From the * Bekesy Laboratory of Neurobiology, Shaker channel mutants, in which the first (R362), second (R365), and fourth (R371) basic residues
in the S4 segment have been neutralized, are found to pass potassium currents with voltage-insensitive kinetics
when expressed in Xenopus oocytes. Single channel recordings clarify that these channels continue to open and
close from Voltage-gated ion channels are responsible for the electrical excitability of cells in heart, muscle, and throughout the nervous system. In the early 1950's, Hodgkin
and Huxley (1952) Structural studies in voltage-dependent Na+, K+, and
Ca2+ channels have revealed an apparent membrane
spanning region, the S4 segment, in which every third
amino acid carries positive charge (Noda et al., 1984 Nevertheless, the transitions closest to the open state
appear relatively, or entirely, voltage insensitive even in
voltage-gated potassium channels. This understanding
has arisen from studies of single channel kinetics (e.g.,
Zagotta et al., 1989 This paper explores the hypothesis that Shaker mutants in which S4 charges were progressively neutralized might lead to reduced or absent voltage sensitivity
in the first latency process. Would the resulting channels continue to open and close via the voltage-insensitive kinetics noted above? Or would these channels be
permanently closed in the absence of normal interaction from S4 charges? When expressed in Xenopus oocytes, two of the mutants studied here yielded macroscopic K+ currents that show no evidence of voltage-sensitive gating across the voltage range from Mutations and Channel Expression
The sequence of the S4 segment in the Shaker K+ channel is
shown in Table I. The seven conserved basic amino acids are indicated in Table I by both sequence numbers (above the sequence) and charge numbers (below the sequence). As noted in
Table I, the mutant in which the first and second conserved
arginines (R) in the S4 sequence were neutralized to glutamine
(Q) will be referred to here as 12Q. Similarly, 124Q means that
first, second, and fourth charges were neutralized to glutamines
(see Table I), etc. The control NH2 terminus deleted Shaker 29-4 construct will be referred to as Sh Table I Department of Genetics and Molecular Biology, School of Medicine, University of Hawaii, Honolulu, Hawaii 96822-2359
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
160 to +80 mV with a constant opening probability (Po). Although Po is low (~0.15) in these mutants, mean open time is voltage independent and similar to that of control Shaker channels. Additionally, these
mutant channels retain characteristic Shaker channel selectivity, sensitivity to block by 4-aminopyridine, and are
partially blocked by external Ca2+ ions at very negative potentials. Furthermore, mean open time is approximately
doubled, in both mutant channels and control Shaker channels, when Rb+ is substituted for K+ as the permeant
ion species. Such strong similarities between mutant channels and control Shaker channels suggests that the pore
region has not been substantially altered by the S4 charge neutralizations. We conclude that single channel kinetics in these mutants may indicate how Shaker channels would behave in the absence of voltage sensor input. Thus,
mean open times appear primarily determined by voltage-insensitive transitions close to the open state rather
than by voltage sensor movement, even in control, voltage-sensitive Shaker channels. By contrast, the low and voltage-insensitive Po seen in these mutant channels suggests that important determinants of normal channel opening
derive from electrostatic coupling between S4 charges and the pore domain.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
first demonstrated the ionic mechanism of the transmembrane action potential in the squid giant axon and proposed that such voltage dependence of the K+ and Na+ conductance is due to
charged particles within the membrane that move in
response to changes in the transmembrane electric
field (Hodgkin and Huxley, 1952
; Hille, 1992
). More
recently, the activation process has been modeled as
voltage-dependent conformation changes in each of
four subunits, followed by cooperative opening transitions with lower voltage sensitivity (Hoshi et al., 1994
; Zagotta
et al., 1994a
,b; Schoppa and Sigworth, 1998a
,b,c). The
voltage dependence of the activation process results from
charge movements within the channel protein, associated with early conformational transitions. These intramolecular charge movements can be directly measured as gating currents (Armstrong, 1981
; Hoshi, 1994).
;
Tanabe et al., 1987
; Tempel et al., 1987
), suggesting
that the S4 serves as the primary voltage sensor in these
channel types (Greenblatt et al., 1985
; Caterall, 1986; Guy and Seetharamulu, 1986
; Noda et al., 1986
). This
hypothesis has been evaluated in potassium channels
using a variety of charge neutralizing mutations in segments S2, S3, and S4 (Liman et al., 1991
; Lopez et al.,
1991
; Papazian et al., 1991
, 1995
; Logothetis et al.,
1992
, 1993
; Tytgat and Hess, 1992
; Aggarwal and
MacKinnon, 1996
; Seoh et al., 1996
). Similarly, gating
charge measurements from S4 mutations have been
widely used to confirm the role of the S4 segment as a
voltage sensor in voltage-dependent activation (Bezanilla and Stefani, 1994
; Perozo et al., 1994
; Seoh et al., 1996
). In addition, cooperative transitions leading to
potassium channel activation have been identified in
Shaker and heteromultimeric mammalian potassium
channels (Hurst et al., 1992
; Tytgat and Hess, 1992
; Bezanilla et al., 1994; Sigworth, 1994
; Zagotta et al., 1994b
;
Smith-Maxwell et al., 1998a
). Furthermore, direct evidence for S4 movement during channel activation has
come from fluorescence and sulfhydryl-reagent binding studies in which specific residues were replaced
with cysteine to probe the environment near the S4 segment in Shaker channels. Such studies indicate that
change in membrane potential can alter the internal
and external accessibility of charged residues in the S4
segment, clarifying that changes in S4 exposure are involved in the initiation of channel opening (Yang and
Horn, 1995
; Larsson et al., 1996
; Mannuzzu et al., 1996
; Yang et al., 1996
; Yusaf et al., 1996
).
; Hoshi et al., 1994
), as well as from
macroscopic potassium currents (Zagotta et al., 1994a
,b)
and gating currents (Bezanilla and Stefani, 1994
). Thus,
mean open times as well as the three closed times noted
from single channel recordings are all essentially voltage insensitive across the physiological range of membrane potentials (Zagotta et al., 1989
; Hoshi et al.,
1994
; Schoppa and Sigworth, 1998a
,b,c). The only parameters that have been unequivocally identified as
voltage sensitive at the single channel level are the first latency distributions and Po(V) curves.
