinnon, 1996
; Seoh et al., 1996
; Noceti et al., 1996
; Sigg
and Bezanilla, 1997
). Total charge moved for channel
opening can be calculated by measuring gating currents and the total number of channels contributing to
the gating currents. Charge per channel measurements,
wherein each charged residue is neutralized in turn,
suggest that the first five positive charges from the NH2-terminal side of the S4 and possibly a negative charge in the S2 provide most of the gating charge (Aggarwal
and MacKinnon, 1996
; Seoh et al., 1996
). In the S4 chimeras we studied, at most one or two of these charged
residues were substituted at a time. The decrease expected from these neutralizations, assuming equal contribution from each of the six implicated charged residues, ranges from ~15 to 35% of the total gating
charge. For Shaw S4, this decrease in gating charge is
potentially even greater due to a charge reversal at the
position of the first basic residue.
We found that neither the net charge content nor
the presence of specific charges in the S4 are predictive
for changes in the slope of the conductance-voltage relation. All S4 chimeras, except Shaw S4, activate with
some delay, providing evidence that these channels activate with multiple voltage-dependent transitions, like
Shaker. Changes in steepness of the conductance-voltage relation can come about either from changing the
total gating charge or from changing cooperativity.
Taken together, our results are most consistent with
subunit cooperativity being the major determinant of
the voltage dependence of the conductance-voltage relation at moderate probability of being open. Because
both charge movement and cooperativity are important in determining the shape and position of the conductance-voltage curve, mutations that change cooperativity could obscure changes in the amount of gating
charge, which could be determined by charge per
channel measurements. However, the shift of the conductance-voltage curves to more positive voltages that
accompanies the decrease in slope cannot be explained
by a decrease in gating charge alone (see also Sigworth,
1993).
Our conclusion that the charge content of the S4
does not necessarily correlate with the voltage dependence of channel opening differs from that reached by
two previous studies of the effects of S4 chimeric substitutions. Logothetis et al. (1993) studied a Shab S4 chimera made in the RCK1 (Kv1.1) background in which the conductance-voltage curve is negatively shifted and
the slope is decreased. These changes follow the predictions for a decrease in gating charge (see Fig. 4).
However, the activation kinetics of the chimera are significantly slower than the kinetics of either parent
channel, which is not consistent with a decrease in gating charge alone. The slow activation kinetics suggest
that channel cooperativity may be altered and that a
more comprehensive analysis is necessary to determine
the relative contributions of cooperativity and charge
movement to activation gating in the RCK1/Shab S4 chimera. Tang and Papazian (1997)
studied two S4 chimeras made by transferring portions of the S4 from the rat
olfactory cyclic nucleotide-gated channel into the S4 of
the eag potassium channel. They suggest that the decrease in charge content of the S4 may be responsible for the decrease in slope of the conductance-voltage
curves. However, a positive shift in the position of the
conductance-voltage curve cannot be explained by simply decreasing the charge content of the voltage sensor,
as predicted from the model in Fig. 4 or the more complete model shown in the next paper (Smith-Maxwell et al., 1998
; see also Sigworth, 1993
; Zagotta et al., 1994
a).
The position of the conductance-voltage curve suggests
that the decrease in slope of the conductance-voltage
relation may be due primarily to a change in subunit cooperativity rather than due to a change in the charge
content of the S4. While some of the results from these
previous studies are very similar to results obtained with our S4 chimeras, our interpretation of the kinetic and
steady state properties of the mutant channels has led
to different conclusions about the effects of S4 mutations on channel gating.
S4 and Cooperative Steps in Gating
The single exponential time course of Shaw S4 activation is significant in light of the very pronounced and
voltage-dependent sigmoidicity of the Shaker parent
channel. In contrast to Shaker, where channel opening
arises from multiple voltage-dependent transitions within
each of the four subunits (Schoppa et al., 1992; Zagotta et al., 1994
a; Bezanilla et al., 1994
), Shaw S4 activates
with a rate-limiting transition in the activation pathway
that is slow compared with other transitions. We have
presented evidence that the rate-limiting transition in
Shaw S4 activation is a cooperative step, implying a role
for the S4 in cooperative interactions between subunits.
The rate of Shaw S4 opening is sufficiently slow that the
rate-limiting cooperative transition could easily mask
the faster transitions that give rise to the sigmoidal time
course of Shaker activation (Smith-Maxwell et al., 1998
).
If the rate-limiting transition in Shaw S4 represents a
transition normally present in Shaker that has been
slowed, further study of Shaw S4 may be useful for understanding the final transition in Shaker opening.
