From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520
Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH2-terminal-truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179-182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in ~40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of ~1.6 e0. The fully open T442S chimeric channel has the conductance sequence Rb+ > NH4+ > K+. The opposite conductance sequence, K+ > NH4+ > Rb+, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb+.
Key words: ion channel gating; conserved sequence; point mutation; patch clamp; mutagenesisThe traditional view of a voltage-gated ion channel has
the voltage sensor, the gate, and the ion permeation
pore as separate protein structures (Hille, 1992). However, structure-function studies have shown that mutations in the pore region can also affect gating (for reviews see Brown, 1993
; Sigworth, 1994
). In the case of
voltage-gated potassium channels, Yool and Schwarz (1991)
reported that mutations of F433 in Shaker shift
the voltage dependence of activation in addition to
changing the ion selectivity. Kirsch et al. (1992)
and De
Biasi et al. (1993a
,b) found two mutations in the pore
region of a Kv2.1-Kv3.1 chimeric channel that affect both ion permeation and deactivation kinetics. Heginbotham et al. (1992)
found that the double deletion of
Y445 and G446 in Shaker results in a nonselective channel that also deactivates very slowly.
In the Shaker channel, mutations of T442 show large
gating effects. This residue is the fourth in the potassium channel signature sequence TXXTXGYGD (Heginbotham et al., 1994) and is conserved among Shaker -like
channels, inward rectifiers, Ca2+-activated potassium
channels, cyclic nucleotide-gated channels, plant potassium channels, and potassium channels with two
pore regions. The only alternative residue at this position, Ser, is present in the eag channel. Yool and
Schwarz (1991)
found that the Shaker T442S mutant
channel has greatly prolonged openings as well as a
negative shift in the voltage dependence of activation.
The 442 position in Shaker is adjacent to residues that
affect ion permeation and block. Although Ala, Ser,
Gly, and Asp substitutions for T442 leave the ion selectivity unchanged, some mutations of the succeeding
residues V443 and the GYG motif disrupt ion selectivity (Heginbotham et al., 1994). A mutation of the preceding residue T441 affects internal tetraethylammonium
(TEA)1 binding (Yellen et al., 1991
). In a recent model
of the potassium channel pore, the backbone carbonyl
oxygen of T442 is proposed to contribute to one of the
potassium ion binding sites (Durell and Guy, 1996
).
The combination of permeation and gating effects of
mutations in this region therefore suggest that channel
gating and ion permeation involve some of the same
channel structures. For this reason we have carried out
a more detailed analysis of the behavior of T442 mutant channels.
Site-directed Mutagenesis and RNA Synthesis
All but one of the constructs used in this study were based on a
Shaker B chimera (Lopez et al., 1994; kindly provided by Dr. L.Y.
Jan, University of California, San Francisco, San Francisco, CA)
in which the S6 sequence was substituted with the corresponding sequence from the mKv3.1 (also known as NGK2) channel, and
in which the NH2-terminal inactivation sequence was removed.
This chimeric channel, denoted SN (see Fig. 1), has a single-channel conductance approximately fourfold larger than Shaker
when expressed in Xenopus oocytes. Cassette insertion mutagenesis was used to introduce mutations into SN. An oligonucleotide
coding for Ser at the 442 position, as well as silent mutations for
restriction sites BstEII, ActII, and SgrAI, was first synthesized
(DNA Synthesis Laboratory, Yale University, New Haven, CT)
and inserted into SN in pBluescript (Stratagene, La Jolla, CA) by
using the restriction sites NsiI and HindIII; the latter site had
been introduced when SN was constructed (Lopez et al., 1994
).
This new construct, denoted SNS, was further used to make other
mutations at the 442 position. Oligos containing codons for Asp,
Cys, Gly, His, Tyr, or Val were synthesized and inserted into SNS
using the BstEII and SgrAI sites. The one construct not based on
the SN chimera was SS (kindly provided by Dr. R. MacKinnon,
Rockefeller University, New York), a Shaker H4 channel having a
T442S mutation and the
6-46 NH2-terminal deletion. The
amino acid sequence of Shaker H4 (Kamb et al., 1988
) is identical
to that of ShB (Schwarz and Jan, 1988
) except for four amino acids in the COOH-terminal region. Mutations were verified by sequencing. The cDNAs were linearized with EcoO109I, and the capped, T3 run-off transcripts were stored at
70°C.
Preparation of Oocytes and RNA Injection
Female toads (Xenopus laevis) were anesthetized by immersion in
water containing 1.5 mg/liter 3-aminobenzoic acid ethyl ester (Sigma Chemical Co., St. Louis, MO), and oocytes were removed through a small abdominal incision. The follicular membranes
were removed by incubating for 2-4 h in 2 mg/ml collagenase
(type 1a; Sigma Chemical Co.) containing OR3 solution (Blumenthal and Kaczmarek, 1992), which consists of 50% Leibovitz's L-15 medium (GIBCO BRL, Gaithersburg, MD), 5 × 104
U/liter nystatin (Sigma Chemical Co.), 20 mg/liter gentamycin (Sigma Chemical Co.), and 15 mM Hepes adjusted to pH 7.3. Defolliculated oocytes were washed repeatedly with OR3 to remove
collagenase. Stage V-VI oocytes were selected and stored in OR3
solution at 20°C, before and after RNA injection. Volumes of 50-
100 nl of cRNA were injected using a microinjector (Drummond
Scientific Co., Broomhall, PA). Concentrations of injected cRNA
were varied to control the level of expression.
