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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Substitution of the S4 of Shaw into Shaker alters cooperativity in channel activation by slowing a cooperative transition late in the activation pathway. To determine the amino acids responsible for the functional changes in Shaw S4, we created several mutants by substituting amino acids from Shaw S4 into Shaker. The S4 amino acid sequences of Shaker and Shaw S4 differ at 11 positions. Simultaneous substitution of just three noncharged residues from Shaw S4 into Shaker (V369I, I372L, S376T; ILT) reproduces the kinetic and voltage-dependent properties of Shaw S4 channel activation. These substitutions cause very small changes in the structural and chemical properties of the amino acid side chains. In contrast, substituting the positively charged basic residues in the S4 of Shaker with neutral or negative residues from the S4 of Shaw S4 does not reproduce the shallow voltage dependence or other properties of Shaw S4 opening. Macroscopic ionic currents for ILT could be fit by modifying a single set of transitions in a model for Shaker channel gating (Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1994. J. Gen. Physiol. 103:321-362). Changing the rate and voltage dependence of a final cooperative step in activation successfully reproduces the kinetic, steady state, and voltage-dependent properties of ILT ionic currents. Consistent with the model, ILT gating currents activate at negative voltages where the channel does not open and, at more positive voltages, they precede the ionic currents, confirming the existence of voltage-dependent transitions between closed states in the activation pathway. Of the three substitutions in ILT, the I372L substitution is primarily responsible for the changes in cooperativity and voltage dependence. These results suggest that noncharged residues in the S4 play a crucial role in Shaker potassium channel gating and that small steric changes in these residues can lead to large changes in cooperativity within the channel protein.
Key words: Shaker; gating; ion channel; patch clamp ![]() |
INTRODUCTION |
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The S4 segment of voltage-gated ion channels participates in sensing the membrane voltage and in the conformational changes leading to channel opening (Yang
and Horn, 1995; Aggarwal and MacKinnon, 1996
; Mannuzzu et al., 1996
; Larsson et al., 1996
; Seoh et al.,
1996
; Yusaf et al., 1996
; Yang et al., 1996
). In the preceding paper, we examined the properties of Shaker
channels with S4 segments substituted from other related channels (Smith-Maxwell et al., 1998
). We found
that the slopes and positions of the conductance-voltage curves in the several S4 chimeras do not correlate
with the nominal charge content of the S4. Instead, we
found that cooperative interactions between subunits
play a major role in determining the voltage dependence of channel activation. A closer examination of
the Shaw S4 chimera and of heterodimers with Shaker
and Shaw S4 subunits revealed that a highly cooperative step in the activation pathway is rate limiting.
These findings are consistent with a role for the S4 in
channel activation that involves not only sensing voltage, but also somehow mediating cooperative interactions between channel subunits.
In this paper, we investigate the molecular basis for
the changes in activation gating observed in the Shaw
S4 substitution. We constructed mutants in Shaker with
combinations of amino acids substituted from Shaw S4
to determine the amino acids in Shaw S4 responsible
for the altered gating behavior. We use the information from changes in the position and slope of the conductance-voltage curve and also changes in kinetics and in
sigmoidicity of the activation time course to interpret
the effects introduced by amino acid substitutions.
Changes in sigmoidicity are diagnostic of changes in
the kinetic mechanism of activation, providing information about changes in the relative contributions of
the many conformational changes in the activation
pathway (Zagotta et al., 1994a; Smith-Maxwell et al.,
1998
). We interpret the changes in gating of mutant
channels in terms of a kinetic scheme developed previously for Shaker that closely fits macroscopic ionic, single channel, and gating currents (Zagotta et al., 1994
a).
We identify a set of transitions that are altered by the
substitutions and show that a single, conservative amino
acid substitution is primarily responsible for the change in cooperativity. Our study shows that steric interactions
of noncharged residues are likely to play an important
role in the activation process and mediate cooperative
interactions between subunits. Preliminary reports of
these findings have been presented in abstract form
(Smith-Maxwell et al., 1993
, 1994
; Ledwell et al., 1997
).
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MATERIALS AND METHODS |
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Molecular Biology
All constructs in the Shaker background were made in a form of
Shaker mutant, ShB6-46, in which fast N-type inactivation was
removed and silent restriction enzyme sites were added (Smith-Maxwell et al., 1998
). Construction of the Shaw S4 chimera was
described in the preceding paper (Smith-Maxwell et al., 1998
).
All other mutations made in the Shaker background were generated by PCR-mediated cassette mutagenesis. DNA fragments with
mutations generated by PCR were inserted between naturally occurring unique restriction enzyme sites in the channel construct,
StyI and NsiI. All mutations were verified by dideoxy termination
sequencing (Sanger et al., 1977
). Single amino acid substitutions
are identified by their residue number. Identification of the substitutions in each multiple mutant is as follows: ESS-R362E,
R365S, K380S; EFFSII-R362E, V363F, I364F, R365S, L366I, V367I;
FIIT-I364F, L366I, V369I, S376T; ILT-V369I, I372L, S376T; IL-V369I, I372L; LT-I372L, S376T; IT-V369I, S376T (Fig. 1). We were
unable to detect currents from two other multiple point mutants
we constructed: FIML-V363F, L366I, F370M, I372L and IMLTS-V367I, F370M, I372L, S376T, K380S. Numbering of amino acids
is from Schwarz et al. (1988)
for the ShB1 potassium channel.
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A highly expressing ILT construct was made by substituting the
mutant ILT sequence into a high expression Shaker (W434F) vector obtained from Ligia Toro (Perozo et al., 1993). DNA from the
original ILT mutant was cut between the BsiWI and SpeI restriction enzyme sites, generating a fragment that includes the ILT S4
and the Shaker wild-type tryptophan residue at position 434. This
piece of DNA was substituted into the W434F Shaker construct,
creating a conducting form of ILT that expressed at high levels.
The VIS/Shaw triple point mutant, constructed in the Shaw
background, was made by annealing sense and antisense oligonucleotides spanning two naturally occurring restriction enzyme
sites in the Shaw sequence, ClaI and StyI. Substitutions for the
point mutations were made in Shaw at the following sites, using
numbering from Butler et al. (1989): VIS/Shaw-I302V, L305I,
T309S (see Fig. 1).
