Mutations throughout the S6 region of the hKv1.5 channel
alter the stability of the activation gate
Thomas C.
Rich1,*,
Sarita W.
Yeola2,*,
Michael
M.
Tamkun3, and
Dirk J.
Snyders2
Departments of 1 Biomedical Engineering, 2 Medicine
and Pharmacology, and 3 Molecular Physiology and Biophysics,
Vanderbilt University, Nashville, Tennessee 37232
 |
ABSTRACT |
First published September 21, 2001; 10.1152/ ajpcell.00232.2001.
The S6 segment of voltage-gated
K+ channels is thought to contribute to the gate that opens
the central permeation pathway. Here we present evidence that mutations throughout the cytoplasmic end of S6 strongly influence hKv1.5 channel
gating characteristics. Modification of hKv1.5 at positions T505, V512,
and S515 resulted in large negative shifts in the voltage dependence of
activation, whereas modifications at position Y519 resulted in negative
(Y519N) and positive (Y519F) shifts. When adjusted for the altered
voltage sensitivity, activation kinetics of mutated channels were
similar to those of the wild-type (WT) channel; however, deactivation
kinetics of mutations T505I, T505V, V512A, and V512M [time constant
(
) = 35, 250, 170, and 420 ms, respectively] were still slower
than WT (
= 8.3 ms). In addition, deactivation of WT channels
was highly temperature sensitive. However, deactivation of T505I and
V512A channels was largely temperature insensitive. Together, these
data suggest that mutations in S6 decouple activation from deactivation
by altering the open-state stability and that residues on both sides of
the highly conserved Pro-X-Pro sequence influence the movement of S6
during channel gating.
potassium channels; structure-function analysis; temperature
dependence of channel gating
 |
INTRODUCTION |
MUCH RECENT WORK has
focused on understanding the mechanisms that allow voltage-gated ion
channels to sense changes in membrane potential. The fourth
transmembrane segment (S4) is considered to be the voltage sensor of
voltage-gated ion channels. Mutations that neutralize charged residues
in this region reduce the voltage sensitivity (9, 17, 19).
However, more conservative mutations that do not alter the number of
charges in S4 also alter the voltage dependence of activation
(17, 23, 25, 26). Recent studies have provided additional
evidence for voltage-dependent conformational changes by monitoring the
relative movements of S4 during the gating process (3, 7).
The S2 region also has been implicated as part of the voltage sensor
(14, 24).
Although these studies have revealed important mechanisms involved in
the voltage sensitivity of K+ channels, much less
information is known about how movement of S4 triggers the opening and
closing of the permeation pathway. Early studies by Armstrong (1,
2) showed that tetraethylammonium blocked the K+
channels of squid axons when the channels were in an activated state.
He proposed that conformational changes in the protein structure made
the tetraethylammonium binding site accessible when the channel was
activated and that the activation gate was located in the inner mouth
of the pore. At the molecular level, the S6 region has been identified
as a potential component of the inner mouth of the pore and the binding
site for quaternary ammonium compounds (4, 13). Liu et al.
(11) introduced cysteine residues at sites in the S6
region of Shaker to examine the accessibility of these
residues to the methanethiosulfonate reagents. They found that several
residues within the S6 region were accessible only when the channel was
in an activated state, suggesting that the movement of an intracellular
gate regulates access to the pore. Consistent with this result,
modification of a cysteine residue in the S6 region of the Kv2.1
channel altered open-state stability and ion permeation
(12).
The understanding of channel permeation and gating has been greatly
advanced by the crystal structure of the bacterial
K+-selective channel KcsA (6). In this channel
the second transmembrane regions (equivalent to S6 in hKv1.5) of the
four subunits intersect to form a teepee-like structure with the tip
near the cytoplasmic end. A diaphragm-like movement of this region
increases the diameter of the permeation pathway of the KcsA channel
(18). Li-Smerin et al. (10) used tryptophan
scanning to study the interactions of the S5-pore helix-S6 region of
the Shaker channel. They found that when mutations that
alter channel gating (found throughout these regions) are mapped onto
the KcsA channel structure, they tend to cluster near the interface
between pore domain subunits. Although the KcsA channel can be used as
a model for the equivalent S5-S6 regions of voltage-gated
K+ channels, there is evidence that, unlike KcsA, there is
a sharp bend toward the cytoplasmic side of the S6 segment of the
Shaker channel (5). Such a bend could be caused
by the Pro-X-Pro sequence that is highly conserved among voltage-gated
K+ channels and probably crucial for formation of
functional homotetrameric channels. Indeed, subunits of the Kv1 through
Kv4 subfamilies display this sequence and form functional
homotetrameric channels, whereas those of the Kv5 through Kv9
subfamilies do not.