Measurements of Macroscopic Currents
Macroscopic currents were recorded in cell-attached membrane
patches using conventional oocyte macropatch techniques (Stühmer et al., 1991) and the Pulse software (HEKA-Electronic, Lambrecht, Germany). Patch pipettes were pulled from Kimax glass
capillaries (Kimble Products, Vineland, NJ) with tip diameters
ranging from 2.5 to 25 µm and coated with Sylgard (Dow Corning Co., Midland, MI). The pipette solution contained 140 mM
K-aspartate, 1.8 mM CaCl2, 10 mM Hepes and the bath solution
contained 130 mM K-aspartate, 10 mM KCl, 1 mM EGTA, 10 mM
Hepes; each was adjusted to pH 7.3. The liquid junction potential at the interface of these two solutions was estimated to be 0.8 mV; no correction was applied. Voltage pulses were applied from
a holding potential of
100 mV, and the current signals were filtered at 2.5 kHz. Data were sampled at 12.5 kHz. For subtraction
of linear leak and capacitive currents, a single P/4 pulse (Bezanilla and Armstrong, 1977
) from a
120-mV leak holding potential accompanied each depolarizing pulse. To increase the signal-to-noise ratio, 5 or 10 sweeps were averaged before the data were
stored. Displayed traces were additionally filtered with a Gaussian
digital filter to the
3 db frequency indicated in the figure legends. Fits of time courses to exponential functions were performed by least-squares using the Igor data analysis program
(WaveMetrics, Lake Oswego, OR).
Measurements of Single-Channel Currents
Single-channel recordings were made in inside-out patches and,
in some cases, in cell-attached patches. We found no difference in the kinetics between data recorded by these two recording
methods. Patch pipettes were pulled from 7052 glass (Garner
Glass, Claremont, CA) with 1-2.5-µm tip diameters and were usually coated with Sylgard. Except as noted, the recording solutions
were identical to those used in the measurements of macroscopic
currents; for the selectivity experiments, Rb+, NH4+, Cs+, Na+,
Li+, or N-methyl-D-glucamine (NMDG) was substituted for K+ in
the pipette solution. Filtering and sampling frequencies were the
same as in macroscopic recordings. Channel activation was measured by depolarizations from a 100-mV holding potential to various voltages from
80 to +70 mV. Deactivation was measured at voltages from
100 to
200 mV after a prepulse whose
amplitude and duration were appropriate to open the channel.
Leak subtraction was performed using an average of the nearest
null traces. Most of the measurements were performed on
patches containing only one channel, as verified by recordings
using a 500-ms voltage ramp from the holding potential to +100
mV. Single-channel patches were always used for measurements
of first latencies. Multichannel patches were used in other cases
(such as tail current experiments) where protocols were designed such that only one channel was activated at a time.
Patches were discarded if endogenous ion channel activity existed, as judged by the conductance, kinetics, and reversal potentials of the channel events.
The analysis of the single-channel recordings was performed
with various user-developed programs in the PowerMod environment (HEKA-Electronic) using the Modula-2 language. Data
were filtered to 0.5-1-kHz bandwidth with a digital Gaussian filter
to achieve an appropriate signal-to-noise ratio. Event detection
was performed using the threshold-crossing analysis method
(Colquhoun and Sigworth, 1995), except here a set of three
thresholds was used to detect transitions among multiple current
levels. An idealized event list was constructed in which the time of
threshold crossing and current level were registered. Dwells with
a duration less than 0.36/fc were ignored, where fc is the filter
3
db frequency.
The dwell times at each current level were fitted with a mixture
of two exponential probability density functions using the maximum-likelihood method with simplex optimization (Colquhoun and Sigworth, 1995). Only dwell times longer than 0.5/fc were fitted. The fit was corrected for this left censor time and for the
right censor time, which was the pulse duration. The maximum-likelihood fitting of dwells at sublevels yielded a fast component
that had an extrapolated area usually <25% (the largest being
40%) of the total and had a time constant of 0.5 ms or less. This
component likely resulted from extra threshold crossings due to
noise or drift in the subconductance levels. The time constant of
the slower component was taken as the mean dwell time
i in
state i. No correction was made for the presence of false events
(represented by the faster component) or missed events because,
in the worst case, these accounted for only 9 and 5% of the total,
respectively. A total of 50-2,500 events was fitted to obtain each
i
value.
Rate constants were estimated as follows: let ij be the state entry probability, the probability that the channel enters state j
when it exits state i, such that
![]() |
Values of ij were estimated from the event list. Then the rate
constant from state i to state j was computed as
![]() |
The partial charge qij associated with each rate constant was estimated by fitting the voltage dependence of each transition rate kij to the exponential function
![]() |
Log-transformed values of kij(V) were fitted by least-squares.