Expression System and Electrophysiology
Xenopus oocytes were used for functional expression of mutant
DNA channel constructs, as described in the previous paper
(Smith-Maxwell et al., 1998). For Shaker and all mutants in the
Shaker background, cRNA was made from KpnI-linearized DNA
with T7 RNA polymerase. For Shaw and VIS/Shaw, cRNA was
made from SacI- or SalI-linearized DNA with T3 RNA polymerase.
Macroscopic ionic currents were measured from inside-out
membrane patches as previously described (Smith-Maxwell et al.,
1998; Hamill et al., 1981
), using Mg++-free intracellular solutions
for constructs requiring large positive voltage steps to activate.
Ionic currents were digitized at 5-50 kHz and low pass filtered
with an eight-pole Bessel filter at 2-9 kHz, depending on the
channel kinetics. Details are stated in the figure legends.
Gating currents were measured using a high performance cut-open oocyte voltage clamp (CA-1; Dagan Corp., Minneapolis,
MN) (Taglialatela et al., 1992). The oocytes were permeabilized
with 0.3% saponin. Agar bridges with platinum iridium wire were
filled with 1 M NaMES (sodium methanesulfonic acid). Microelectrodes were filled with 3 M KCl and had tip resistances of <1
M
. The following solutions were used for measurement of gating currents. The internal solution included (mM): 110 KOH, 2 MgCl2, 1 CaCl2, 10 EGTA, 5 HEPES, pH 7.1 with MES. The external solution included (mM): 110 NaOH, 2 KOH, 2 MgCl2, 5 HEPES, pH 7.2 with MES. Nonlinear capacitive currents of comparable amplitude and decay rate were never observed in uninjected oocytes.
All experiments were carried out at 20 ± 0.2°C.
Data Analysis
The voltage dependence of channel opening was estimated from
the normalized conductance calculated two different ways as outlined in Smith-Maxwell et al. (1998). Normalized chord conductance was used for ShB
6-46, R362E, ESS, EFFSII, F370M, V369I,
and S376T. Normalized conductance was calculated from isochronal measurements of tail currents after the peak current for
FIIT, ILT, Shaw S4, IL, LT, IT, I372L, VIS/Shaw, and Shaw. Conductance-voltage curves were fit with first or fourth power Boltzmann functions as follows:
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with n equal to 1 or 4. For a first power Boltzmann function, V1/2 is the voltage at which channel opening is half maximal. For a fourth power Boltzmann, V1/2 approximates the voltage at which each subunit is activated half maximally. The slope factor is equal to RT/zF.
Activation kinetics were quantified by fitting the activation
time course of macroscopic ionic currents with the following exponential function: I(t) = A[1 e (
t + d)/
].
I(t) is the current at time t, A is the scale factor for the fit, is
the time constant, and d is the delay or amount of time required
to shift the single exponential curve along the time axis to obtain
an adequate fit of the activation time course. The time course of
most currents could be fit with this function from a beginning
current level of 2-5% up to the maximum current level. Large
sigmoidal delays such as those seen with Shaker cause currents to
deviate significantly from single exponential behavior early in activation, making it necessary for single exponential fits to begin
at 20-50% of the maximum current. Time constants for deactivation were obtained from fits of a single exponential to tail currents measured at negative membrane potentials. Tail currents
were generally well fit by a single exponential function.
Analysis of the sigmoidicity in activation kinetics was carried
out as described previously (Zagotta et al., 1994a, 1994b; Smith-Maxwell et al., 1998
). Briefly, currents are first scaled so that all
traces reach the same maximum value. Then the scaling of the
currents along the time axis is changed so that the slope at the
half maximal current level is the same for all traces. This method
of analysis allows comparison of the delay in the activation kinetics relative to the overall time course of channel opening. When
channel opening follows a single exponential time course with
no delay, the relative time to half maximum current (thmx) is defined as equal to one. Relative thmx values greater than one indicate the presence of a delay in channel opening (see Fig. 4 C).
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Simulations of macroscopic currents were carried out as described previously (Zagotta et al., 1994b; Smith-Maxwell et al., 1998
). Rate constants in the 15-state model used here to describe ILT and I372L macroscopic currents are taken from a model described previously for the Shaker potassium channel (see Fig. 7
and Table I in Zagotta et al., 1994
a). Only transitions between
the last closed and open states are altered to simulate mutant
macroscopic currents.
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RESULTS |
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Substitution of S4 segments in Shaker can decrease the
apparent voltage dependence of channel opening by
changing cooperative interactions between the subunits, separate from changes due to decreased gating
charge (Smith-Maxwell et al., 1998). The most pronounced changes in channel activation were introduced
by substitution with the S4 of Shaw. Shaw S4 activation
is shifted ~+120 mV to more positive voltages and activates with 2.7-fold less apparent voltage dependence
than Shaker (Fig. 2, Table I). The rate of Shaw S4
opening is much slower than the rate of Shaker opening and is shifted along the voltage axis in the same direction as the probability of channel opening. Shaw S4
activation kinetics follow a single exponential time
course over a wide voltage range, unlike Shaker, which
activates at most voltages with a large sigmoidal delay.
All of these changes in macroscopic currents can be accounted for qualitatively by making a single cooperative transition in the activation pathway rate limiting
(Smith-Maxwell et al., 1998
).
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In this paper, we determine which amino acids in the S4 sequence of Shaw S4 are responsible for the functional differences between Shaw S4 and Shaker. As shown in Fig. 1, the S4 sequences of Shaw and Shaker differ in 11 of 21 positions. We generated several mutant Shaker potassium channels with single or multiple point mutations, substituting residues from the S4 of Shaw into Shaker in various combinations (Fig. 1). Representative macroscopic current traces and normalized conductance-voltage curves from several patches for each mutant are illustrated in Fig. 2. Results obtained from analysis of the conductance-voltage curves for each channel species are summarized in Table I.
While the 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
), it does not reflect the contributions to
channel gating from interactions between subunits
(Sigworth, 1993
; Zagotta et al., 1994
a). Since we are interested in all effects of S4 substitutions on channel
opening, we focused our attention on the slope of the
conductance-voltage relation at moderate open probabilities where contributions from both charge and cooperativity are reflected in channel opening. We use
"slope" as distinguished from "limiting slope" to refer
to the steepness of the conductance-voltage relation in
the range of moderate open probabilities.