To further examine the role of S6 in channel gating, we made a series
of mutations near the Pro-X-Pro sequence (P509-V510-P511) of the hKv1.5
voltage-gated K+ channel (Fig.
1). When adjusted for shifts in
voltage-dependent gating, the activation kinetics of mutated channels
were similar to those of the wild-type (WT) channel. Interestingly,
even when adjusted for these shifts, the deactivation kinetics of
T505I, T505V, V512A, and V512M channels were dramatically slower than those of WT, and those of Y519F were faster. We also tested for differences in the temperature dependence of activation and
deactivation among WT channels and two of the slowly deactivating
mutations, T505I and V512A. Activation of all three channels and
deactivation of WT channels were highly sensitive to temperature.
Surprisingly, deactivation of T505I and V512A was largely insensitive
to temperature (20°
T
40°C). These data provide evidence
that residues on both sides of the Pro-X-Pro sequence greatly affect
the stability of the activation gate and that mutations to these
residues can decouple channel activation from deactivation.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1.
Amino acid sequence of the S6 region of hKv1.5.
A: alignment of the S6 region of several K+
channels. In the region of S6 examined, sequence variability occurs at
T505, L508, V510, V512, S515, N518, and Y519. Except for V510 and N518,
these residues can be aligned into an -helical projection of the
putative pore region (29). Asterisks indicate
cysteine-substituted residues in Ref. 11 (homologous to hKv1.5 at
positions I506, V510, P511, V512, and I513). B:
two-dimensional schematic of hKv1.5 structure. The relevant amino acids
in S6 are given as one-letter codes. Mutations that did not express
functional channels were T505D, T505K, V512E, V512D, I513A, and
F517V.
|
|
 |
METHODS |
Cell preparation.
The PCR-based method for site-directed mutagenesis in the S6 region and
the mutations used to form stable cell lines expressing the mutated
channels (29) and cell culture procedures
(27) have been described elsewhere. Briefly, mouse
Ltk
cells stably expressing WT or mutated hKv1.5 channels
were cultured in DMEM supplemented with 10% horse serum and 0.25 mg/ml
G418. The cultures were passed every 4-5 days by using a brief
trypsin treatment. Before experimental use, cultures were incubated
with 2 µM dexamethasone for 12-24 h to induce efficient channel
expression. The cells were removed from the dish with a rubber
policeman, stored at room temperature, and used within 12 h.
Electrical recording.
Recordings were obtained using an Axopatch-200A patch-clamp amplifier
(Axon Instruments, Foster City, CA) and the whole cell patch-clamp
technique. Pipettes were pulled from starbore borosilicate glass
(Radnoti, Monrovia, CA) and heat polished. Bath temperature (11°
T
45°C) was maintained within ±0.5°C of the set point by
using a Peltier device. To ensure adequate voltage control in the whole
cell configuration, pipette resistance was limited to 3 M
and
averaged 2.4 ± 0.1 M
(n = 120). Voltage
offsets were zeroed with the pipette in the bath solution. No
additional corrections were done for the small liquid junction
potential difference, which is estimated to be <5 mV with the
solutions used (15). Pipettes were then lowered onto the
cells, and gigaohm seals were formed by applying light suction
(9.9 ± 0.7 G
). After whole cell configuration was achieved,
capacitive transients were elicited by applying 20-mV steps from the
holding potential (
80 mV) and recorded at 40 kHz (filtered at 10 kHz)
for calculation of access resistance and input impedance. Capacitance
and series resistance compensation were then optimized to achieve 80%
compensation. We calculated the residual resistance and excluded those
experiments in which the voltage error due to series resistance
exceeded 5 mV. Current records were sampled at 2-10 times the
anti-alias filter setting, stored on a personal computer, and archived
on an optical disk. Software for acquisition that allowed custom protocols (both stimulation and sampling) not available in commercial packages was designed in house (20, 27).
Solutions.