Fast Solution Switching
A fast solution-switching experiment was carried out to compare the sublevel currents carried by different permeant ions. Solution switching was achieved by moving the interface between continuously flowing, parallel K+ and Rb+ solution streams relative to a stationary outside-out patch. The solution streams flowed from a pulled theta-glass capillary with a tip diameter of ~300 µm, whose movement was driven by a linear stepper motor (model SF 77; Warner Instrument Co., Hamden, CT). The flow velocity of the solutions was 75-150 µm/ms, chosen to form a sharp solution interface while minimizing the chance of breaking the patch. Mechanical vibration artifacts were reduced by decreasing the stepper motor drive voltage to reduce its speed, and the residual artifacts were removed by subtracting averaged null traces from the trace with channel activity. After each experiment, the patch was broken by pressure applied to the pipette, and the rate of the solution switching was measured by monitoring the current steps resulting from changes in the liquid junction potential. In a typical experiment, the time between the 10 and 90% amplitude points of the junction potential change was 1.1 ms.
Simulations
To verify the single-channel analysis, artificial data were generated by a discrete-time algorithm as follows. At each time point,
the program determines whether a transition is made out of the
present state i by using a uniformly distributed random number.
Letting t be the sample interval, the probability of a transition is
taken to be
![]() |
If a transition does occur, the exit state j is chosen with probability
![]() |
by using a second uniformly distributed random number. Gaussian random numbers were added to the simulated data to produce noise at the same signal-to-noise ratio as seen in the experimental data. After digitally filtering to the same bandwidth as used in the actual measurement, the simulated data were processed by the analysis programs for comparison with the experimental results. Statistical quantities are given as mean ± SEM.
Expression of Mutations at the 442 Position
We characterized mutations of T442 in the background
of an NH2-terminal-truncated Shaker construct or in
the Shaker-NGK2 chimera SN (Fig. 1). We first tested
the expression of these mutations by two-electrode voltage clamp of Xenopus oocytes injected with each cRNA.
The substitution of Ser for T442 in either background resulted in robust currents (Fig. 2), as did the Gly substitution in the SN background. On the other hand,
substitutions of Cys, Asp, His, Val, or Tyr in the SN
background resulted in no ionic current, even when
concentrated cRNA from two separate batches was injected. We were also unable to detect gating currents
from these mutants, which implies that the lack of current does not result from a simple blockage of the
pore.
Mutations of T442 Slow Channel Deactivation but Have Little Effect on Channel Activation
Macroscopic currents from each channel type are shown in Fig. 2, A and B. All the three mutant channels showed activation time courses similar to SN, but the tail currents of the mutants decayed with a much slower time course. The G-V curves (Fig. 2 C) show that the activation voltage dependence of the T442 mutants is steeper and is shifted toward negative voltages. The change in voltage dependence can be explained by the stabilizing effect of the mutations on the channel open state, as suggested by single-channel recordings (see below).
The T442 mutations seemed to have little effect on
the activation kinetics. The activation time course was
characterized by fitting a single exponential function to
the macroscopic current starting at the time when the
current is 50% of its final value. The "activation time
constant" a is taken to be the time constant of the fitted exponential, while the "activation delay"
a was obtained by extrapolating the fitted exponential to the
current baseline. Schoppa and Sigworth (manuscript
submitted for publication) have shown that, at depolarized potentials,
a approximates the rate of the slowest
activation step, while
a approximates the sum of dwell
times in the other states in the activation pathway. Fig.
2, D and E, shows that the mutations have very little effect on the magnitude and voltage dependences of
these parameters at depolarized voltages.
When fitted with a single exponential, the deactivation time course of the SN channel has a time constant
of 1.8 ms at 100 mV (Fig. 2 B); the same value is obtained in Shaker channels under these conditions (Zagotta et al., 1994a
; Schoppa, N.E., and F.J. Sigworth,
manuscript submitted for publication). All of the T442 mutations resulted in much slower deactivation. The
time constant at
100 mV was 310 ms for SS, which has
the Ser mutation in the Shaker background. For mutations in the SN background, the time constants were
similar: 590 ms for SNS and 250 ms for SNG. The voltage dependence of decay kinetics is essentially unchanged by the mutations, however, representing a
partial charge movement of 0.8 to 0.9 eo in each case
(Fig. 2 F).
Fig. 3 shows representative single-channel currents
from each mutant. The SN channel has a large conductance, about four times that of wild-type Shaker channels
(Lopez et al., 1994); we measure a slope conductance
of 95 pS near
100 mV with an external solution containing 140 mM K+. The SNS and SNG mutants have
lower conductances of 44 and 41 pS, respectively. SS
has a conductance of 49 pS. All three T442 mutants
have remarkably long channel open times, especially in view of the negative potentials (
60 and
70 mV) at
which the recordings were made. The mutant channels
also have a reduced frequency of brief closures compared to SN channels. The long-lasting openings are
consistent with the slow deactivation seen in the macroscopic currents.
In view of the prolonged open times, it is surprising
that the activation kinetics of mutant channels are similar to those of SN channels. One possibility would be
that the only kinetic effect of the mutations is on the
stability of the open state. If so, then the latency to first
opening should be unaffected by the mutations. We
therefore measured the first latencies of SN and SNS
single-channel currents at various voltages (Fig. 4). Despite the large difference in their behavior after first
openings, the two-channel types showed very similar
first-latency distributions at each voltage (Fig. 4, B and
D). The median first latency at 60 mV for SN was 38 ± 2 ms (n = 4); for SNS the value was 32 ± 4 ms (n = 3).
Thus, it appears that the main kinetic difference between SNS and SN channels is the prolongation of the
channel open times.
Zagotta et al. (1994b) showed that the stabilization of
the open state, for example through a decreased channel closing rate, leads to cooperative behavior and a
steeper voltage dependence of channel activation.