Substitution of S4 Basic Residues
Extensive evidence in the literature supports the hypothesis that the S4 in voltage-gated cation channels is
part of the voltage sensor and that S4 basic residues
provide much of the gating charge (Yang and Horn,
1995; Aggarwal and MacKinnon, 1996
; Seoh et al., 1996
; Larsson et al., 1996
; Mannuzzu et al., 1996
; Yusaf
et al., 1996
; Yang et al., 1996
). Because the basic residues play an important role in channel activation, we
focused first on three amino acid differences between
Shaker and Shaw S4 that occur at positions where
Shaker has basic residues and Shaw S4 does not. Two of
the substitutions exchange neutral serines for the basic
residues at positions 2 and 7 in the S4 of Shaker and
the other substitution exchanges, such as negatively
charged glutamate for the positively charged arginine
at position 1 (see Fig. 1). We studied several mutants in
which one or more of these three basic residues were substituted, including R362E, ESS, and EFFSII (Fig. 2,
Table I). The ESS mutant has substitutions at all three
basic residues, which results in a decrease in nominal
net charge of the S4 from +7 in Shaker to +3 in ESS.
Such a large decrease in charge content of the S4
might be expected to alter profoundly the voltage dependence of channel gating. Surprisingly, changes in
channel opening introduced by the ESS mutant are relatively modest compared with the changes introduced
by the total S4 of Shaw. The voltage range of activation is shifted only +10 mV and the slope of the conductance-voltage curve is only slightly decreased. In fact,
for all three of the mutants (R362E, ESS, EFFSII), the
slope is roughly the same, regardless of whether one,
two, or three basic residues are substituted. Thus, there
is no correlation between the nominal charge content of the S4 and the position and slope of the conductance-voltage curve. All three mutant channels activate
quickly, like Shaker but unlike Shaw S4.
These results show that the substituted basic residues
in Shaw S4 are not responsible for the large changes
observed in the voltage-dependent behavior of the
macroscopic ionic currents. The results could be interpreted to mean that basic amino acids at positions 1, 2, and 7 of the S4, lying at either end of the S4 segment,
contribute minimally to the activation process of Shaker
potassium channels compared with the four remaining
basic residues within the core of the segment. This does
not seem to be the case, however, because experiments
measuring the individual contribution to the gating
charge of each of the S4 basic residues in Shaker suggest that basic residues at positions 1-5 contribute substantially to the gating charge (Aggarwal and MacKinnon, 1996; Seoh et al., 1996
). An alternative interpretation is suggested by the results and conclusions in our
preceding paper. The decrease in S4 charge in R362E,
ESS, and EFFSII may be masked because the shape and
position of the conductance-voltage curve are dominated by cooperativity between subunits.
Substitution of S4 Nonbasic Residues
We studied several mutants with substitutions of nonbasic residues from Shaw S4 into Shaker. One mutant, EFFSII, in addition to substitution of the first two basic residues in the S4, includes four hydrophobic substitutions in the NH2-terminal half of the S4. The changes in activation introduced by the substitutions in the EFFSII mutant are small compared with those introduced in the Shaw S4 chimera. This result suggests that the NH2-terminal half of the S4 is not responsible for the major differences between the functional properties of Shaw S4 and Shaker.
There are five residues in the COOH-terminal half of the S4 that differ between Shaw S4 and Shaker. Several mutant channels were constructed with overlapping combinations of amino acid substitutions in this region. Among these mutants, only FIIT, F370M, and ILT expressed functional channels. The FIIT mutant opens with a steady state voltage dependence intermediate between Shaker and Shaw S4 (Table I), but it activates relatively rapidly and with a sigmoidal time course similar to Shaker.
The F370M mutant was generated by the substitution
of methionine for a phenylalanine normally present in
the middle of the Shaker S4. As shown in Fig. 2, activation kinetics are fast and sigmoidal, like Shaker. This is
the only mutant we examined where the probability of
channel opening is shifted to more negative voltages.
All of the other mutations reported here, like most S4
mutations in the literature, shift the probability of
channel opening to more positive voltages. However, a
few S4 mutations that negatively shift activation have
been reported (Lopez et al., 1991; Papazian et al., 1991
;
Liman et al., 1991
; Logothetis et al., 1992
, 1993
). The
F370M mutant also differs from most other S4 mutants
in that C-type inactivation is dramatically affected. The
channel inactivates much more rapidly than Shaker
and recovers from inactivation very slowly (data not
shown). Also, tail currents measured during channel
closing are very slow compared with Shaker (data not
shown).
Of the subsets of Shaw S4 amino acid replacements, only ILT had macroscopic ionic currents similar to Shaw S4 (Fig. 2, Table I). Activation kinetics of ILT are slow and single exponential over a wide range of voltages, like Shaw S4. The voltage range and slope of the conductance-voltage curve appear very similar to Shaw S4 as well. Since none of these three substitutions change the basic residues in the S4, the decrease in the slope of the conductance-voltage curve cannot be the result of decreasing the charge content of the S4. Our results are consistent with the idea that the S4 substitutions, V369I, I372L, and S376T, are responsible for the change in cooperativity of the activation process seen in Shaw S4.
Substitutions of Subsets of ILT
Individual substitution of any one of the amino acids in the ILT mutant is not sufficient to cause Shaker to behave like ILT. Representative currents from the V369I, I372L, and S376T mutants along with normalized conductance-voltage curves from several patches are shown in Fig. 3. Boltzmann fits to the conductance- voltage curves are summarized in Table I. Considerable changes in the voltage range of activation and the slope of the conductance-voltage curve are seen with each of the three point mutants, but all activate with relatively fast kinetics compared with Shaw S4 and ILT. Notably, records in Fig. 3 show that the V369I and S376T mutants both exhibit sigmoidal activation kinetics similar to Shaker while the I372L mutant appears to activate with much less sigmoidicity (see below).