The intracellular pipette filling solution contained (in mM) 110 KCl,
10 HEPES, 5 K4-BAPTA
(1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid),
5 K2ATP, and 1 MgCl2 and was adjusted to a pH
of 7.2 using KOH. This gave a final K+ concentration of
~145 mM. The bath solution contained (in mM) 130 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose and
was adjusted to pH of 7.35 with NaOH. The pH of the solutions decreased
~0.1 units with a 10°C increase in temperature due to the
temperature dependence of the buffer. Thus the pH of the solutions at
high temperature remained within 0.2 pH units of the pH at room
temperature. Such small changes in pH did not significantly affect
gating at room temperature.
Analysis.
Results were assessed by calculating time constants of activation and
deactivation and then calculating current-voltage relationships. All
software for the analysis presented was written in house using Visual
Basic, FORTRAN, and MATLAB. The curve-fitting procedure to calculate
time constants (and their relative amplitudes) used either a nonlinear
least-squares (Gauss-Newton) algorithm or a simplex algorithm. The
number of exponentials required and goodness of fit were evaluated by
inspecting the residuals for nonrandom trends and comparing
2 values statistically. All gating models were optimized
with software written in MATLAB by using the least-squares error criterion.
 |
RESULTS |
To examine the role of the inner pore in channel gating, we
scanned the putative pore lining residues of the hKv1.5 channel with a
series of single amino acid substitutions to the cytoplasmic end of the
S6 region (Fig. 1). Currents elicited from functional mutations at
positions T505, L508, V512, S515, and Y519 (capitalized residues in
Fig. 1) were examined and compared with those of WT. Although these
mutations were in the putative pore region, they did not significantly
alter the reversal potential; thus K+ selectivity was
maintained (Table 1). Two mutations that
introduce charged residues at positions T505 (T505D and T505K) and V512 (V512E and V512D) as well as the mutations I513A and F517V failed to
yield functional channels.
Effects of S6 mutations on deactivation.
To examine the time course of deactivation, we monitored current decay
at membrane potentials between
30 and
110 mV following 50-ms
depolarizations to +50 mV. In most cases deactivation was best fit with
two exponentials (Fig. 2). In general,
the amplitude of the dominant time constant was >70% of the total
amplitude (Table 1). The deactivation of WT channels proceeded in a
voltage-dependent manner with a dominant time constant of 8.3 ms at
100 mV (27). The deactivation of the mutated channels
also proceeded in a voltage-dependent manner (Fig.
3). The deactivation of some mutations
was similar to that of WT; however, the deactivation of other mutations
was dramatically altered (Fig. 3 and Table 1). At position T505, the
deactivation of T505S was 2-fold faster than that of WT, whereas the
deactivation of T505A was almost 2-fold slower. The deactivation of
T505I and T505V were 4- and 30-fold slower than that of WT, respectively. At position L508, L508M deactivation kinetics were similar to those of WT. Examination of L508A was limited by the low
level of expression; however, deactivation kinetics appeared faster
than those of WT (data not shown). At position V512, the mutations
V512A and V512M both displayed a marked slowing of deactivation (20- and 50-fold slower, respectively). At position S515, introduction of
both a positive (S515K) or a negative charge (S515E) was tolerated at
this point, but currents for S515K were too small for detailed analysis. The deactivation kinetics of S515E were slower than those of
WT at
50 mV; however, at more hyperpolarized potentials, the
deactivation kinetics were similar to those of WT. At position Y519,
both Y519F and Y519N displayed slower deactivation kinetics than WT.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
Exponential fits to deactivating currents of T505I
channels measured at 50 mV after a 50-ms depolarization to +50 mV.
Top: residual errors for the monoexponential (1 exp) and
biexponential (2 exp) fits: monoexponential, = 315 ms;
biexponential, 1 = 322 ms, 2 = 51 ms, A 1 (amplitude of 1 component) = 80%. Bottom: fits to deactivating current (I)
in semilogarithmic format. The two components of a biexponential fit
are shown; the slow component is superimposed on the original data, and
the fast component is superimposed on data points calculated by
subtracting the slow component. Systematic deviations were absent from
the biexponential fits.
|
|

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3.
Slowing of channel closing by mutations in S6. Top left
panel shows currents of wild-type (WT) channels from the entire pulse
protocol (inset). Remaining panels show expanded views of
the deactivating currents of WT and mutant channels measured at
potentials of 50, 70, 90, and 110 mV. The mutations T505I,
T505V, V512A, and V512M caused substantial slowing of channel closing.