Thus, the increase in steepness of the activation curves
of the T442 mutants (Fig. 2 C) is not unexpected, given
the remarkable stabilization of the open state by the
T442 mutations. The negative shift of the activation
curves can also be understood from the stabilization of
the open state, which tends to shift the gating equilibrium toward channel opening.
There Are at Least Two Subconductance Levels in SNS
Besides the lengthening of open times, all three channel types having T442 mutations showed intermediate
levels of conductance. Some examples of dwells in
these sublevels are marked with arrows in Fig. 3. In SNS
single-channel records elicited by depolarizations in
the range 60 to
90 mV, dwells at one or two distinct
conductance levels are typically seen before the channel reaches the fully open state. During channel deactivation, a "staircase" of conductance steps is also observed. The same behavior is seen in SNG channels. The SS channels also show the same subconductance
behavior in some sweeps, but in others there are long
periods of very rapid flickering; an example of each of
these "modes" is shown in Fig. 3 B. We chose the SNS
channels for further analysis because they do not show
this complicated behavior.
As reported by Hoshi et al. (1994), wild-type Shaker
channels show subconductance levels, but this activity
seems to be of a different kinetic nature. We observe
lower conductance levels that persist typically for tens
of milliseconds and occur randomly throughout the recording (Fig. 3 B), rather than being visited mainly
upon activation or deactivation as is seen with the T442 mutants. From a large set of recordings, Schoppa and
Sigworth (manuscript submitted for publication) estimated that of the total open time of NH2-terminal-
truncated Shaker channels, 17% is spent in conductance levels below the main level. We observed occasional periods of this kind of subconductance activity
also in SN channels.
To characterize the sublevels in SNS, we made amplitude histograms of the single-channel current by accumulating all the data points in the portion of the
record when the channel was making opening or closing transitions (Fig. 5 A). Representative histograms for
activation at 70 mV and deactivation at
140 mV are
shown in Fig. 5 B. At
70 mV, only a single sublevel
could be distinguished, but at more negative potentials
a smaller sublevel is also apparent. We shall call these
sublevels sub2 and sub1, respectively. As shown in Fig. 5
C, the amplitude of each current level showed a linear
voltage dependence in the voltage range tested. Sublevel currents were generally noisier than the fully
open channel current, as indicated by the increased
width of their Gaussian components in the fits to the
histograms.
When channels make many transitions, all-points amplitude histograms of this sort can be distorted, and the
peaks broadened, by the data samples that represent
transitions between current levels. One approach is to
exclude from histograms the samples near identified
transitions (Colquhoun and Sigworth, 1995; Chapman et al., 1997
). In the present case, the number of transition points is sufficiently small that this correction is
not required. For example, each histogram in Fig. 5 B
represents 380 sweeps and contains more than 200,000 entries. Of these, we calculate that fewer than 2,500 entries arise during current transitions.
SNS Sublevels Correspond to Intermediate Steps in the Activation Process
The question we asked next was whether each of the
sublevels is necessarily traversed when a channel passes
from the resting closed state to the final open state. We
addressed this question by measuring from single-channel activation time courses at 70 mV the dwell times
in sub1 and sub2 using a set of three thresholds, as
shown in Fig. 6 A. The threshold values were chosen to
be the midpoints between the various current levels of
the channel (0, 0.37, 0.70, and 1.0 times the fully open
channel current) as derived from the fits in Fig. 5 C.
Measurements were made only during the initial activation of the channel, i.e., between the time of first leaving the closed current level and the time of first entering the fully open level. Assuming that each current
level represents a single state, the dwell times at a given
level are expected to have a single-exponential distribution whose time constant is determined by the sum of
the rates of transitions leading away from that state. Although at this potential the sub1 peak is not visible in
amplitude histograms, substantial dwells in sub1 were
detected by the threshold analysis. The dwell times at
sub1 could not, however, be described by a single-exponential distribution (Fig. 6 B). The excess of short
events in the sub1 distribution accounted for ~60% of
the total.
The excess of short events in the distribution might
be expected to arise from a second component of the
dwell time distribution. Another possibility, however, is
that they arise from an artifact of the analysis procedure. When an instantaneous step in current occurs,
the recorded time course will show a slow transition
from one level to another because of the effect of filtering. A large transition, say from zero to the sub2 current level, will result in a finite time delay between
crossing the lower and upper thresholds that bracket
the sub1 current level (1 and
2 in Fig. 6 A), even in
this case when the dwell time in sub1 is zero. To test
whether the short events in the sub1 distribution might
arise from this mechanism, we ran a simulation using a
kinetic scheme like that shown in Fig. 6 C with various
values for the parameters. The dotted curve in Fig. 6 B
is the distribution of apparent dwell times at sub1 from
the model parameters shown. The distribution is well
described by the model, which has 59% of the channel
openings occurring as transitions from a closed state
(close2) directly to the sub2 state. On the other hand,
the distribution of apparent dwells at sub2 is well described by a simple exponential distribution of dwells,
although parameters corresponding to the case in
which 10% of openings proceed directly to the open
state also provides an acceptable fit (Fig. 6 D). In view
of the fact that this small fraction is probably not significant, in the final model (Fig. 6 C) we allow channels to
open only after passing through the sub2 state. We conclude that during activation, an SNS channel has an
~40% chance of traversing the sub1 state, and essentially always passes through the sub2 state, on its way to
the fully open state.