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Since none of the single point mutants activate like ILT, we next tried to determine if any pairwise combination of the three substitutions, V369I, I372L, and S376T, was sufficient to produce ILT-like activation. Representative currents and normalized conductance- voltage curves for several patches with IL, LT, and IT mutants are also shown in Fig. 3. Values from Boltzmann fits to the conductance-voltage curves are presented in Table I. As with the single amino acid substitutions, there are considerable changes in the voltage range of activation and the slope of the conductance- voltage curve. The double mutants containing the I372L substitution activate more slowly than the V369I, S376T, and IT mutants, but not nearly as slowly as ILT, nor do they operate in the same voltage range nor with the same voltage dependence of opening (see Fig. 9). As with the single I372L point mutant, the two double mutants containing the I372L substitution appear to activate with less sigmoidicity than Shaker and the IT mutant. The conclusion from these experiments is that all three amino acid substitutions, V369I, I372L, and S376T, are necessary to make Shaker channels activate like Shaw S4 channels.
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This result is particularly interesting since all three substitutions (valine to isoleucine, isoleucine to leucine, and serine to threonine) are very conservative. Two of the three substitutions (V369I and S376T) add a methyl group to the end of an amino acid side chain, making it larger and more hydrophobic. The third substitution, I372L, simply translocates a methyl group by one carbon, changing the branchpoint of the aliphatic side chain. The I372L substitution results in little or no change in hydrophobicity or size of the residue, though it does result in a change in shape of the side chain. The profound effects of the substitutions in ILT on channel opening suggest that steric interactions of hydrophobic and polar side chains in the COOH-terminal portion of the S4 play an important role in the cooperative transition in the activation pathway. The large functional effects due to small physical and chemical changes introduced by these three substitutions suggest that residues in the COOH-terminal portion of each S4 are in a closely packed region of the channel protein.
These interactions do not seem to be as important in the gating of the S4 donor channel Shaw. The converse triple mutant VIS/Shaw (see Fig. 1; isoleucine to valine, leucine to isoleucine, threonine to serine in Shaw) does not appreciably alter the rate, time course, or voltage dependence of Shaw activation (data not shown). Perhaps the VIS substitutions in Shaw are better tolerated because the packing of amino acid side chains in the vicinity of the S4 is less constrained in Shaw. Alternatively, these substitutions may affect analogous transitions in the Shaw channel, but their effects are not evident due to other transitions dominating Shaw activation.
Within the Kv1 subfamily of Shaker homologs, the S4
is highly conserved across many species (Chandy and
Gutman, 1995). The wild-type amino acids at the ILT
positions (V369, I372, and S376) in Shaker are conserved among both Shaker and Shal homologs. Among rat Shaw homologs, the S4 is also highly conserved, as
are the amino acids I369, I372, and T376 at the equivalent Shaker positions. In contrast, the S4 of Drosophila
Shaw differs from its rat Shaw homologs in 11 of 21 amino acids. While the amino acids I369 and T376 in
Drosophila Shaw are the same as in the rat Shaw homologs, the residue at the Shaker equivalent position
372 in Drosophila Shaw is a leucine rather than the isoleucine present in the rat homologs. The leucine in
Shaw at this position replaces a highly conserved isoleucine that is present in most other channels. The fact
that residues at these three positions, and in particular I372, are highly conserved within and across subfamilies is consistent with the large functional consequences to mutations at these sites in Shaker.
Comparison of ILT and Shaw S4 Activation
While macroscopic ionic currents from ILT are very
similar to currents from Shaw S4, there are differences
between them (Fig. 4). ILT channels open and close a
little faster than Shaw S4 channels (Fig. 4 B). However,
the voltage dependence of the kinetics and steady state
activation are very similar. The equivalent charge associated with channel opening and closing was estimated
assuming that the opening and closing kinetics of ILT
and Shaw S4 are dominated by a single rate-limiting
transition (see legend, Fig. 4). This assumption is reasonable since the opening and closing kinetics of both
Shaw S4 and ILT are well fit by single exponential functions over a wide range of voltages. The equivalent
charge associated with channel opening is 0.78 and
0.85 electronic charge for Shaw S4 and ILT, respectively. These values are similar to each other and are
significantly larger than the value of 0.42 electronic charge calculated for opening transitions late in the activation pathway of Shaker (Zagotta et al., 1994b). Values for equivalent charge associated with the closing
transitions are estimated to be 0.86 and 0.90 electronic
charge for Shaw S4 and ILT, respectively. Again, the
values for ILT and Shaw S4 are similar, though they are
slightly less than the 1.1 electronic charges calculated
for Shaker channel closing (Zagotta et al., 1994
b).
Like Shaw S4, ILT activation is well fit by a single exponential function over a wide range of voltages. However, unlike Shaw S4, ILT activation deviates from single exponential kinetics at very positive voltages. We
compared deviations from single exponential behavior
in the two channel species by measuring the delay in activation relative to the rate-limiting transitions in the
activation pathway. The scaling procedure outlined in
MATERIALS AND METHODS allows us to compare the delay in activation at different voltages and between different channel species, under conditions where the
current magnitude and the rates of activation vary (see
also Smith-Maxwell et al., 1998; Zagotta et al., 1994
a,
1994b). A channel opening with no delay and after a
single exponential time course will have a relative time
to half maximum current, thmx, equal to one. A value
greater than one indicates the presence of delay in
channel opening due to multiple voltage-dependent
transitions between closed states in the activation pathway, none of which is rate limiting. For Shaw S4 and
ILT, the scaled currents superimpose at voltages between +30 and +140 mV and the relative thmx of the scaled currents is approximately one (Fig. 4 C), suggesting that a single voltage-dependent transition is rate
limiting over this entire voltage range. Above +140
mV, there is a small but progressive increase in the relative delay in the activation time course of ILT that is
not nearly as pronounced in Shaw S4. This increase in
relative delay at more positive voltages indicates that
ILT activation includes multiple voltage-dependent
transitions between closed states before the cooperative
step that is rate limiting at more negative voltages. The
transitions between closed states emerge at more positive voltages because the voltage dependence of the
rate-limiting transition is steeper than that of many or all of the transitions between closed states.