160 to
+80 mV. However, single channel recordings show that
these mutant channels still open and close, albeit in a
voltage-insensitive manner, while retaining normal potassium selectivity and normal mean open times. Using
these mutations, it becomes possible to study channel
properties in the apparent absence of voltage-sensitive
input from the S4 segment.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
. Since the amino acid sequence of Sh
is identical to the Shaker B sequence from S1
through S6, standard Shaker B residue numbering is used
throughout. Glutamine-substituted charge-neutralizing mutations within the S4 segment (12Q, 124Q, 1247Q, 127Q, and
147Q) were introduced in an NH2 terminus deleted Shaker 29-4 construct (Iverson and Rudy, 1990
; McCormack et al., 1994
) by
Dr. K. McCormack and were generously made available for this
study by Dr. F.J. Sigworth (Yale University, New Haven, CT). An additional mutant, 24Q, was constructed in this laboratory by the synthesized oligomer method, also in NH2-terminus-deleted Shaker
29-4, and assembled in pGEM-9zf(
) vector. Mutation was verified
by restriction enzyme digestion and subsequent sequencing.
Sequence of the S4 Segment in Shaker Potassium Channels
Plasmids were linearized with NotI and mRNA was transcribed in vitro using mMESSAGE and mMACHINE kits (Ambion, Inc.). mRNA was microinjected into Xenopus oocytes in developmental stage V or VI at a concentration ranging between 0.005 and 0.1 µg/µl (total volume per oocyte, 50 nl). The injected oocytes were incubated in ND96-PGH broth containing (mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.2, plus 2.5 pyruvate, 50 mg/ml gentamicin, and 1% horse serum, at 18°C for 1-7 d before electrophysiological recordings.
Electrophysiology
Channel expression levels in Xenopus oocytes were tested by two-electrode voltage clamp (TEV),1 using a CA-1 High Performance
Oocyte Clamp (Dagan Corp.). TEV electrodes were filled with
2 mM KCl and have 0.1-0.3 M resistance. Patch-clamp recordings were obtained using EPC-9 patch clamp amplifier (HEKA
Elektronik). Patch pipettes were fabricated from aluminum silicate (for macropatch pipettes) and coated with dental wax to reduce electrode capacitance, or from borosilicate glass (for single
channel recordings) and sylgard coated. Macropatch pipettes yield resistances between 0.5 and 2 M
in standard solutions. Pipettes for single-channel measurement had resistances between 6 and 20 M
in symmetric K+ solutions (115 mM K+). All single-channel currents were recorded from isolated patches in the inside-out configuration. Traces were recorded in steps from holding potential to different test potentials (from
160 to +80 mV)
using different pulse durations and sample intervals to address a
potentially wide range of channel kinetics. 1-s traces were recorded with a 200-µs sample interval using a 1.25-kHz Bessel filter. 100- and 50-ms traces were recorded at 10-µs sample intervals with a 4-kHz Bessel filter. The digit filter was set ~1-2 kHz. Data
acquisition was controlled using the Pulse+PulseFit software package (HEKA Elektronik). Experiments were carried out at
room temperature (between 20° and 22°C).
All data reported here were obtained without P/n subtractions, except where specifically indicated. Such P/n subtractions are commonly used to distinguish ionic current from both capacitance and leak currents by subtraction of "leak pulses" obtained in voltage steps, and from a leak holding potential, at which voltage-gated channels remain closed. This method is not appropriate for channel types that show maintained currents across the full range of experimentally accessible membrane potentials.
Data Analysis
Data analysis was performed using software from PulseFit, PulseTools (HEKA Elektronik), IgorPro (Wave Metrics), Mathematica (Wolfram Research, Inc.), and MacTAC (Skalar Instruments). Single channel openings were identified using the half-amplitude
threshold-crossing method (Colquhoun and Sigworth, 1983).
Analysis of single-channel fluctuations was carried out following
the methods described by Llano et al. (1988)
and Perozo et al.
(1991)
.
Single channel open probability was determined from the formula A/(A + B), where A is the total time spent in the open state and B is the total time spent in the closed state. This formula is
usable only when a patch shows no evidence of a second channel being present in any of the data traces at all test potentials. However, it is easier to obtain traces with two or three channels present in the patch. We have used a method for estimating
channel Po in patch records where the total number of channels,
n, can be shown to be no more than three. Statistically, the probability of being either open or closed is equal to 1, thus Pclosed = 1 Po.
In multiple channel patch data, it is easy to sum the time spent in closed states, and hence to calculate the probability of no channel being open. Presuming each channel gates independently, the mean probability of any one channel being open and closed is Po and Pclosed, respectively. In multiple channel patches, the probability of all channels being closed is B, and the probability of at least one channel being open is Bo, then we have:
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This final equation was used to calculate the mean probability of a single channel being open (Po) at different test potentials in symmetric K+ solutions. This equation was checked by analyzing multiple channel data and comparing results with analysis of single channel data from the same channel-type. Since similar results were obtained, some multiple channel traces from the 124Q and 1247Q mutants were analyzed by this method, provided that no more than three channels were detected in any one patch.
Averaged results are reported as mean ± SD (n), where n is the number of patches.
Solutions
For all patch clamp experiments, in addition to monovalent chloride salt, external solutions, referred to as Ringer solutions, always contained 1.8 mM CaCl2 and 10 mM HEPES, pH 7.2. Internal solutions, named EGTA solutions, contained 1.8 mM EGTA and 10 mM HEPES, pH 7.2. The solutions were named according to the content of monovalent cations (mM): normal frog Ringer, 115 NaCl, 2.5 KCl; K-Ringer, 115 KCl; Tris-Ringer, 115 TrisCl; TMA-Ringer, 115 TMA; K-EGTA, 115 KCl; Tris-EGTA, 115 TrisCl. To test channel ion selectivity, K+ was replaced completely by Rb+, NH4+, Cs+, Na+, respectively, in the internal and external solutions.