When a channel enters directly into sub2 on activation, does it pass from the same closed state that leads to sub1? This question cannot be answered from our experiments. It is possible, and we believe likely, that the two sublevels are entered from different closed states; we have drawn the scheme In Fig. 6 C reflecting this possibility.
Transitions between States Are Voltage Dependent
The activation of a Shaker potassium channel involves
many intermediate transitions, each of which is voltage
dependent and therefore involves charge movement
(Bezanilla et al., 1994; Zagotta et al., 1994a
). We were
therefore interested in testing whether transitions between the conductance states in the SNS channel were
voltage dependent. From single-channel recordings at
various membrane potentials, we again used a set of
three thresholds to measure the dwell times in each
current level and to determine the exit state following
each dwell; this is illustrated for tail currents in Fig. 7.
We found that the dwell times at each conductance level are voltage-dependent, as shown in Fig. 8. The
open time becomes shorter at hyperpolarized voltages,
with a mean value of 50.5 ms at
120 mV. The dwell
times at sub1 and sub2 have their maximum values of
5.3 and 9.4 ms at
100 and
120 mV, respectively. At
more hyperpolarized and more depolarized voltages,
the mean dwell time at each sublevel is smaller. There
is also a class of brief closures that completely interrupt
the current in the open state (see for example Fig. 7
A). These are also voltage dependent, having a smaller
mean duration at more hyperpolarized voltages.
The estimates of rate constants obtained in the voltage range from 70 to
140 mV are summarized in
Fig. 9 along with a kinetic scheme. In this scheme,
again, the SNS channel can open via two paths: one
passes through both substates while the other traverses only the sub2 state. A distinct closed state, closed3, follows the open state to account for the brief closures.
According to the scheme, the sub1-sub2 and sub2-
open transitions involve substantial charge movements,
1.56 and 1.62 eo, respectively. Although its parameters
were obtained from measurements at more negative
voltages, the scheme accounts quite well for the measured dwell times in the sub2 state at
40 and
30 mV
(Fig. 8). At 0 mV the dwell times in sub1 and sub2 are
expected to be quite short, 0.31 and 0.82 ms, respectively.
SNS Substates Have Different Ion Selectivities
Most potassium channels are permeable to Rb+ and
NH4+ in addition to K+. Two simple measures of the selectivity of a channel are the so-called permeability ratio, obtained from the reversal potential under biionic
conditions, and the conductance ratio, comparing current carried by the various ions. These quantities have
been determined by Heginbotham and MacKinnon
(1993) and by Perez-Cornejo and Begenisich (1994)
for Shaker channels. By both measures, K+ is the most
permeant ion of the three; the permeability ratios have
the sequence K+ > Rb+ > NH4+, but the conductances
follow the sequence K+ > NH4+ > Rb+. We list values
for these parameters from the work of Heginbotham and MacKinnon (1993)
in Table I. We performed similar measurements on SN and SNS channels in inside-out patches, although under somewhat different ionic
conditions. We first tested the selectivity of SN under
biionic conditions with 140 mM Rb+ or NH4+ in the pipette solution and 140 mM K+ in the bath. The reversal
potentials were
10 and
56 mV, yielding the permeability ratios given in the table and a sequence identical to that of Shaker, K+ > Rb+ > NH4+. To evaluate the
conductance ratios, Heginbotham and MacKinnon (1993)
measured conductances near 0 mV in symmetrical solutions; we used a less rigorous approach, comparing the
single-channel currents under biionic conditions at
120 mV, where current is assumed to be carried predominantly by the test ion. In SN channels, the conductance sequence so obtained was K+ > NH4+ > Rb+,
again the same as in Shaker.
Table I. Comparison of the Selectivities of SN, SNS, and Shaker |
SNS channels also conduct Rb+ and NH4+ currents,
but no inward current was detected from SNS at the
single-channel level when Na+, Li+, Cs+, or NMDG ion
was used in the pipette solution. The reversal potentials under biionic conditions were 5 mV (Rb+/K+) and
45 mV (NH4+/K+), yielding the same permeability
sequence as Shaker. The conductance sequence was different, however. Both NH4+ and Rb+ currents had
larger single-channel conductances, with the sequence Rb+ > NH4+ > K+ (Table I).
NH4+ and Rb+ currents through the SNS channel
(Fig. 10 A) are similar to K+ current in that they both
show long, stable openings. They also show sublevels.
Like those in K+ current, the sublevels in Rb+ and
NH4+ currents are brief compared to the open levels
and are traversed as the channel activates and deactivates. Fig. 10 B shows a representative single-channel
tail current time course at 140 mV for each permeant ion. Amplitude histograms obtained at
140 mV (Fig.
10 C) show clear peaks for two or three sublevels in
NH4+ and Rb+, respectively. We performed a kinetic
analysis of the sublevels in Rb+, like that shown in Fig.
6. We found that the intermediate (S2
) sublevel was
traversed almost every time, while the small (S1
) sublevel was frequently skipped during deactivation at
120 mV, a phenomenon also seen with K+ currents.