The different voltage-dependent patterns of sigmoidicity observed for ILT and Shaw S4 could have one of
several possible explanations. The rate-limiting transition of the Shaw S4 channel is a little slower than in
ILT and, therefore, more positive voltage steps may be
required to make the transition sufficiently fast to unmask delay due to closed-state transitions. Another possible explanation is that Shaw S4 activation involves
fewer or faster closed-state transitions or perhaps involves just a single transition between one closed state
and the open state. In contrast, the voltage-dependent pattern of delay in activation is much different for
Shaker. At low depolarizations there is little delay,
while at more depolarized voltages the amount of delay
increases progressively, and then saturates (see Fig. 8;
Zagotta et al. 1994a, 1994b). This pattern of delay in
Shaker can be explained by the large number of transitions between closed states with similar voltage dependences and by the nonindependent nature of the first
closing transition between the open state and the last
closed state (Zagotta et al., 1994
a, 1994b).
A Kinetic Model for ILT
To understand further the changes in the ionic currents introduced by the substitutions in ILT, we modified an existing multi-state model developed for Shaker
(Zagotta et al., 1994a). The rationale for this is as follows. First, the multi-state model is quite successful at
accounting for the kinetic and steady state properties of wild-type Shaker activation gating at the level of gating currents, macroscopic ionic currents, and single
channel currents (Zagotta et al., 1994
a). Second, since
the ILT mutant channel was constructed from three
very conservative amino acid substitutions, it seems reasonable to assume that the mutant channel would activate in fundamentally the same way as Shaker, but with
changes in one or more of the existing transitions.
Third, the delay in ILT activation kinetics observed
above +140 mV reveals the existence of other transitions in the activation pathway besides the rate-limiting transition.
The 15-state model for Shaker activation, shown in
Fig. 5, is an extended representation of a kinetic
scheme wherein each subunit of a channel tetramer
must undergo two sequential transitions before the
channel can open (Zagotta et al., 1994a). All horizontal transitions in the 15-state model represent the first independent and identical transition undergone by each
of the four channel subunits. All vertical transitions
represent subsequent identical transitions in each subunit, with the exception that the transition between the
last closed state and the open state has special properties. The first closing transition is slower than expected
for a purely independent process and is formally equivalent to cooperative stabilization of the open state.
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We were able to account successfully for the functional properties of ILT ionic currents by changing the
rates and voltage dependences of a single set of transitions between the last closed state and the open state.
We focused on this set of transitions for several reasons.
First, the single exponential time course of ILT channel opening implies that a single transition is rate limiting. Since the single exponential time course of activation is the result of identical mutations in each of four
channel subunits, a single cooperative or concerted transition must be altered by the mutations (Smith-Maxwell
et al., 1998). If instead, the mutations slow the forward
rate of either set of independent transitions in the 15-state model for Shaker, the activation time course would be sigmoid and not single exponential. Second, ILT
channels are much slower to open and close than Shaker
channels. If the last transition in ILT activation is sufficiently slower than the preceding transitions, the slow
transition would dominate the overall time course and
make a multi-step activation process, like the 15-state Shaker model, appear to be a single exponential process.
Third, slowing the forward rates in either set of independent transitions will not produce the large decrease
in slope observed for ILT. We showed in our previous
paper (Smith-Maxwell et al., 1998) that slowing the forward rate of a set of independent transitions in a six-state model shifts the conductance-voltage relation to more positive voltages but does not decrease the slope
of the curve. However, in the 15-state model for
Shaker, there are two sets of independent transitions
that have different voltage dependences. While slowing
the rate of the forward transitions for either set of independent transitions shifts the conductance-voltage relation to more positive voltages, the slope of the curve
is differentially affected. Slowing all forward rates of the
first set of independent transitions has no effect on the
slope of the conductance-voltage relation because
these transitions dominate opening in the 15-state Shaker model over a wide range of voltages. Slowing all
forward rates in the second set of independent transitions decreases the slope because this second set of
transitions now dominate activation and they move a
little less charge than the first set of transitions. However, this decrease in slope is very modest compared
with the dramatic decrease predicted by slowing a single forward transition or compared with the large decrease in slope observed with ILT. Therefore, altering
either set of independent transitions in the 15-state
model cannot explain our results.
Values for the rate constants and charge associated
with the last set of transitions in the activation pathway
for the Shaker and ILT models are given in Table II.
Values for the earlier transitions in the multi-state ILT
model are identical to those used for Shaker (see Fig. 7
and Table I in Zagotta et al., 1994a). To approximate the ILT ionic currents, the rate constant for the final
opening transition, ko(0), is made much smaller than
for Shaker, while the rate constant for the first closing
transition, kc(0), is larger than for Shaker. The net effect of these modifications is to shift the probability of
channel opening to more positive voltages and to slow
activation kinetics substantially. The charge associated
with the final opening transition in ILT is 1 electronic charge, three times larger than the 0.32 electronic
charge associated with the same transition in Shaker.
The charge associated with the first closing transition
in ILT of 0.80 electronic charge is slightly smaller than
the 1.10 electronic charges used for the same transition
in Shaker. The charge incorporated into these transitions in the ILT model is comparable to the equivalent
charge calculated independently in the previous section from the voltage dependence of the time constants
of activation and deactivation of ILT, where 0.85 electronic charge is associated with channel opening and
0.90 electronic charge with channel closing.
The modified Shaker model successfully reproduces
the voltage range and kinetics of activation and the
slope of the conductance-voltage curve observed for
the ILT mutant (Figs. 5 and 6). Although the predictions more closely follow the more negative conductance-voltage results, this discrepancy represents only a
small energetic difference (0.4 kcal mol1, calculated
from
G = z
V1/2, where z is the amount of charge moved for the final transition and
V1/2 is the difference in the half activation voltage between the model
and the ILT data). The time constants from single exponential fits to the simulated ionic currents are also
very similar to those from fits of currents from the mutant ILT channel, as is the voltage-dependent pattern of sigmoidicity (Fig. 6).
|
Since ILT opening and closing kinetics are well fit by
a single exponential function at voltages between 90
and +140 mV, we also simulated currents using a simple two state scheme as in Fig. 5. Values for the rate
constants and associated charge are the same as those
used for the final transition in the multi-state model for
ILT given in Table II. The simulated currents from the two-state model are very similar to simulated currents
from the multi-state ILT model. The steady state probability of channel opening is the same for the two models and there is little difference in the time course of activation. The two models differ only in their prediction
for whether or not there is a small delay in activation at
higher voltages. When the currents are scaled to measure the relative delay in activation, there is no delay in the two-state model. The relative accuracy with which
the two state model approximates the steady state properties and gating kinetics of the larger ILT model illustrates the dominance of the final rate-limiting step.