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RESULTS |
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The basic amino acids in the S4 segments of Shaker
channels are not functionally equivalent and may make
different contributions to the mechanism of channel
activation (Liman et al., 1991; Lopez et al., 1991
; Papazian et al., 1991
; Tytgat and Hess, 1992
; Logothetis et al.,
1993
). Thus, single neutralizations of the even numbered charges (R2 or R4) shift the opening probability
[Po(V)] curve to the left along the voltage axis, whereas
single neutralizations of R1 or R3 (but not K7) shift the
Po(V) curve to the right (Papazian et al., 1991
; Logothetis et al., 1992
). On the other hand, K5 and R6 play an
important role in the proper folding and maturation of
the channel protein (Perozo et al., 1994
; Papazian et al., 1995
). Finally, although Aggarwal and MacKinnon
(1996)
have shown that neutralization of K7 does not
alter charge per channel, an additional 7Q neutralization might affect S4 stability in combination with other
charge neutralizations.
Miller and Aldrich (1996) showed that double mutants involving neutralizations of the second and fourth
charges induce larger left shifts than would be predicted from additive effects of the two single mutations.
Furthermore, their macroscopic current traces from
the NH2 terminus-deleted versions of these mutants
(see Fig 2 B, Miller and Aldrich, 1996
) seemed to show
little evidence of activation and deactivation kinetics.
These findings were strongly supportive of our own
preliminary findings (Bao et al., 1996
), and suggested
that a more extensive study of S4 charge neutralizations, using single channel methods, might help to elucidate the role of the first four charges in Shaker channel gating.
|
Effects of S4 Mutants 12Q, 127Q, 147Q, and 24Q on Shaker K+ Channel Voltage Sensitivity
The multiple charge-neutralizing mutations 12Q, 127Q,
and 147Q were tested using TEV and patch clamp measurements (Fig. 1 A). These mutants all show voltage-sensitive gating with similar thresholds for channel
opening of about 60 mV. Fig. 1 A shows macropatch
currents at two different voltages (
30 and +80 mV) from mutants 12Q, 127Q, and 147Q, as well as from the
control Sh
channel. These traces were recorded in
symmetric K+ solutions (115 mM) from a holding potential of
80 mV to different test potentials for 100 ms, and then back to a hyperpolarization potential
(
100 mV) for another 50 ms. On-line leak and zero
subtraction were carried out using a P/4 protocol with
the leak holding potential at
120 mV. The peak tail
currents (Itail) obtained at different testing potentials
were normalized (as Itail/Itail, max) and plotted against
test potential to provide macroscopic Po(V) curves for each mutant. None of these mutations shows any
marked shift in the voltage dependence of channel
opening in comparison with Sh
(Fig. 1 C). The midpoints of the Po(V) curves for 12Q, 127Q, and 147Q are
35,
45, and
30 mV, respectively, as compared with approximately
45 mV in Sh
.
|
The mutant 24Q was also found to show voltage-sensitive gating as demonstrated by the fast tail currents indicating channel closing on return to the holding potential (Fig. 1 B). Threshold opening occurs about
150 mV, and the midpoint of the macroscopic Po(V)
curve is close to
100 mV (Fig. 1 C). Thus 24Q
(R365Q:R371Q) shows strongly left-shifted voltage dependence, although the shift seen here is considerably
smaller than the
180-mV midpoint found by Miller
and Aldrich (1996)
for their 24 neutralization (R365N: R371I). The traces shown in Fig. 1 B were obtained in
steps from a holding potential of
160 mV to the indicated test potentials, with the leak holding potential adjusted to
160 mV, and with negative-going P/4 pulses.
No significant current was seen in the step to
140 mV.
Activation and deactivation kinetics are readily visible
in Fig. 1 B, although apparently at least 80 mV left-shifted by comparison with control Sh
channels.
Voltage-insensitive Gating in 124Q and 1247Q S4 Mutants
Recordings from 124Q- or 1247Q-injected oocytes, using the two-electrode voltage clamp system with normal
frog Ringer as the bathing solution (not shown) did not
demonstrate either inward tail currents indicative of
time-dependent gating, or voltage-sensitive activation kinetics at any test potential from 100 to +100 mV. Furthermore, large holding currents (by comparison with
those seen in uninjected oocytes) were evident in symmetric K+ solutions at
80 mV. Thus, TEV recordings
showed no clear evidence of normal voltage-sensitive
gating, despite the presence of "leak" currents significantly larger than those seen in uninjected oocytes. However, it seemed possible that rapid voltage-sensitive
gating might have been overlooked due to the relatively
slow clamp speed (~0.5 ms) of the TEV system.
Inside-out patch clamp recordings from 124Q and
1247Q channels in symmetric K+ solutions (115 mM
K+) were used to assess this possibility (Fig. 2 A). In this
figure, which shows records obtained from the 124Q
mutant from a holding potential of 0 mV without P/n
subtraction, no activation or deactivation kinetics were
observed for steps to potentials from +80 to approximately 80 mV. However, the current-voltage (I-V)
curve shows progressive reduction of inward current in
steps to potentials more negative than
80 mV (Fig. 2
B), as if channels deactivate rapidly at negative potentials in the range
80 to
160 mV.
A possible explanation for this finding would be that
significant Ca2+ block was occurring in these channels
at very negative potentials. This possibility was explored
(see Fig. 2 B) by comparing I-V curves obtained with
and without Ca2+ in the external solution. When the
normal 1.8 mM external Ca2+ was substituted by 1.8 mM
EGTA, the I-V curve is effectively linearized. A Woodhull model for channel block (Woodhull, 1973) was
used to fit these data, suggesting a Ca2+ block position
~40% of the distance from the extracellular end of
the channel, with an apparent kD of ~50 mM Ca2+ (at
0 mV).
Thus, the apparent deactivation kinetics of macroscopic currents in the 124Q mutant, as well as equivalent findings for 1247Q (not shown), can be fully explained by calcium block occurring at very negative potentials. This finding suggests that these channels may
be essentially voltage-insensitive from 160 to +100 mV.