The general kinetic behavior suggested that S1
and S2
sublevels in NH4+ currents and the S1
and S2
sublevels in Rb+ correspond to the same kinetic states
as the sub1 and sub2 levels in K+ current. However, in
the amplitude histogram of Rb+ current, there is a
third peak, S3
, at a current level that is larger than the
S2
sublevel. In single-channel recordings, this current
level appears with a short dwell time, as illustrated in
Fig. 10 B.
The existence of an extra current level in Rb+ places
in some doubt our assignment of the sublevels. As a further test of the identity of the sublevels in NH4+ and
Rb+ that correspond to sub1 and sub2 in K+ current,
we performed rapid solution-switching experiments.
During recordings of single-channel deactivation, the
solution bathing an outside-out patch was rapidly
changed. Through the accumulation of many trials, recordings were obtained in which the permeant ion was
changed between K+ and Rb+ while the current was in
each conducting level. Fig. 11 A shows representative
sweeps from these experiments, which show changes in
current level consistent with the identification of S2
in Rb+ with sub2, and S1
with the sub1 conductance
level. A comparison of the current levels before and after the solution switching in many sweeps (Fig. 11 B)
confirms the identification. The additional level S3
appears to arise from a short-lived, degenerate state whose conductance level is indistinguishable from sub2
in K+ but becomes visible in Rb+ currents.
It is interesting to note in Fig. 11 A that upon switching from K+ to Rb+, the current in the open state increases in magnitude, while the two substate currents
are seen instead to decrease. A comparison of the conductances when K+, Rb+, or NH4+ is the permeant ion
shows in fact that the sublevels have a reversed conductance sequence. That is, while the SNS open state has
the conductance sequence Rb+ > NH4+ > K+, the sub1
and sub2 states have the sequence K+ > NH4+ > Rb+
(Table I). This phenomenon has been seen at each
voltage tested, ranging from 70 to
140 mV; Fig. 12
plots the current levels with the various permeant ions
at
120 mV. We conclude that the SNS channel has
different ion selectivities when it is in different conductance states and preferably selects against the larger
NH4+ and Rb+ ions at the lower conductance levels.
The Shaker potassium channel is highly selective for K+
over Na+ and other monovalent ions. Heginbotham et
al. (1994) found that the GYG motif in the P region of
the Shaker potassium channel is important for this ion
selectivity. When some residues in and near this motif
are mutated, the resulting channels lose their selectivity
for K+. These observations, along with experiments involving toxin blockade and chemical labeling of mutant channels (MacKinnon and Miller, 1989
; MacKinnon and Yellen, 1990
; Lü and Miller, 1995
), have confirmed that residues in the P region form part of the
ion permeation pore. T442 is two residues upstream
from the GYG motif, and although the substitution of
Ala, Gly, Asn, or Ser for T442 does not disrupt channel
selectivity (Heginbotham et al., 1994
), mutations at the neighboring position V443 can render the channel
nonselective. The preceding residue T441 has been associated with internal TEA binding (Yellen et al., 1991
)
and ion selectivity (Yool and Schwarz, 1995
, 1996
). In a
recent structural model of the Shaker potassium channel (Durell and Guy, 1996
), the T442 residue is located
at the inner end of the pore, with its backbone carbonyl oxygen forming one of the K+ ion binding sites.
Within the K+ channel superfamily, T442 is as highly
conserved as the GYG motif itself, with the only alternative at this position being the conserved substitution of
Ser in the eag channel (Warmke and Ganetzky, 1994).
We found that the conservative mutations to Ser or Gly
result in functional channels that have, however, altered single- channel conductance and very large changes
in channel kinetics. Large changes in the side-chain
structure at the 442 position, such as the charged side-chain of Asp, the imidazole ring of His, the phenyl
group of Tyr, or the sulfhydryl group of Cys result in no
detectable ionic or gating currents. Even a Val substitution is lethal, even though it is similar to Thr in volume
and side-chain structure. These results are consistent with the view that T442 forms an important part of the
pore structure.
Mutations at T442 Increase the Stability of the Open State
It has been reported that mutations at T442 decrease
the rate of deactivation and change the voltage dependence of activation (Yool and Schwarz, 1991; Yellen et
al., 1991
; Heginbotham et al., 1994
). These are among
the most dramatic changes in gating that have been observed as a result of mutations in the pore region. Our
results confirmed that both Ser and Gly mutations at
this position made deactivation extremely slow, both in
the background of an NH2-terminal truncated Shaker
H4 clone (yielding the SS mutant) and in the background of a Shaker -NGK2 chimera (SN, yielding the
mutants SNS and SNG). Single-channel recordings
from these mutants showed that the open state is
greatly stabilized, such that the open time of the mutant channels is two or three orders of magnitude
longer than that of the wild-type channel. This stabilization of the open state can also explain the negative shift
of the G-V relationship observed in the mutant channels. We used the SNS channel for the detailed characterization of single-channel properties because its single-channel behavior was simpler; the corresponding SS channel showed an additional, rapid bursting mode
of activity that is difficult to analyze.
It appears that the mutations of T442 leave the earlier steps in the activation process unaffected. We draw this conclusion from both the macroscopic activation time courses and the first latencies of the single-channel currents. The macroscopic currents of the T442 mutants and the background channel all show comparable activation time constants, suggesting that the rate-limiting step in the activation pathway is not affected by the mutations. The activation delay time, which reflects the overall rate of other gating steps, is also unchanged. At the single-channel level, we demonstrated that despite the very different open times, the SN and SNS channels have very similar first-latency distributions. These results are consistent with the idea that the T442 mutations affect the gating predominantly through the stabilization of the open state.