The multi-state model successfully predicts the delay
in activation observed at voltages above +140 mV, seen
for the ILT mutant (Figs. 4 and 5). Delay emerges at
higher voltages because the final transition into the
open state is no longer rate limiting. In the model, this
occurs because the equivalent charge associated with
the transition into the open state, zo, is four times
greater than the charge associated with the next slowest
rate constant, . Because of the steeper voltage dependence of the ko transition, the rates of the early and late
transitions approach each other at large positive voltages, and the early transitions produce a delay in activation. As can be seen in Fig. 6 C, the relative delay and
the voltage dependence of the delay from the multi-state-simulated ILT currents superimpose on results
from the ILT mutant channel. These results show that
even though macroscopic ionic currents from the ILT
mutant can be approximated by a simple two-state model over a wide voltage range, a multi-state model is
required to simulate the functional properties of ILT
currents over a more extended voltage range. Thus, we
are able to reproduce successfully the essential features
of the macroscopic ionic currents of ILT by making a
very simple modification of an existing multi-state model for Shaker channel activation.
ILT Gating Currents
Our conclusion that the activation pathway of the ILT
mutant resembles that of Shaker, except for the final
transition, makes certain predictions about the movement of gating charge in ILT. The gating charge
should move in a voltage range more negative than the
voltages required to open the channel and, at voltages where the channel opens, gating currents should be detected well before the ionic currents. As shown in Fig.
7, ON gating currents from the ILT mutant follow both
of these predictions. At voltage steps to between 100
and +20 mV, large gating currents are observed with
no detectable ionic currents. At more positive voltages
where the channels open, between +40 and +200 mV,
the gating currents are quite rapid compared with the
time course of channel opening. These results are consistent with the prediction from the altered Shaker
model that activation of ILT involves multiple voltage-dependent transitions that precede a final rate-limiting
step. More detailed analysis of ILT gating currents during activation and deactivation will be used to refine
the model for ILT (Ledwell, J.L., and R.W. Aldrich,
manuscript in preparation).
The Importance of the I372L Substitution
The I372L substitution changes the rate and voltage dependence of channel opening and the amount of sigmoidal delay in the activation time course. In fact, currents from all mutants containing the I372L substitution activate with less sigmoidal delay than mutants without the I372L substitution. The sigmoidicity of the single and double point mutants of ILT is analyzed in Fig. 8, where the relative time to half maximum, thmx, of scaled currents is plotted as a function of voltage. All mutants with the I372L substitution are represented by filled symbols, while all other mutants are represented by open symbols. The plot shows very clearly that all mutants with the I372L substitution activate with much less sigmoidal delay than Shaker and mutants without the I372L substitution, including the mutants V369I, S376T, and IT.
Time constants from all mutants with the I372L substitution are generally slower than those without the I372L substitution and are positively shifted along the voltage axis, as are the conductance-voltage curves (Fig. 9; see also Fig. 3). The slowest time constants for mutants with the I372L substitution, between 15 and 20 ms, are still much faster than the slowest time constant for ILT, ~44 ms at +50 mV. For mutants lacking the I372L mutation, the slowest time constant is nearer 10 ms, similar to Shaker. The slower, positively shifted kinetics of the I372L-containing mutants could explain the decrease in sigmoidicity if the I372L mutation slows the final step in activation while having little effect on the relatively rapid preceding transitions. These channels would then require larger positive voltage steps to open and there would be a large discrepancy between the rate of the slower final step and the rates of the faster preceding transitions. This would make the final step rate limiting and decrease the sigmoidicity of I372L channel activation, suggesting that the I372L substitution may be the dominant mutation changing cooperativity of the final transition in all I372L-containing mutants, including ILT.
The I372L mutation also alters the voltage dependence of channel kinetics. Equivalent charge estimates
for activation and deactivation kinetics from Shaw S4,
Shaker, ILT, and all single and double point mutants of
ILT are compared in Table III. For the I372L mutant,
0.90 electronic charges are associated with channel
opening and 1.17 electronic charges are associated
with channel closing. The charge associated with opening of the I372L mutant is similar to charge estimates
for Shaw S4 and ILT and is more than twofold larger
than the charge estimated for late transitions in the activation of Shaker (Zagotta et al., 1994b). The equivalent charge for closing is not significantly different from wild type. For all mutants with the I372L substitution, the charge associated with channel opening is at
least twofold larger than for the other mutants and
Shaker, which all contain the conserved isoleucine at
position 372. This analysis suggests that the I372L mutation changes a transition in the activation pathway in
each of the I372L-containing mutants, making the transition rate limiting and the voltage dependence steeper.
|
A Kinetic Model for I372L Activation
Activation of I372L mutant channels can be explained
by modifying the rate constants and charge associated
with the final transition in the multi-state model for
Shaker (Fig. 5). As with the ILT model, all transitions
between closed states were assigned values identical to
the original Shaker model (see Fig. 7 and Table I in
Zagotta et al., 1994a). Results from analysis of simulated currents using three different sets of parameters
for the final transition are superimposed on results
from analysis of I372L mutant ionic currents (Fig. 10).
All three sets of parameters have the same 0-mV rate
constants, but differ by the amount of charge assigned
to the forward and backward transitions. Simulations from all three sets of parameters fit the normalized
conductance-voltage curve for the I372L mutant reasonably well, but differ in how well they fit the time
constants for opening and closing.
|
The best fits to the I372L mutant data set the equivalent charge associated with the final opening transition to the value of 1.00 electronic charge taken from the ILT model, while the worst fits use the value of 0.32 electronic charge from the Shaker model. Our analysis of I372L mutant activation kinetics estimates an equivalent charge of 0.95 electronic charge associated with channel opening, which is similar to that obtained for ILT (Table III). For the first closing transition, there is less difference between the equivalent charge used for the Shaker model of 1.1 electronic charges and for the ILT model of 0.8 electronic charge. This small difference makes it more difficult to determine whether or not the I372L substitution is responsible for the decrease in charge in the ILT mutant. Also, there is very little difference in how well the I372L mutant data are fit using charge values for the first closing transition from the models for Shaker or ILT.