Nevertheless, we chose to complete this study using
Ca2+-containing external solutions rather than Ca2+-free external solutions. We note (Fig. 2 B) that Ca2+
block is minimal at potentials greater than
80 mV.
Furthermore, as will be seen from single channel recordings (Fig. 3), the Ca2+ block at negative potentials
is a fast "flickering block" that has little effect on the apparent kinetics of idealized records obtained using the
half-crossing method. However, we report here detailed kinetic analysis of closed time distributions only
from potentials greater than
80 mV (see Fig. 5),
where fast closings are unlikely to have been contributed by Ca2+ block.
|
|
Single Channel Analysis of the 124Q and 1247Q Mutants
Since macroscopic currents had shown that Po appears
constant across voltage, single channel recordings were
needed to assess whether these mutant channels were
locked in a permanently open state, or whether they
might be opening and closing but with voltage-insensitive kinetics. In the latter case, single channel measurements could be used to evaluate the rate-limiting steps
for channel gating as well as the potential voltage dependence of transitions between kinetic states. Important conclusions can be drawn from analysis of single-channel behavior even in the absence of a complete kinetic model (Zagotta et al., 1989).
Single 124Q and 1247Q channels open and close in a voltage-insensitive manner.
Single channel currents were recorded from excised patches in an inside-out configuration in symmetric potassium solutions. Preliminary
studies of these patches indicated that Po was not affected by changes in either holding or test potentials
(from 160 to +80 mV). The apparent absence of any
effect of holding potential on Po suggests that these
channels do not enter or recover from C-type inactivation at any of the test potentials assessed here. Thus, it
should be possible to obtain long continuous recordings by holding at the required "test" potential. Nevertheless, subtle effects might occur from changes in
C-type inactivation, and we chose to use repetitive pulse
recordings with 5-s intervals between successive traces
to maximize the independence between data traces. However, tests with pulses of different durations (50, 100, 200, and 500 ms) all indicated substantial numbers
of long openings that outlasted the pulse duration.
Hence, 1-s pulses were used to characterize these longest openings, while shorter pulses were used to address
brief opening and closing events (see below). Patches
were routinely held at reversal potential (0 mV in symmetric K+ solutions) to minimize holding currents and
prolong patch lifetime.
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Single channel mean open time is voltage independent in
124Q and 1247Q, but is affected by permeant ion species.
Open time distribution histograms were obtained from
40 idealized 1-s traces (recorded at 1.2 kHz) and concatenated to yield an equivalent 40-s recording. Amplitude and open-time histograms were fitted using a simplex algorithm. Amplitude histograms were fitted to a
sum of Gaussian functions using Igor software. Single channel currents were derived from the Gaussian fits to
amplitude histograms. Steady state dwell times for
1247Q single channels in symmetric K+ solutions, including amplitude histograms and distribution of open
times, are demonstrated in Fig. 5 A. Our data was best
fitted as the sum of two exponential distributions, yielding mean open times across the full voltage range from
160 to +80 mV of 2.0 ± 0.4 and 16.3 ± 3.8 ms (n = 6 patches, with data from each patch obtained at eight
different test potentials) for 124Q, with these open
states having relative probabilities of ~85 and ~15%,
respectively. Mean open times for 1247Q were 2.5 ± 0.4 and 18.3 ± 2.6 ms (n = 6 patches, each yielding
data at six test potentials). These findings suggest the
presence of two separable open states. However, amplitude histograms give no indication that the longer
open state differs in conductance from the shorter, primary, open state (see Fig. 5 A). Furthermore, 124Q and
1247Q data are not statistically distinguishable.
Single channel closed times are voltage independent in
124Q and 1247Q.
Closed time distributions were also
obtained from the same traces as were used for open
time distributions (see Fig. 5, A and B). Best results
were obtained from three exponential fits to data from
1-s traces in the 124Q and 1247Q mutant channels (Fig. 5 A). This process was repeated for analysis of 100- and 50-ms pulses recorded using a 4 kHz Bessel filter
and sampled at shorter intervals. Mean closed time constants were (ms) 0.11 ± 0.02 (n = 7), 0.32 ± 0.05 (n = 23), 3.1 ± 0.4 (n = 25), and 21.1 ± 4.6 (n = 17 patches) for 124Q. Results for 1247Q channels were
again not statistically distinguishable from those reported here for 124Q, and similar results were also obtained from our control Sh channels, except that we
were unable to resolve the longer ~20-ms closed time
in Sh
. We conclude that our findings differ from results obtained in wild-type Shaker channels (see also
Schoppa and Sigworth, 1998a
) by addition of the
slower ~20-ms rate. Although this 20-ms time constant
might not have been identified in the 20-ms test pulses
used by Schoppa and Sigworth (1998a)
, it should have
been readily detectable in our Sh
data, given the 100-ms pulses used here and the slow onset of C-type inactivation in high external K+ solutions (López-Barneo et
al., 1993
). We conclude that this ~20-ms time constant
is not present in control Shaker channels.
Ionic Selectivity in Voltage-insensitive 124Q and 1247Q Mutants
The similar mean open times for Sh channels and for
the 124Q and 1247Q mutants might suggest that channel structure has not been significantly affected by the
apparent loss of voltage sensor input. Additionally, it is
already clear from the data reported above that these
mutant channels, like Sh
channels, conduct both K+
and Rb+ ions, giving single channel currents in the pA
range. We here report a more complete study of the selectivity of these mutant channels, demonstrating their
substantial equivalence to Sh
controls, both from
measurements of reversal potentials (see below) and from the relative conductances observed under simplified ionic conditions (Fig. 6 A). Mutant 124Q and
1247Q have similar properties; therefore, only the
ionic selectivity of 124Q is shown here.
|
Ionic selectivity can be determined using the permeability ratio calculated from the reversal potential seen
under biionic conditions with the test cation on one
side of the membrane and the reference cation (K+)
on the other side. For this study, reversal potentials
were evaluated by measuring magnitudes of single
channel currents across a voltage range from 120 to
80 mV in inside-out configuration, and plotting the observed currents as functions of test potential. The internal solutions contained 115 mM K+ as the reference
ion with the external cation being presented in the
same concentration. Erev was 0 mV for external K+ solutions,
5 mV for external Rb+,
30 mV for external
NH4+, and less than
100 mV for external Na+. These
findings indicate a highly K+-selective channel, in
which PX/PK = 0.82 in Rb+; 0.31 in NH4+ and <0.01 in
Na+. Clearly, selectivity as defined by the PX/PK ratio is
very similar to that found for control Shaker channels,
although the ratio for NH4+ seen here is two- to threefold greater than the 0.13 found by Heginbotham et al.