The fact that substitution of Ser or Gly at the 442 position stabilizes the open state suggests that the Thr residue at this position participates in a specific interaction that destabilizes the open state. It is not clear what
kind of interaction this may be, although it is tempting
to imagine that the hydroxyl group may form a specific
hydrogen bond when the channel is in closed states.
The increase in channel open time and open probability with the mutations corresponds to a moderate free
energy change G of about
1 kcal/mol for each
subunit.
Subconductance States in Shaker and Other Channel Types
Chapman et al. (1997) have recently characterized subconductance states in the wild-type Kv2.1 channel and
in two mutants. A total of four subconductance levels
were seen; they were observed most often before the
channel reached the fully open state during a depolarizing pulse. In the Kv2.1 channel, the sublevels were prominent only at small depolarizations, although in a
slowly activating mutant, named drk1-LS, the sublevels
were readily seen at large depolarizations as well. With
this mutant, dwells in sublevels were seen to precede
full channel openings in 70 to 80% of the single-channel activation time courses. The authors concluded
that the sublevels reflect intermediate states in the activation process.
The mutant Shaker channels studied here show very
similar behaviors. We observed only two main subconductance levels, but the relatively long dwell times at
these levels allowed us to characterize their kinetics in
the SNS channel. The subconductance levels are preferentially visited during channel activation and deactivation. Their dwell times are strongly voltage dependent, having the largest values near 100 mV. With a
probability approaching 100%, an activating channel
passes through the larger subconductance state sub2
before entering the open state and passes back through sub2 during deactivation as well.
There are some differences between the behavior of
the channels studied by Chapman et al. (1997) and the
SNS channels studied here. First, the SNS subconductance states are relatively noisy, showing larger current
fluctuations than the open state does. It is possible that
the fluctuations mask (or even arise from) transitions
among additional subconductance states, having closely spaced conductance levels. An example of a poorly resolved conductance level is S3
, a low-occupancy state
that can be resolved in Rb+ but that is not visible with
other permeant ions, presumably because it cannot be
distinguished from sub2. In contrast, the current levels
in Kv2.1 and its mutants are more stable and well defined. Second, there are differences in kinetics of transitions, such that in SNS the unidirectional state sequences closed
sublevel
open and open
sublevel
closed are seen quite often, while in the Kv2.1
channels the transitions among closed and sublevel states appear to be much more reversible. Third, we
find the rates of transitions among sub1, sub2, and the
open state to be quite voltage dependent. The dwell
times in sublevels of Kv2.1 appear to be similarly voltage dependent, but the drk1-LS mutant is remarkable in having nearly voltage-independent dwell times in
sublevels, when the traces presented for depolarizations to 0 and +40 mV are compared. This voltage independence might be related to the fact that the drk1-LS channel also has a nearly voltage-independent activation time course.
Subconductance states have been reported in many
other channel types (for review see Fox, 1987). Ferguson et al. (1993)
observed in rat muscle calcium-activated K+ channels a behavior not unlike that described
here, in which channels enter or leave the open state
from short-lived subconductance states. The dwell times
in the substates are very brief, on the order of 50 µs,
and somewhat less than half of the opening or closing
transitions appear to occur to substates rather than directly to fully open or closed states. Schneggenburger
and Ascher (1997)
have described novel subconductance behavior in a mutant NMDA receptor channel.
The substate has altered ion selectivity and, most interestingly, is seen to have transition rates that depend on
the electrochemical gradients of ions, leading to a violation of microscopic reversibility in gating. We did not
see irreversible gating in SNS channels, but it should be
kept in mind that the limitations in voltage range imposed by voltage-dependent gating make it more difficult in this case to manipulate the electrochemical potential of ions. Finally, it should be kept in mind that
wildtype Shaker channels also show a kind of subconductance behavior (Hoshi et al., 1994
; Schoppa, N.E.,
and F.J. Sigworth, manuscript submitted for publication; Fig. 3 B), but unlike that seen here in SNS channels, the transitions to subconductance states appear to
be very weakly coupled to activation.
Origin of Sublevels
Activation of the Shaker potassium channel involves
many kinetic transitions, each of which is voltage dependent (Bezanilla et al., 1994; Zagotta et al., 1994a
).
We find that the voltage dependences of the transitions
from sub1 to sub2 and from sub2 to the open state
each correspond to a charge movement of about 1.6 e0.
The fact that the charge movements in the two transitions are essentially equal suggests that these transitions arise from structurally equivalent conformational
changes, for example equivalent motions occurring in
separate subunits. The magnitude of these charge
movements is larger than that of any individual step in a model of Shaker channel activation from our laboratory (Schoppa, N.E., and F.J. Sigworth, manuscript submitted for publication), but it is quite similar to the
value of 1.42 e0 proposed for the final steps leading to
channel opening in a simpler activation model proposed by Zagotta et al. (1994b)
.