Our analysis suggests that the I372L mutation changes the rates and voltage dependence of a cooperative transition late in the activation pathway, that we identify in our model as the transition between the last closed state and the open state. When other mutations accompany the I372L substitution, as in the ILT and Shaw S4 mutants, gating is modified further.
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DISCUSSION |
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---|
Substituting the S4 of Shaw into Shaker changes cooperativity in the activation pathway, decreasing the voltage dependence of channel opening and making a single cooperative transition rate limiting (Smith-Maxwell
et al., 1998). In this paper, we show that simultaneous
substitution of only three noncharged residues within
the S4 of Shaker (V369I, I372L, S376T), creating the
ILT mutant, is sufficient to reproduce this change in
cooperativity. Ionic currents from ILT are very similar
to currents from Shaw S4, while currents from the
other substitution mutants that we tested are more similar to Shaker. Of particular interest is the fact that simultaneous substitution of three charged residues in
the S4 of Shaker with residues from Shaw S4 (ESS) fails
to reproduce the large decrease in voltage sensitivity of
Shaw S4 opening. Instead, the ILT substitutions that
mimic Shaw S4 activation are very conservative. While
there is no change in the electrostatic charge or chemical reactivity of the residues, there are changes in hydrophobicity, size, and shape of the substituted side
chains. Closely packed regions of proteins are especially sensitive to small changes in size and shape of the
amino acid side chains (Eriksson et al., 1992
, 1993
;
Creighton, 1993
). Therefore, the changes in gating are
likely to be mediated by differences in steric interactions.
Previous work has also shown that substitution of hydrophobic residues in or near the S4 can greatly affect channel activation (Lopez et al., 1991
; McCormack et al.,
1991
, 1993
; Hurst et al., 1992
; Schoppa et al., 1992
; Aggarwal and MacKinnon, 1996
; Tang and Papazian, 1997
).
The substitution of leucine for isoleucine is responsible for the single exponential activation kinetics and
the increase in the charge movement associated with
the final opening transition. Yet, it is seemingly the
most conservative of the three substitutions in the ILT
mutant. The only structural difference between isoleucine and leucine is the point of attachment of a single
methyl group along an otherwise identical aliphatic
side chain. Leucine and isoleucine are both hydrophobic and have the same molecular weight and van der
Waals volume. They are, however, slightly different in degree of hydrophilicity, the extent to which they tend to be
found buried within proteins or on the protein surface,
and the extent to which they stabilize protein secondary
structures (Creighton, 1993). In spite of the small differences between the amino acids, the I372L substitution
seems to be critically important for changing the properties of the final transition in the activation pathway.
The opposite substitution of isoleucine for leucine at
other positions in or near the S4 has been reported
previously to have large effects on steady state activation in Shaker (Hurst et al., 1992; McCormack et al.,
1993
). Substitution of isoleucine for leucine has been
reported to have significant effects on the energetics of
other proteins as well. In a study of T4 lysozyme, a leucine within the core of the protein was replaced in turn
with isoleucine, valine, methionine, and phenylalanine
(Eriksson et al., 1992
, 1993
). The isoleucine and valine
substitutions decrease the stability of the folded protein
by 1.4 and 2.3 kcal mol
1, respectively, much more
than expected from consideration of hydrophobicity
and cavity formation alone. The large decrease in stability is due to steric strain introduced by replacing the leucine with a residue of a different shape. The seemingly less conservative substitutions of leucine with methionine and phenylalanine are less destabilizing than
the isoleucine and valine substitutions.
Our analysis shows that the I372L substitution is responsible for the increase in equivalent charge movement associated with the final opening transition. One question that arises from this result is how substituting one uncharged amino acid for another could increase the charge movement associated with a voltage-dependent transition. The substitution could alter the movement of the voltage sensor by increasing the fraction of the electric field through which the charged residues move to open the channel or by increasing the number of charged residues that interact with the electric field. Alternatively, the final opening transition in Shaker could have the same voltage dependence as in the I372L mutant but be so fast as to be indistinguishable as a separate component in the kinetic measurements.
Cooperative Interactions in Activation
Subunit cooperativity seems to be a central feature in
potassium channel activation. Among several kinetic
models that have been proposed, there is general
agreement that potassium channel activation involves
transitions between multiple voltage-dependent closed
states and that one or more transitions in the activation pathway involve cooperative interactions (Schoppa et
al., 1992; Tytgat and Hess, 1992
; Sigworth, 1993
; Zagotta et al., 1994
a; Bezanilla et al., 1994
; McCormack et
al., 1994
; Hurst et al., 1995
; Starkus et al., 1995
). However, many details of the models differ, including the
number of states, the number of independent transitions, the connectivity of the states, and the way in
which the cooperativity is implemented. Cooperative
interactions within proteins can come about in a number of different ways. Further analysis will be required
of wild-type and mutant channels to determine the
physical mechanisms underlying cooperativity in channel gating. However, simple modifications of a reasonably successful model for Shaker gating explain the effects of the ILT mutation on channel gating quite well.
Of the many transitions in the kinetic model for wild-type Shaker, it is necessary to modify only a single cooperative step to fit the ILT ionic currents, making the transition rate limiting. To model ILT, we must decrease the forward rate constant, increase the backward rate constant, increase the equivalent charge for the forward (opening) transition and decrease the charge for the backward (closing) transition. These changes are sufficient to explain the slowing of channel opening kinetics, the lack of sigmoidicity in the activation kinetics, and the changes in the voltage dependence of opening and closing kinetics compared with Shaker. These changes can also explain the large decrease in apparent voltage dependence of ILT channel opening that occurs even though there is little change in the total charge associated with channel opening. The model for wild-type Shaker channels has a total of 13.08 electronic charges, while the model for ILT has 13.46 electronic charges.
The ability of the 15-state Shaker model to explain
the large differences in ILT gating with only small
changes further supports the validity of the model for
Shaker. A mutant such as ILT, in which a single transition is rate limiting, is useful for study of the transition
in relative isolation. Since most of the transitions underlying wild-type channel activation have similar rates
and voltage dependences, it is difficult to study individual transitions independently of one another (Zagotta
et al., 1994a).