Vertical scale bar represents 300 pA. Note the changes in time scale
for the various mutations.
|
|
The hKv1.5 channel is a strong outward rectifier; therefore, to more
readily observe deactivation at potentials below
60 mV, we examined
deactivation of some mutated channels in 145 mM external K+
(Fig. 4). Under these conditions large
inward tails were observed. Deactivation of WT and mutant channels at
110 mV could be largely described by single exponentials with the
following time constants: WT, 5 ms; T505I, 83 ± 5 ms
(n = 6); T505V, 770 ± 110 ms (n = 5); V512A, 360 ± 20 ms (n = 4); and V512M,
730 ± 30 ms (n = 8). A problem with the analysis
of deactivation kinetics for mutations T505I, T505V, V512A, and V512M
was that the extremely slow deactivation time constants were similar to
the voltage-independent inactivation time constants (240 and 2,700 ms)
previously described (20, 27). Thus it is likely that both
deactivation and slow inactivation contributed to the time course of
channel closure, and the deactivation rates may be slower than
indicated by the time constants.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4.
Representative traces of T505I (A), T505V
(B), V512A (C), and V512M (D) currents
measured in high extracellular K+
([K+]o = 145 mM). The currents were
elicited by 50-ms depolarizations to +50 mV followed by
hyperpolarizations to 110 mV. Dashed lines depict scaled WT currents.
Under these conditions, the slowing of channel closing caused by these
mutations is obvious even at strong hyperpolarizations.
|
|
Effects of S6 mutations on the midpoint of activation.
Activation of WT channels displayed a sigmoidal voltage dependence with
a midpoint of activation (Eh) of
14 mV in the
presence of the Kv
subunit endogenously expressed in mouse
Ltk
cells (27, 28). We observed that the
voltage dependence of activation of several of the mutations was
similar to that of WT, whereas the voltage dependence of other
mutations was dramatically altered (Table 1). T505A and T505S caused
small shifts in the voltage dependence of activation (<5 mV). T505I
caused a slightly larger shift (
8.5 mV). However, T505V caused a
shift of
36 mV in the voltage dependence of activation. L508M
displayed a shift of +8.5 mV in the voltage dependence of activation.
L508A also caused a positive shift, but the current amplitudes were too
small for accurate assessment of this shift. Both V512A and V512M
caused large negative shifts (greater than
27 mV) in the voltage
dependence of activation. The shift of
12 mV in the voltage
dependence of S515E activation was opposite to that expected from a
local charge effect on the voltage sensor. It is unlikely that this
residue is located at a position extracellular to the voltage sensor; it seems more likely that this residue is not in close proximity to
voltage sensor (e.g., it may be pointed toward the permeation pathway).
This shift was much smaller than that caused by hydrophobic substitutions at nearby residues. Interestingly, mutations at position
Y519 caused opposite shifts in Eh: +12 and
13.5 mV for Y519F and Y519N, respectively.
The kinetics of channel activation were characterized by a dominant
exponential component with a time constant of ~2 ms (+60 mV) for WT
channels. Figure 5, A-C,
shows the time constant of the dominant exponential of channel kinetics
for both WT (solid line) and mutant (dotted lines with symbols)
channels. For each of the mutations the time constants of activation
decreased with increasing membrane potential
(Em). When the membrane potential was adjusted
for shifts in the midpoint of activation,
Em
Eh (Fig. 5,
D-F), it became apparent that, for each mutation, the activation time constants displayed a similar voltage dependence to WT.
Furthermore, the changes in activation kinetics at strong depolarizations were strongly and linearly correlated with shifts in
Eh (Fig.
6A). Although the mutations
with the slowest deactivation kinetics displayed negative shifts
in Eh, the substantial slowing was
disproportional to the observed shift in Eh
(Fig. 6B).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 5.
Voltage dependence of dominant activation and
deactivation time constants ( ). Time constants at membrane
potentials above the midpoint of activation (Eh;
see Table 1) were obtained by exponential fits of the time course of
the activating current at indicated potentials (4 n 10). Time constants at membrane potentials below
Eh were obtained from deactivating currents (see
Fig. 4) at indicated potentials. Solid line represents WT kinetics.