Chapman et al. (1997) have pointed out that subconductance levels can arise in two different ways. One
possibility is that they represent distinct, partially open
conformations of the channel pore. In Fig. 13 A, we
present a hypothesis of this sort, based on the model of
Zagotta et al. (1994b)
. In this model, each channel subunit can undergo two voltage-dependent transitions,
and the channel's open state results when each subunit
is in its final, permissive state. In Fig. 13 A, the two main
sublevels described here correspond to the configurations in which two or three subunits are in the permissive state, resulting in a partially conducting pore. In
this model, the effect of the T442S mutation would be to stabilize the permissive states; this would lengthen
the open times and make dwells in the sublevels long
enough to be observable. Since each subunit undergoes identical transitions, the model predicts the same
charge movement for the sub1
sub2 and sub2
open transitions. Another prediction of this model is
that the channel must traverse the state having three
permissive subunits (state 13 in Fig. 13, corresponding
to sub2) before it can reach the final open state 14. On
the other hand, there exist two alternative pathways by
which the channel can reach state 13; one is through the sub1 state 11, while the other is through the closed
state, 12. Thus the topology of this scheme can account
for our observations that sub2 is tightly coupled to the
open state, while sub1 is frequently skipped when the
channel activates and deactivates.
The other possibility is that subconductance levels
arise from rapid switching between fully open and fully
closed states. A mechanism like this has been documented for sublevels arising from blockers of the Ca-activated K+ channel (Moss and Moczydlowski, 1996).
A scheme Incorporating this idea is shown in Fig. 13 B,
where transitions from nonconducting to conducting
states of the channel involve a distinct allosteric transition with very fast rates. The sublevels seen in single-channel recordings would then result from the averaging effect of filtering on the rapidly flickering current,
so that the apparent conductance would depend on
the ratio of forward to reverse rates of the allosteric transition, which is different for the different states.
(The presence of rapid flickering in the fully open state
of the channel could explain the excess noise seen in
Shaker's open state as well.) The effect of the T442S mutation would be to modify the equilibria of the allosteric transition, making opening much more favorable
and thereby rendering the sub1 and sub2 conducting
states visible. Similarly to the other scheme, this model
predicts the identical charge movements to occur in
the sub1
sub2 and sub2
open transitions and predicts that the sub2 state will be visited on the way to and
from the final open state.
Of these two schemes, we favor the alternative shown
in Fig. 13 A. This scheme Is attractive because the distinct open states provide a simple explanation for the
different ion selectivities of the sublevels. Chapman et
al. (1997) also argued against the alternative class of
schemes involving rapid switching because of the lack
of excess noise in their sublevel currents. Nevertheless, there are disagreements in detail between the kinetics
we observe and what would be predicted by the scheme
In Fig. 13 A. The forward rates we measure for the steps
sub1
sub2 and sub2
open are much more voltage
dependent than the rate constant
in the model of
Zagotta et al. (1994b)
. Furthermore, from this scheme
one expects that dwells in sub1 and sub2 should be visible in wildtype Shaker channels. Based on the rate constants of Zagotta et al., the dwell time in state 13 at 0 mV is about 300 µs, which would result in brief but
readily visible sublevels. Such behavior has not been reported in Shaker channels, but we are presently investigating the possibility of its existence.
Subunit Effects on Permeation
Each subunit appears to contribute to the ion permeation pore of the Shaker channel, which is located in the
center of the tetrameric protein (Li et al., 1994) and is
formed in part by the four P loops (MacKinnon, 1995
).
This idea is supported by the fact that each subunit has
an additive effect on the affinity of the pore blocker
TEA (Heginbotham and MacKinnon, 1992
; Kavanaugh et al., 1992
). It has also been found that in related
channel types, including Na+ channels (Heinemann et
al., 1992
), Ca2+ channels (Ellinor et al., 1995
), and cyclic nucleotide-gated channels (Liu et al., 1996
), the
selectivity properties are influenced by individual subunits or protomeric domains, such that a mutation in a
single subunit or domain can dramatically alter the ion
selectivity properties.
Because of the particular voltage dependence of
dwell times in the sublevels, we were unable to characterize the selectivity of the subconductance states
through the measurement of reversal potentials. However, measurements of inward currents through SNS channels showed differing ion selectivities, consistent
with the idea that the pore structure changes as the
channel activates. We therefore speculate that transitions among the conductance states represent voltage-dependent conformational changes in each subunit,
which affect the pore structure. It is interesting to note
that the lower-conductance states of the channel tend
to exclude the large Rb+ ions. This effect is reminiscent
of state-dependent selectivity in the alamethicin channel,
where the lowest-conductance state does not pass Ca2+
or Tris ions (Hanke and Boheim, 1980). The subconductance state in a mutant NMDA receptor (Schneggenburger and Ascher, 1997
) is also more selective for the
smaller Na+ ions over Cs+ ions.
Original version received 10 February 1997 and accepted version received 20 May 1997.
Address correspondence to F.J. Sigworth, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. FAX: 203-785-4951; E-mail: fred.sigworth{at}yale.edu
1 Abbreviations used in this paper: NMDG, N -methyl-D-glucamine; TEA, tetraethylammonium.We thank Dr. R. MacKinnon for suggesting a closer look at the T442S mutant and for providing the SS construct. We thank Dr. L.Y. Jan (Yale University School of Medicine) for the SN construct, V. Pantani (Yale University School of Medicine) for help with the solution-exchange stepper, Y. Yan (Yale University School of Medicine) for assistance with mutagenesis, and Dr. J. Neyton (Ecole Normale Supérieure, Paris, France) for reading the manuscript.
This work was supported by National Institutes of Health grant NS-21501.