(1994)
. Apparently, multiple charge neutralizations can
remove channel voltage sensitivity without necessarily altering the ionic selectivity of the channels, as if the structure of the selectivity filter has been no more than minimally affected by the S4 charge neutralizations.
However, as pointed out by Hille (1992), permeability sequences obtained from reversal potentials are not
necessarily identical to those seen from relative single
channel conductances. Therefore, single channel data
are included where the test ion was presented in the internal solution with the impermeant ion Tris in the external solution, or vice versa (Fig. 6 A). Amplitudes of
single channel currents at constant voltages were compared for the following test cations: K+, Na+, Rb+,
NH4+, and Cs+. With Tris Ringer as the external solution
and the test ion presented in the internal solution, all
single channel currents were outward at +40 mV. Alternatively, when Tris-EGTA was used as the internal reference cation, only inward single channel currents were
seen at
40 mV. At both potentials, our results correspond to the permeability sequence seen in the reversal
potential data: K+ > Rb+ > NH4+ > Cs+ and Na+.
Sensitivity to channel block by 4-aminopyridine (4-AP; McCormack et al., 1994) was used as an additional
test indicating the structural integrity of the ion permeation pathway in the 124Q and 1247Q channels. In
whole-cell two-electrode voltage clamp, ionic currents
of 124Q decreased dramatically after changing the bath solution from normal K-Ringer to K-Ringer containing 10 mM 4-AP on the same oocyte (Fig. 6 B).
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DISCUSSION |
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This study has shown that two multiple charge-neutralizing mutations that involve the first, second, and
fourth S4 charges, produce channels that conduct K+
currents without evident voltage sensitivity. Nevertheless, at the single channel level, these mutant channels
continue to open and close with a low (0.15) but constant Po across a wide range of test potentials. Their
mean open times are not distinguishable from those of
control Sh channels. Closed time distributions in
these channels are also indistinguishable from those of
Sh
channels, except that there is an additional long
closed time (~20 ms) that cannot be identified in the
Sh
controls. Furthermore, these 124Q and 1247Q mutants retain the high K+ selectivity of the parent channel, as if the structure of the selectivity filter has been
little affected by these S4 charge neutralizations.
Effects of S4 Charge Neutralization
As initially shown by Papazian et al. (1991), Shaker
channels appear tolerant of single neutralizations of all
charges other than K5 and R6, which were subsequently found to form electrostatic interactions with
negatively charged residues in other segments and appear vital for normal folding of the channel (Papazian
et al., 1995
). Thus, the multiple charge neutralizing
mutations used here avoid charges 5 and 6 and were selected to provide detailed controls for the voltage-
insensitive mutants rather than to provide additional
information as to the functional roles of each charge in
normal Shaker channels. All multiple neutralizations of
charges 1, 2, 4, and 7 studied here, other than 124Q
and 1247Q, have been shown to give voltage-gated
channels. Neither single nor double neutralizations of
the first, second, and fourth charges can recreate the
effects found when all three of these charges are simultaneously neutralized.
Nevertheless, the mechanisms controlling right and
left shifting of the Po(V) curve have not yet been fully
elucidated. Thus, two different mutations, both involving neutralization of the second and fourth charges,
show substantial quantitative differences in Po(V) midpoint. The R365N:R371I mutant studied by Miller and Aldrich (1996) gave a midpoint of
180 mV, compared
with the
100-mV midpoint found here for 24Q (R365Q:
R371Q). Po(V) midpoint, which presumably reflects the
stability of the S4 in relation to surrounding pore structures, is known to be affected by the size and hydrophobicity of substituted S4 residues (see Sigworth, 1994
).
Such properties may be more important than the presence or absence of S4 charges.
Are the 124Q and 1247Q Channels C-Type Inactivated?
We have pointed out that these mutants do not enter
C-type inactivation during applied test pulses, and this
is true both for channels held at 0 and 160 mV (data
not shown). However, since the mutant channels open
with similar probabilities at all reasonable potentials, it
seems clear that they should have reached an equilibrium with C-type inactivation at some time long before their experimental evaluation. Thus, a question arises
as to the nature of this equilibrium. Three major possibilities exist.
(a) Mutant channels may be in equilibrium with a nonabsorbing C-type-inactivated state with a life time of ~20 ms. Since this C-type (or P-type) state appears "closed" in our channels, the normal Shaker selectivity seen in our data would reflect the properties of noninactivated channels while the inactivated channels would be K+ impermeant. In this case, the low Po,max presumably arises from sequestration of channels in the inactivated state.
(b) Mutant channels may be incapable of C-type inactivation. Loots and Isacoff (1998) have shown that fluorescent probes inserted at residue 359 report involvement of the region around charge 1 in the P-type conformational change that precedes C-type inactivation. Neutralization of the first, second, and fourth charges
could well be sufficient to block this P-type conformational change. Thus, this hypothesis would remove
C-type inactivation as a cause of the low Po,max found in
our 124Q and 1247Q mutants, suggesting that the reduced Po may be a direct result of the S4 charge neutralizations. Within a model such as the 3+2' model
proposed by Schoppa and Sigworth (1998c)
, the S4
CN-1
ON transitions (Fig. 7) are presumed to be independently voltage sensitive, accounting for voltage-sensitive changes in the frequency of channel opening events at different test potentials. However, it seems
likely that the electric field to which the S4
CN-1 and
CN-1
ON voltage sensors respond would be substantially modified by neutralization of three nearby S4
charges, possibly reducing Po,max in these neutralization
mutants.
|
(c) Mutant channels may be permanently C-type inactivated, opening and closing without leaving C-type
states. Shaker channels have been found to remain capable of ion conduction while in C-type states (Starkus et al.,
1997), although with markedly changed selectivity.