Curves in A (T505V, T505I, T505A, and T505S), B
(V512M and V512A), and C (Y519N, S515E, L508M, and Y519F)
show the activation and deactivation time constants of mutated channels
vs. the actual membrane potential (Em). Curves
in D-F (same mutations as in A-C) show
the time constants vs. a shifted membrane potential
[Eh of each mutation subtracted from
Em (Em Eh)]. It is apparent that the impaired closing
of mutations T505I, T505V, V512A, and V512M was not due to a pure shift
in the voltage dependence of activation but, rather, that the shift in
the voltage dependence of activation of these mutants was in part due
to the slowed deactivation kinetics.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Relationship between dominant activation and deactivation time
constants and voltage dependence of activation. A: dominant
time constants of activation obtained from exponential fits to currents
measured at depolarizations to +60 mV ( +60). The
activation time constants varied linearly with the midpoint of
activation (slope = 0.033 ± 0.007, intercept = 2.8 ± 0.2, P = 0.0028). The strong linear trend indicates
that changes in forward transitions along the activation pathway are
not responsible for observed shifts in the voltage dependence of
activation. Eh was estimated by fitting the
Boltzmann equation to currents obtained at 30 mV ( 60 mV for T505V,
V512A, and V512M) following 250-ms depolarizing steps to potentials
between 80 and +60 mV. B: dominant time constants of
deactivation were estimated from exponential fits to currents obtained
at 100 mV ( 100) following 50-ms depolarizations to
+50 mV (note the logarithmic scale of the vertical axis). Each point
represents the mean ± SE (n 4) for both
Eh and .
|
|
Effects of S6 mutations on the temperature dependence of activation
and deactivation.
To further examine the relationship between channel activation and
deactivation, we measured the temperature dependence of WT, T505I, and
V512A kinetics. We used the dominant time constants of channel
activation and deactivation rather than trying to dissect entropic and
enthalpic contributions of individual transitions because of the
difficulty in separating the effects of temperature on individual
transitions. At increased temperature we observed that WT channels
activated at more negative potentials. The midpoint of activation
shifted from
12.8 ± 0.9 mV at 22°C to
25.1 ± 2.3 mV
at 32°C. The dominant activation time constant also was temperature dependent (at +50 mV:
22°C = 2.0 ± 0.1,
32°C = 0.52 ± 0.04 ms). At temperatures
above 28°C and strong depolarizations (+50 mV). the time constant of
activation was best fit with a single exponential. As with WT, both
T505I and V512A activated at more negative potentials at elevated
temperatures. For T505I, the midpoint of activation shifted from
21.5 ± 1.5 mV at 22°C to
36.5 ± 1.6 at 32°C.
Similarly, for V512A, the midpoint of activation shifted from
40.4 ± 1.0 mV at 22°C to
56.0 ± 3.0 at 29°C. The
dominant activation time constants of T505I and V512A were also
temperature dependent (T505I at +50 mV:
22°C = 2.6 ± 0.3 ms,
32°C = 0.64 ± 0.10 ms;
V512A at +50 mV:
22°C = 2.4 ± 0.2 ms,
32°C 0.98 ± 0.02 ms). The activation time constants of WT, T505I, and V512A decreased in a log-linear fashion as
temperature increased (Fig. 7,
A-C). Thus the overall temperature dependence of the
activation kinetics of the T505I and V512A channels was similar to that
of WT. Deactivation of WT channels also was temperature dependent (at
60 mV:
22°C = 21.7 ± 3.4 ms,
37°C = 6.0 ± 0.4 ms). As with the
activation time constant, the WT deactivation time constant decreased
in a log-linear fashion with increased temperature (Fig.
8A). However, unlike the WT
deactivation, T505I deactivation displayed little or no temperature
dependence between 22 and 37°C (Fig. 8B). Deactivation of
V512A also appeared temperature independent between 22 and 30°C (at
110:
22°C = 140 ± 31 ms,
28°C = 143 ± 12 ms). Although a more
complete assessment of the temperature dependence was precluded by the very negative threshold of activation at elevated temperatures (especially of V512A), these data demonstrate that T505I and V512A channels close slowly, in a largely temperature-insensitive
manner.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 7.
Temperature dependence of channel activation.
A: temperature dependence of WT hKv1.5 activation time
constants ( act) fit to data measured at depolarizations
to +50 mV. Line represents an exponential fit to data with a
"temperature coefficient" of 4.3°C 1 (corresponding
to a Q10 of 3.3 for the activation rate constant 1/ ).
The holding potential was 80 mV for 10 < T < 28°C, 90
mV for 28 < T < 37°C, and 100 mV for T > 37°C,
where T is temperature. B: temperature dependence of T505I
act fit to data measured at depolarizations to +50 mV.
Line represents an exponential fit to data with a temperature
coefficient of 4.8°C 1 (corresponding to a
Q10 of 3.2). The holding potential was 80 mV for 10 < T < 28°C, 90 mV for 28 < T < 32°C, and 100
mV for T > 32°C. C: temperature dependence of V512A
act fit to data measured at depolarizations to +50 mV.