Results of other studies with S4 mutations in Shaker
and related potassium channels can also be interpreted
in terms of changes in cooperativity. Mutations at positions equivalent to 372-376 in Shaker made in eag potassium channels have been reported to cause changes
in channel function similar to those observed with ILT,
including a large positive shift in the voltage range of channel activation and a decrease in the slope of the
conductance-voltage curve (Tang and Papazian, 1997).
These results can be interpreted as a change in the cooperativity of a transition in the activation pathway and
suggest that the COOH-terminal half of the S4 may serve
a similar function in eag and Shaker potassium channels, mediating cooperative interactions between channel subunits. And in another Shaker homolog, Kv1.1, using concatenated heterotetramer constructs, Hurst et
al. (1992)
showed that an isoleucine substituted for a
wild-type leucine present in the COOH-terminal half of
the S4 alters cooperative interactions between subunits
during activation.
Comparison of ILT with the hydrophobic substitution mutant, V2, located at the border between the S4
and the S4-S5 linker, provides insights into how mutations of hydrophobic residues in the S4 can alter channel activation (McCormack et al., 1991). In the V2 mutant, substitution of valine for leucine shifts the probability of channel opening to much more positive voltages and decreases the apparent voltage sensitivity of channel
opening, changes similar to those observed with ILT.
However, V2 and ILT have very different effects on activation kinetics. ILT activates much more slowly than
Shaker and activates with a single exponential time
course, whereas the time course of V2 activation is relatively rapid and sigmoid, like the time course of Shaker
activation. In all, three different classes of Shaker model
have been modified to explain the functional behavior
of V2 (Schoppa et al., 1992
; Sigworth, 1993
; McCormack
et al., 1994
). All include several independent transitions
and at least one cooperative transition late in the activation pathway. For all three models, it is necessary to alter the properties of a cooperative transition to simulate the V2 mutation. The specific details of the models
may, therefore, be less important than the fact that it is
necessary to change cooperativity to simulate the effect
of the mutation.
Even though the apparent voltage dependence of
channel opening is decreased, the total amount of
charge moved to open the V2 channel is not changed.
Gating currents from the V2 mutant show that while
most of the gating charge moves in a voltage range similar to the wild-type channel, there is a small component of gating charge, between 18 and 19% of the total
(as calculated from the model), that moves at more
positive voltages (Schoppa et al., 1992). This small, positively shifted component of gating charge corresponds
to the charge movement associated with the final voltage-dependent transitions. The apparent decrease in the voltage dependence of the probability of channel
opening reflects the isolation of the final rate-limiting
step in the activation pathway.
A similar small, positively shifted component of gating charge, comprising 13-14% of the total charge, is
predicted for the ILT mutant as well. ILT gating current measurements confirm that gating charge moves
in a negative voltage range where the channel does not
open. Since the kinetics of the rate-limiting step in ILT
activation are voltage dependent, there must be another component of gating current in the voltage
range of channel opening (Ledwell et al., 1997).
Separation of charge components along the voltage
axis has been observed with other S4 mutations as well.
The mutant Sh10, studied by Aggarwal and MacKinnon (1996)
, substitutes the nonbasic and mostly hydrophobic residues from the S4 of Kv2.1 (drk1) into Shaker. The charge separation this generates is even
greater than that found with V2. Mutations causing separation of gating charge are not confined to hydrophobic substitution mutations but also have been observed
with neutralization of positive charges in the S4, including glutamine substitutions of the second and third basic residues (Perozo et al., 1994
; Aggarwal and MacKinnon, 1996
; Seoh et al., 1996
). However, substitution of
the third basic residue with asparagine does not cause
separation of gating charge components.
Channel Structure and Conformational Changes
The folded structure of proteins is maintained by multiple
interactions, including hydrogen bonds, van der Waals
interactions, and hydrophobic interactions (Creighton,
1993). Since the S4 has a mix of hydrophobic, polar,
and basic residues, many different types of interactions
may contribute to stabilizing the S4 segment within different conformational states of the channel protein. Several lines of evidence suggest that upon activation
many residues in the S4 experience a change in their
relative exposure to hydrophobic and aqueous environments and, therefore, may encounter different types of
interactions along the activation pathway (McCormack et al., 1993
; Starkus et al., 1995
; Yang and Horn 1995
;
Yang et al., 1996
; Larsson et al., 1996
; Mannuzzu et al.,
1996
; Yusaf et al., 1996
).
While the secondary structure of the S4 segment is
not yet known, there is evidence that the secondary
structure of the COOH-terminal half of the S4 is important in channel activation. In the rat Shaker homolog,
Kv1.1, substitution of hydrophobic residues at two different sites within the COOH-terminal half of the S4
with proline, an amino acid known to destabilize both
alpha helices and beta sheets (Richardson and Richardson, 1989), alters the ability of channels to activate
(Hurst et al., 1995
). These results support findings by
McCormack et al. (1993)
from hydrophobic substitutions in the S4-S5 that secondary structure in this region, which includes the overlapping structural motifs
of periodic charged residues throughout the S4 and a
leucine heptad repeat in the COOH-terminal portion
of the S4, plays an important role in channel activation. The valine to isoleucine and isoleucine to leucine substitutions, as found in the ILT mutant, promote stabilization of alpha helical conformations, whereas the
serine to threonine substitution does not (Creighton,
1993
). If the COOH-terminal half of the S4 is alpha helical, all three substitutions would lie on the same face of the alpha helix, along a 10-11 Å stretch of the helical axis. The three residues could comprise a surface
that interacts with other regions of the channel. If the
S4 is alpha helical and the charged residues of the S4
interact with the charged residues of the S2 and S3, as
has been suggested (Papazian et al., 1995
; Seoh et al.,
1996
; Planells-Cases et al., 1995
; Tiwari-Woodruff et al.,
1997
), the helical face of the S4 with the ILT substitutions would be directed away from the S2 and S3 segments.
![]() |
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.
We thank Rob Taylor, Melinda Prtezak, Chris Warren, and Joan Haab for help with the molecular biology and Ligia Toro for the kind gift of the W434F construct of Shaker. We thank Max Kanevsky and Tom Middendorf for helpful comments on the manuscript. We thank Rob Taylor and Max Kanevsky for participation in the early experiments.
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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