This hypothesis would require the selectivity of the inactivated state to have been drastically modified by the charge neutralizations, since wild-type Shaker channels
become effectively impermeant to K+ ions when C-type
inactivated. Thus, the apparently normal Shaker K+ selectivity seen in these mutants would be highly abnormal for C-type inactivated channels. In this case, the
low observed Po,max could be caused by changes in
opening probability resulting from conformational
changes associated with C-type inactivation.
Unfortunately, the question posed in the heading of this section cannot yet be answered. We can neither force channels to close nor detect kinetic changes that would unequivocally report onset or recovery from C-type inactivation. Thus, alternative approaches will be required to further evaluate these competing hypotheses.
Single Channel Properties of the Voltage-insensitive Mutants 124Q and 1247Q
Despite the uncertainties noted above, the results reported here have considerable implications. The voltage-insensitive behavior seen in 124Q and 1247Q arises
when charge 1 is neutralized in addition to charges 2 and 4. We presume that charge 1 must contribute in
some substantial way to the normal coupling between S4
movement and voltage-dependent channel gating. Such
coupling appears to be a cooperative process (Tytgat
and Hess, 1992), which occurs when all four S4 segments
have been repositioned by the voltage field (Smith-Maxwell et al., 1998a
,b) in control Shaker channels.
Recently, a detailed and comprehensive study of
Shaker channel kinetics has been carried out based on
macroscopic currents, gating currents, and single channel data in both Sh channels and a well-studied S4
mutant (Schoppa and Sigworth, 1998a
,b,c). The resulting data was shown to be best fitted by a specific model (see 3+2' model in Schoppa and Sigworth, 1998c
) that
delineates the parameters of S4 movement and the kinetic form of the coupling between S4 movement and
channel gating, while including the previously identified
processes that reflect channel closings to states outside the primary activation pathway (Hoshi et al., 1994
). Thus,
it seems particularly important to determine how our
results fit within this conceptual framework.
The 3+2' model is shown in Fig. 7, and its relevance to
our data should be readily apparent. Our mutants show
the same mean open times and three closed times as
noted by Schoppa and Sigworth (1998a,c) in control Sh
channels. Additionally, we see a fourth closed time (~20
ms) that may represent equilibration with a nonabsorbing inactivated state, as noted above. Nevertheless, the
3+2' model identifies two independently voltage-sensitive steps between the S4 and ON states, which contribute
a total of 1.7 eo to the voltage sensitivity of the Shaker
channel. These voltage-sensitive S4
CN-1
ON transitions, together with the effects of S4 movement, determine the voltage sensitivities of first latency distributions and single channel Po(V) curves in control Sh
channels.
By contrast, in the 124Q and 1247Q mutants, all traces of
independent voltage-sensitivity are lost. Even if all S4 input was removed by these S4 charge neutralizations, the
1.7 e0 reported for the non-S4 voltage sensors of the S4 to
ON transitions should have been readily measurable in
our Po(V) data. Thus, the low and voltage-insensitive Po in 124Q and 1247Q mutant channels implies additional
changes in these S4 to ON transitions caused by direct or
indirect effects of the S4 charge neutralizations.
Implications for Ion Channel Gating Mechanisms
The results presented here provide further support for a separation of the channel gating mechanism into three distinct components. Components from the single channel perspective are listed below.
The voltage-insensitive transitions close to the open state.
These transitions seem quantitatively unaffected by the
neutralization of S4 charges in the 124Q and 1247Q
mutants. However, they are affected to similar extents
by transfer from K+ to Rb+ solutions in both control
and mutant channels, suggesting that they are substantially determined by interactions between the pore and
its permeating ions (see also Swenson and Armstrong, 1981; Matteson and Swenson, 1986
; Zagotta et al.,
1994a
). In view of the tight relationship between these
voltage-insensitive kinetics and permeant ions, we presume that these processes involve the "pore gate" proposed by Zheng and Sigworth (1998)
to account for effects of the T442 residue on intermediate conductances.
The voltage-sensitive transitions that determine the first-
latency and Po(V) distributions.
The voltage-sensitive input,
represented by the S4 ON transitions in the 3+2'
model, appears to be lost after neutralization of the
first, second, and fourth S4 charges. These transitions have been associated with a "main" activation gate
formed by rearrangements of the S6 helices (Liu et al.,
1997
) that constrict the inner mouth of the channel in
the KcsA structure (Doyle et al., 1998
). However,
Zheng and Sigworth (1998)
point out that both the
pore gate and the main gate must be coupled in normal functioning of the Shaker channel. Clearly, such
coupling has been broken in the 124Q and 1247Q mutants studied here, as if the S4
ON transitions (absent
in 124Q and 1247Q) provide a parallel drive to both
the pore gate and the main gate during normal Shaker
channel activation.
The inherently voltage-insensitive transitions involved in
the P-type and C-type slow inactivation mechanisms.
Recent
work by Loots and Isacoff (1998) has clarified that slow
inactivation involves two distinct conformational changes (P- and C-type), both of which occur around the narrow tunnel region of the permeation path in the Doyle
et al. (1998)
structure. Like the transitions close to the
open state, the kinetics of these transitions appear to be
substantially determined by interactions with permeating ions (López-Barneo et al., 1993
; Levy and Deutsch, 1996
; Starkus et al., 1997
). Unfortunately, as noted
above, it is not yet clear to what extent these transitions
may be affected by S4 charge neutralizations, despite
the demonstrated involvement of the outer section of
the S4 segment in the C-type conformation change
(Loots and Isacoff, 1998
).
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FOOTNOTES |
---|
Address correspondence to Martin D. Rayner, Bekesy Laboratory of Neurobiology, Pacific Biomedical Research Center, 1993 East-West Rd., University of Hawaii, Honolulu, Hawaii 96822-2359. Fax: 808-956-6984; E-mail: martin{at}pbrc.hawaii.edu
Original version received 22 July 1998 and accepted version received 3 November 1998.