Line represents an exponential fit to data with a temperature
coefficient of 6.6°C 1 (corresponding to a
Q10 of 3.0). The holding potential was 90 mV for 10 < T < 28°C, 100 mV for 28 < T < 37°C, and
110 mV for T > 37°C.
|
|

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 8.
Temperature dependence of channel closing. A:
temperature dependence of WT hKv1.5 time constants
( deact) fit to the deactivation time course measured at
60 mV. Line represents an exponential fit to data with a temperature
coefficient of 8.3°C 1 (corresponding to a
Q10 of 2.7). B: temperature dependence of T505I
deact fit to the time course of channel closing measured
at 60 ( ) and 110 mV ( ). Lines
represent exponential fits to data (for comparative purposes only; the
temperature range is far too small for accurate fits) with temperature
coefficients of 39°C 1 and 300°C 1
(corresponding to respective Q10 values of 1.2 and 0.35) at
60 and 110 mV, respectively. Holding potentials were as described
in Fig. 7 legend.
|
|
 |
DISCUSSION |
We have observed several interesting results: 1)
modifications of residues in the S6 region of the hKv1.5 channel
dramatically slow the time course of channel closing; 2)
these modifications alter the voltage dependence of activation;
3) the effects were similar for mutations on both sides of
the conserved Pro-X-Pro sequence; 4) when shifts in the
voltage dependence of activation are accounted for, the mutations did
not cause significant changes in the activation time-course but still
caused drastic changes in the deactivation time course; and
5) although the temperature dependence of activation of both
T505I and V512A was similar to that of WT, the primary time constant of
channel closing of these two mutations displayed little or no
temperature dependence. These results suggest that the mutations alter
a transition (or transitions) late in the activation pathway that,
depending on the mutation, either facilitates or hinders channel
opening and closing. Furthermore, they indicate that T505I and V512A
cause a selective "deactivation failure" in the face of a preserved
(near WT) activation gating.
Mutations to the S6 region alter channel gating.
Several of the point mutations in the S6 region of hKv1.5 caused
shifts in the voltage dependence of activation and dramatically altered
the deactivation time course. These effects were dependent on both the
amino acid position and the specific amino acid that replaced the WT
residue. For example, the conservative substitution T505S resulted in
faster deactivation than WT. However, when T505 was replaced by the
smaller alanine, deactivation was slightly slower than that of WT,
indicating that 1) the slowing of deactivation due to
mutations at T505 was not a result of steric hindrance, and
2) the hydroxyl side chains of threonine and serine
facilitated channel closing. When T505 was replaced by either valine or
isoleucine, deactivation was dramatically slowed. The effects of the
mutations at T505 were similar to those reported at the equivalent
position of other channels (16, 30). Similarly,
deactivation of V512A and V512M channels was dramatically slowed. These
changes were not linearly correlated to shifts in the voltage
dependence of activation. Thus the results suggest that the slowing of
deactivation was, at a minimum, due to a stabilization of the open
state and not a pure shift in voltage-dependent gating. This
deactivation failure demonstrates that channel closing can be largely
uncoupled from channel opening by altering the molecular properties of S6.
The ability of mutations before and after the Pro-X-Pro sequence to
uncouple activation from deactivation suggests that this section of S6
is an integral part of the activation gate. As such, these results
agree with the previous work by Liu et al. (11) demonstrating functionally that S6 comprises part of the activation gate of Shaker channels. They showed that
cysteine-substituted residues (homologous to hKv1.5 at positions I506,
V510, P511, V512, and I513; indicated by asterisks in Fig.
1A) were accessible to bulky thiol-specific reagents when
channels were in an open state. In another study, Li-Smerin et al.
(10) used tryptophan-scanning mutagenesis to show that
modification of residues in S6 alters the gating properties of
Shaker. Interestingly, when residues that altered
Shaker channel gating were mapped onto the KcsA structure, the locations of these residues tended to cluster near the interface between pore domain subunits.
It has been difficult to dissect the molecular nature of the gating
interactions that lead to channel opening and closing. This is due, in
part, to the lack of homology in the S6 region between voltage-gated
K+ channels and the crystallized KcsA channel. Also,
investigations of the molecular interactions with site-directed
mutagenesis are for the most part limited to 20 different natural amino
acid side chains. However, some attempts have been made to probe the
interactions of individual residues within their molecular environment.