It is also possible that the Shaw S4 mutation alters channel gating more profoundly by decreasing the number of closed states in the activation pathway, possibly to a single closed state. In this case, Shaw S4 would not activate like the Shaker channel by undergoing multiple transitions between closed states, but instead would undergo a single transition between the closed state and the open state. Both kinetic schemes can produce opening kinetics that follow a single exponential time course with no delay. Further experiments will be needed to determine which kinetic scheme more accurately describes Shaw S4 activation gating. However, both kinetic schemes require inclusion of a cooperative step in the activation pathway.
Transfer of Gating Characteristics by S4 Substitutions
The Shaw S4 chimera shares some functional properties with each of its parent channels, Shaw and Shaker. The Shaw S4 substitution makes a single transition in the activation pathway rate limiting, giving rise to single exponential activation kinetics. This shifts the voltage range of channel activation to more positive voltages, compared with the Shaker parent channel, and decreases the steepness of the conductance-voltage curve. These characteristics of Shaw S4 activation are changed in the direction of the S4 donor, Shaw. Conversely, functional characteristics of the Shaw S4 chimera not directly involved in activation gating, such as single channel conductance and the rate of C-type inactivation, closely resemble properties of the Shaker parent channel.
Our results from Shaw S4 are consistent with our results from all S4 substitutions in that changing the S4
sequence alters activation, but the S4 alone is not sufficient to transfer all the functional properties of activation between channels (see also Logothetis et al., 1993;
Tang and Papazian, 1997
; Koopmann et al., 1997
).
Regions of the channel required to transfer the full activation phenotype may be quite extensive. For Shaker
potassium channels, evidence exists for interactions between the S4 and residues in S2 and S3 (Papazian et al.,
1995
; Seoh et al., 1996
; Tiwari-Woodruff et al., 1997
;
see also Planells-Cases et al., 1995
), effects on activation
kinetics from mutations in the S3-S4 linker (Mathur et
al., 1997
; Tang and Papazian, 1997
), and changes in
voltage dependence of channel activation from mutations in the S4 through the S5 (Gautam and Tanouye,
1990
; Zagotta and Aldrich, 1990
; Lichtinghagen et al.,
1990
; Isacoff et al., 1991
; Papazian et al., 1991
; McCormack et al., 1991
, 1993
; Lopez et al., 1991
; Perozo et al.,
1994
; Shieh et al., 1997
).
It is clear from this and previous work that substitutions of all types of amino acids in the S4, including polar, nonpolar, and hydrophobic amino acids, can lead
to dramatic changes in the slope of the conductance-
voltage relation and the voltage range of channel activation (McCormack et al., 1991, 1993
; Lopez et al.,
1991
; Liman et al., 1991
; Schoppa et al., 1992
; Logothetis et al., 1992
; Tytgat and Hess, 1992
). Additionally,
substitutions of S4 basic residues can cause changes in
the voltage dependence that cannot be explained by
changes in charge alone (Liman et al., 1991
; Logothetis
et al., 1992
; Aggarwal and MacKinnon, 1996
). We suggest that it is likely that these observed changes in activation gating, from substitution of charged and uncharged
residues alike, include changes in subunit cooperativity.
In the next paper, we examine the relative contributions of the charged and uncharged S4 residues to the
cooperative interactions in activation gating.
![]() |
FOOTNOTES |
---|
Address correspondence to Dr. Richard W. Aldrich, Dept. of Molecular and Cellular Physiology, Stanford University, Beckman Center, B171, Stanford, CA 94305-5426. Fax: 650-725-4463; E-mail: raldrich{at}leland.stanford.edu
Received for publication 5 September 1997 and accepted in revised form 15 December 1997.
The authors thank L. Salkoff for kindly sending cDNA for Shaw in the bacterial vector pBSK (Butler et al., 1989) and R. MacKinnon for the kind gift of the A and B protomers for constructing dimers. We also thank Melinda Przetak, Joan Haab, Peter Drain, and Adrienne Dubin for help with making the mutant channel constructs. We thank Tom Middendorf, Max
Kanevsky, and Gargi Talukder for helpful comments on the manuscript.
This work was supported by a grant from the National Institutes of Health (NS-23294). R.W. Aldrich is an investigator with the Howard Hughes Medical Institute. J.L. Ledwell was supported by an NSERC 1967 Science and Engineering Scholarship from the Natural Sciences and Engineering Research Council of Canada.
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