Portions of this work were previously published in abstract form (Bao, H., A. Hakeem, K. McCormack, M.D. Rayner and J.G. Starkus. 1996. Biophys. J. 70:A189).We thank K. McCormack for providing us with Sh, and F.J. Sigworth for making available the 12Q, 124Q, 127Q, 147Q, and
1247Q mutations.
This study was supported in part by National Institutes of Health Grant RO1-NS21151, and by Pacific Biomedical Research Center Bridging Funds (to J.G. Starkus). J.G. Starkus and M.D. Rayner were further supported by Grants-in-Aid, while A. Hakeem also received a Predoctoral Fellowship, all from the American Heart Association (Hawaii Affiliate). M.D. Rayner received additional support from the Queen Emma Foundation.
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Abbreviations used in this paper |
---|
4-AP, 4-aminopyridine; I-V, current- voltage; TEV, two-electrode voltage clamp.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Aggarwal, S.K., and R. MacKinnon. 1996. Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron 16: 1169-1177 [Medline]. |
2. |
Armstrong, C.M..
1981.
Sodium channels and gating currents.
Physiol. Rev.
61:
644-683
|
3. | Bao, H., A. Hakeem, K. McCormack, M. Rayner, and J. Starkus. 1996. Voltage-insensitive gating in Shaker B S4 mutants. Biophys. J. 70: A189 . |
4. | Bezanilla, F., and E. Stefani. 1994. Voltage-dependent gating of ionic channels. Annu. Rev. Biophys. Biomol. Struct 23: 819-846 [Medline]. |
5. | Catterall, W.A.. 1986. Molecular properties of voltage-sensitive sodium channel. Annu. Rev. Biochem 55: 953-985 [Medline]. |
6. | Colquhoun, D., and F.J. Sigworth. 1983. Fitting and statistical analysis of single-channel records. In Single Channel Recording. B. Sakmann and E. Neher, editors. Plenum Publishing Corp., New York. 505 pp. |
7. |
Doyle, D.A.,
J.M. Cabral,
R.A. Pfuetzner,
A. Kuo,
J.M. Gulbis,
S.L. Cohen,
B.T. Chait, and
R. MacKinnon.
1998.
The structure of
the potassium channel: molecular basis of K+ conduction and selectivity.
Science
280:
69-77
|
8. | Greenblatt, R.E., Y. Blatt, and M. Montal. 1985. The structure of the voltage-sensitive sodium channel. Inferences derived from computer-aided analysis of the Electrophorus electricus channel primary structure. FEBS Lett 193: 125-134 [Medline]. |
9. | Guy, H.R., and P. Seetharamulu. 1986. Molecular model of the action potential sodium channel. Proc. Natl. Acad. Sci. USA. 83: 508-512 [Abstract]. |
10. | Heginbotham, L., Z. Lu, T. Abramson, and R. MacKinnon. 1994. Mutations in the K+ channel signature sequence. Biophys. J. 66: 1061-1067 [Abstract]. |
11. | Hille, B. 1992. Ionic Channels of Excitable Membranes. 2nd ed. Sinauer Associates, Inc., Sunderland, MA. 59-82. |
12. | Hodgkin, A.L., and A.F. Huxley. 1952. A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. (Lond.). 117: 500-544 [Medline]. |
13. | Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1994. Shaker potassium channel gating I: transitions near the open state. J. Gen. Physiol 103: 249-278 [Abstract]. |
14. |
Hurst, R.S.,
M.P. Kavanaugh,
J. Yakel,
J.P. Adelman, and
R.A. North.
1992.
Cooperative interactions among subunits of a voltage-dependent potassium channel.
J. Biol. Chem
267:
23742-23745
|
15. | Iverson, L.E., and B. Rudy. 1990. The role of divergent amino and carboxyl domains on the inactivation properties of potassium channels derived from the Shaker gene of Drosophila. J. Neurosci 10: 2903-2916 [Abstract]. |
16. | Larsson, H.P., O.S. Baker, D.S. Dhillon, and E.Y. Isacoff. 1996. Transmembrane movement of the Shaker K+ channel S4. Neuron. 16: 387-397 [Medline]. |
17. | Levy, D.L., and C. Deutsch. 1996. Recovery from C-type inactivation is modulated by extracellular potassium. Biophys. J. 70: 798-805 [Abstract]. |
18. | Liman, E.R., P. Hess, F. Weaver, and G. Koren. 1991. Voltage-sensing residues in the S4 region of a mammalian K+ channel. Nature 353: 752-756 [Medline]. |
19. | Liu, Y., M. Holmgren, M.E. Jurman, and G. Yellen. 1997. Gated access to the pore of a voltage-dependent K+ channel. Neuron 19: 175-184 [Medline]. |
20. | Llano, I., C.K. Webb, and F. Bezanilla. 1988. Potassium conductance of the squid giant axon. J. Gen. Physiol 92: 179-196 [Abstract]. |
21. | Logothetis, D.E., B.F. Kammen, K. Lindpaintner, D. Bisbas, and B. Nadal-Ginard. 1993. Gating charge differences between two voltage-gated K+ channels are due to the specific charge content of their respective S4 regions. Neuron 10: 1121-1129 [Medline]. |
22. | Logothetis, D.E., S. Movahedi, C. Satler, K. Lindpaintner, and B. Nadal-Ginard. 1992. Incremental reductions of positive charge within the S4 region of voltage-gated K+ channel result in corresponding decreases in gating charge. Neuron 8: 531-540 [Medline]. |
23. |
Loots, E., and
E.Y. Isacoff.
1998.
Protein rearrangements underlying slow inactivation of the Shaker K+ channel.