Zhou et al. (30) showed that changes in the rate of
deactivation due to mutations at T529 in rKv1.4 (T505 in hKv1.5)
correlated with the amino acid side chain partition energies of soluble
proteins. They speculated that this might indicate the residue is
exposed to the hydrophobic protein interior when the channel is closed and that, upon channel opening, the residue enters the more hydrophilic channel lumen. Our results do not indicate such a clear-cut correlation between hydrophobic interactions and channel deactivation. For example,
if hydrophobic interactions at T505 were the primary determinants of
changes in deactivation rate, then, on the basis of the partition
energies (8), deactivation of T505I would be expected to
be slower than that of T505V. However, at
100 mV the deactivation
time constant of T505I was 35 ms, whereas the deactivation time
constant of T505V was 250 ms. Although hydrophobic interactions may
contribute to the changes in deactivation observed when T505 is
modified, other interactions such as local helix flexibility and
packing modifications caused by point mutations also may contribute. No
correlation between partition energy and deactivation rate existed for
mutations at position V512. It also is unlikely that changes in the
side chain volume were a determining factor. Neither shifts in midpoint
of activation nor deactivation time constants correlated with changes
in side chain volume for mutations at T505 or V512 (using side chain
volumes from Ref. 31).
Kinetic modeling of changes in open-state stability.
We have demonstrated that mutations to S6 dramatically alter
channel activation and deactivation. Can these results be described quantitatively by altering the stability of the activation gate? Consider a two-state model, C
O. Stabilization of the open state would not only slow channel deactivation, it would also shift the
voltage dependence of activation toward more negative potentials, consistent with the results described above. However, such a simple model cannot describe a majority of voltage-gated K+
channel kinetics.
To determine whether changes in the stability of the final
closed-to-open transition step can account for the data, we used the
following kinetic model (Eqs. 1 and 2)
|
(1)
|
where
,
,
, and
are the voltage-dependent rate
constants described by
|
(2)
|
where W is the activation energy (in units of
kT), z is the net charge moved during the
transition, X is the fraction of the membrane field through
which the charge moves, e is the elementary charge unit,
V is the membrane potential, T is the absolute
temperature, k is the Boltzmann constant, and h
is the Planck constant. Model parameters used to describe WT kinetics
are given in Table 2. This model
describes a majority of hKv1.5 activation and deactivation kinetics
(the voltage dependence of activation and deactivation, the dominant
activation and deactivation time constants). However, it does not
reproduce the second component of activation or the multiple components
of inactivation previously described for hKv1.5 (20).
These aspects of hKv1.5 gating were neglected to illustrate the general
results of the mutations to the S6 region.
Figure 9A shows that the model
describes the overall deactivation time course of WT channels. We first
examined whether simply stabilizing the open state (increasing the
magnitude of W
) could explain both the shift
in the voltage dependence of activation and the slow deactivation time
course of T505I and V512A. Both the shift in the voltage dependence of
T505I activation and the time course of channel closing were well fit
by modifying W
(Table
3 and Fig. 9B). However,
neither the shift in the voltage dependence of V512A activation nor the
time course of channel closing could be fit by modifying
W
(Table 3).

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 9.
Model fits (solid line) to the time course of channel
closing (dot plots). Currents were elicited by 50-ms depolarizations to
+50 mV followed by 500- (WT) or 5,000-ms (T505I and V512A) steps to
membrane potentials of 50, 70, 90, and 110 mV. A:
model fits to WT hKv1.5 deactivation kinetics. The model describes both
activation (model Eh = 14 mV) and
deactivation of WT channels. Parameters for this model are given in
Table 2. B: model fits with stabilized open position of the
activation gate describe T505I activation (model
Eh = 20 mV) and deactivation. This model
does not describe either V512A activation (model
Eh = 20 mV) or deactivation well (see text).
C: model fits with stabilized open position, destabilized closed
position of the activation gate describe V512A activation (model
Eh = 40 mV) and deactivation. Although it
is uncertain whether the closed position of T505I is destabilized, it
seems likely that the closed position of V512A is destabilized.
Parameters for this model are given in Table 3.
|
|
We next examined whether stabilizing the open state as well as
destabilizing the preceding closed state (decreasing the magnitude of
W
) could describe the effects of V512A. In
this case, the model fit the time course of channel closing and also
fit the shifts in the voltage dependence of activation of V512A (Fig. 9C and Table 3). Thus the large negative shifts in the
voltage dependence of activation of V512A (as well as that of V512M and T505V) may have been due to both the destabilization of a closed-state prior channel opening and stabilization of an open state, relative to WT.