J. Gen. Physiol
112:
377-389
|
24. | Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1991. Hydrophobic substitution mutations in the S4 sequence alter voltage-dependent gating in Shaker K+ channel. Neuron 2: 327-336 . |
25. | López-Barneo, J., T. Hoshi, S.H. Heinemann, and R.W. Aldrich. 1993. Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Receptors Channels 1: 61-71 [Medline]. |
26. | Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Direct physical measure of conformational rearrangement underlying potassium channel gating. Science. 271: 213-216 [Abstract]. |
27. | Matteson, D.R., and R.P. Swenson Jr.. 1986. External monovalent cations that impede the closing of K+ channels. J. Gen. Physiol. 87: 795-816 [Abstract]. |
28. | Miller, A.G., and R.W. Aldrich. 1996. Conversion of a delayed rectifier K+ channel to a voltage-gated inward rectifier K+ channel by three amino acid substitutions. Neuron 16: 853-858 [Medline]. |
29. | McCormack, K., W.J. Joiner, and S.H. Heinemann. 1994. A characterization of the activation structural rearrangements in voltage-dependent Shaker K+ channels. Neuron. 12: 301-315 [Medline]. |
30. | Noda, M., T. Ikeda, T. Kayano, H. Suzuki, H. Takeshima, M. Kurasaaki, H. Takahashi, and S. Numa. 1986. Existence of distinct sodium channel messenger RNAs in rat brain. Nature 320: 188-192 [Medline]. |
31. | Noda, M., T. Tanabe, T. Takai, T. Kayano, and T. Ikeda. 1984. Primary structure of the Electrophorus electricus sodium channel deduced from cDNA sequence. Nature. 312: 121-127 [Medline]. |
32. | Papazian, D.M., X.M. Shao, S.A. Seoh, A.F. Mark, Y. Huang, and H. Wainstock. 1995. Electrostatic interactions of S4 voltage sensor in Shaker K+ channel. Neuron 14: 1293-1301 [Medline]. |
33. | Papazian, D.M., L.C. Timpe, Y.N. Jan, and L.Y. Jan. 1991. Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence. Nature. 349: 305-310 [Medline]. |
34. | Perozo, E., L. Santacruz-Toloza, E. Stefani, E. Bezanilla, and D.M. Papazian. 1994. S4 mutations alter gating currents of Shaker K channels. Biophys. J 66: 345-354 [Abstract]. |
35. | Perozo, E., C.A. Vandenberg, D.S. Jong, and F. Bezanilla. 1991. Single channel studies of the phosphorylation of K+ channels in the squid giant axon. J. Gen. Physiol. 98: 1-34 [Abstract]. |
36. |
Schoppa, N.E., and
F.J. Sigworth.
1998a.
Activation of Shaker potassium channels I. Characterization of voltage-dependent transitions.
J. Gen. Physiol
111:
271-294
|
37. |
Schoppa, N.E., and
F.J. Sigworth.
1998b.
Activation of Shaker potassium channels II. Kinetics of the V2 mutant channel.
J. Gen. Physiol.
111:
295-311
|
38. |
Schoppa, N.E., and
F.J. Sigworth.
1998c.
Activation of Shaker potassium channels III. An activation gating model for wild-type and
V2 mutant channels.
J. Gen. Physiol
111:
313-342
|
39. | Seoh, S.A., D. Sigg, D.M. Papazian, and F. Bezanilla. 1996. Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. Neuron. 16: 1159-1167 [Medline]. |
40. | Sigworth, F.J.. 1994. Voltage gating of ion channels. Q. Rev. Biophys. 27: 1-40 [Medline]. |
41. |
Smith-Maxwell, C.J.,
J.L. Ledwell, and
R.W. Aldrich.
1998a.
Role of
the S4 in cooperativity of voltage-dependent potassium channel
activation.
J. Gen. Physiol.
111:
399-420
|
42. |
Smith-Maxwell, C.J.,
J.L. Ledwell, and
R.W. Aldrich.
1998b.
Uncharged S4 residues and cooperativity in voltage-dependent potassium channel activation.
J. Gen. Physiol
111:
421-439
|
43. |
Starkus, J.G.,
L. Kuschel,
M.D. Rayner, and
S.H. Heinemann.
1997.
Ion conduction through C-type inactivated Shaker channels.
J.
Gen. Physiol
110:
539-550
|
44. | Swenson, R.P., and C.M. Armstrong. 1981. K+ channels close more slowly in the presence of external K+ and Rb+. Nature 291: 427-429 [Medline]. |
45. | Tanabe, T., H. Takeshima, A. Mikami, V. Flovkerzi, H. Takahashi, K. Kangawa, M. Kojima, T. Hirose, and S. Numa. 1987. Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature 328: 313-318 [Medline]. |
46. | Tempel, B.L., D.M. Papazian, T.L. Schwarz, Y.N. Jan, and L.Y. Jan. 1987. Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila. Science 237: 770-775 [Medline]. |
47. | Tytgat, J., and P. Hess. 1992. Evidence for cooperative interaction in potassium channel gating. Nature 359: 420-423 [Medline]. |
48. |
Woodhull, A.M..
1973.
Ionic blockage of sodium channels in nerve.
J. Gen. Physiol.
61:
687-708
|
49. | Yang, N., A.L. George, and R. Horn. 1996. Molecular basis of charge movement in voltage-gated sodium channels. Neuron 16: 113-122 [Medline]. |
50. | Yang, N., and R. Horn. 1995. Evidence for voltage-dependent S4 movement in sodium channels. Neuron 15: 213-218 [Medline]. |
51. | Yusaf, S.P., D. Wray, and A. Sivaprasadarao. 1996. Measurement of the movement of the S4 segment during the activation of a voltage-gated potassium channel. Pflügers Arch 433: 91-97 [Medline]. |
52. | Zagotta, W.N., T. Hoshi, J. Dittman, and R.W. Aldrich. 1994a. Shaker potassium channel gating II: transitions in the activation pathway. J. Gen. Physiol 103: 279-319 [Abstract]. |
53. | Zagotta, W.N., T. Hoshi, J. Dittman, and R.W. Aldrich. 1994b. Shaker potassium channel gating III: evaluation of kinetic models for activation. J. Gen. Physiol. 103: 321-362 [Abstract]. |
54. | Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1989. Gating of single Shaker potassium channels in Drosophila muscle and in Xenopus oocytes injected with Shaker mRNA. Proc. Natl. Acad. Sci. USA. 86: 7243-7247 [Abstract]. |
55. |
Zheng, J., and
F.J. Sigworth.
1998.
Intermediate conductances during deactivation of heteromultimeric Shaker potassium channels.
J. Gen. Physiol
112:
457-474
|