Decoupling activation from deactivation.
Thus far we have provided experimental evidence suggesting that
mutations in the S6 region of hKv1.5 decouple activation from deactivation and have presented a kinetic model consistent with this
data. To further isolate the gating transitions that were affected by
these mutations, we examined the temperature dependence of WT, T505I,
and V512A activation and deactivation. We found that temperature
influenced both the voltage dependence of activation and the activation
time course of WT, T505I, and V512A to a similar extent. The
temperature dependence of channel closing was markedly different
between WT and the T505I and V512A channels. WT deactivation was highly
temperature dependent, whereas deactivation of T505I and V512A was
largely temperature independent. In fact, at increased temperatures,
changes in the rates of channel closing were, in part contaminated by
slow inactivation. These results suggest that T505I and V512A did not
significantly alter temperature-dependent components of activation,
but, rather, they stabilized the open state such that any temperature
dependence of deactivation was masked. Thus these results are
consistent with the hypothesis that mutations in the S6 region alter
transitions late in the activation pathway (e.g., C
O and O
C)
and that these transitions occur after the temperature-dependent
activation transitions. These results also are consistent with the
kinetic model presented above. In this model, the closed-to-open
transition(s) would have little or no temperature dependence, whereas
the open-to-closed transition(s) would have substantial temperature
dependence. In studies of the temperature dependence of
Shaker K+ channel gating, Rodriguez and
coworkers (21, 22) provided strong evidence for such a
model. They also observed that a net decrease in enthalpy occurs when
the channel enters the open state from the final closed state,
indicating that in some sense the channel becomes more ordered. In
light of the data presented here, it seems likely that residues in S6
are crucial in this rearrangement. Although the exact nature of the
molecular interactions remains unclear, the results did not correlate
with changes in side chain volume or hydrophobic interactions.
In conclusion, we have shown that 1) mutations in the S6
region of hKv1.5 led to a shift of the open-closed equilibrium as a
function of voltage; 2) these mutations caused a marked
slowing of the deactivation of the channel; and 3) two of
these mutations, T505I and V512A, have little effect on the temperature
dependence of activation but dramatically reduce the temperature
dependence of deactivation. The effects of these mutations suggest that
this region of the S6 segment comprises part of the activation gate and
that mutations in this region alter the stability of both the open and
closed conformations of the gate. The effects of these mutations can be
described quantitatively as changes in the energy barrier between the
open and closed states. Thus these data are consistent with a scheme in
which changes in membrane potential cause movement of the voltage
sensor that, in turn, triggers a rearrangement of the inner mouth of
the pore, allowing ion permeation. Residues throughout S6 are involved
in this process, and changes in these residues lead to changes in the
open-closed equilibrium. Once these channels are activated, they are
locked into an open state, effectively decoupling activation from deactivation.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grants HL-59689 and HL-49330 and by Flanders Interuniversity Institute for Biotechnology Grant VIB0055.
 |
FOOTNOTES |
*
T. C. Rich and S. W. Yeola contributed equally
to this work.
Present addresses: T. C. Rich, Dept. of Physiology and Biophysics,
University of Colorado Health Sciences Center, Denver, CO 80262;
S. W. Yeola, Bristol Myers Squibb, Wallingford, CT 06492; and M. M. Tamkun, Dept. of Physiology, Colorado State University, Ft. Collins, CO 80523.
Address for reprint requests and other correspondence: D. J. Snyders, Laboratory for Molecular Biophysics, Physiology, and Pharmacology, Dept. of Biomedical Sciences, Univ. of Antwerp, Universiteitsplein 1-T4.21, B-2610 Antwerp, Belgium (E-mail:
dirk.snyders{at}ua.ac.be).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 May 2001; accepted in final form 17 September 2001.
 |
REFERENCES |
1.
Armstrong, CM.
Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons.
J Gen Physiol
58:
413-437,
1971[Abstract/Free Full Text].
2.
Armstrong, CM.
Time course of TEA+-induced anomalous rectification in squid axons.
J Gen Physiol
54:
553-575,
1966[Abstract/Free Full Text].
3.
Cha, A,
Snyder GE,
Selvin PR,
and
Bezanilla F.
Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy.
Nature
402:
809-813,
1999[ISI][Medline].
4.
Choi, KL,
Mossman C,
Aube J,
and
Yellen G.
The internal quaternary ammonium receptor site of Shaker potassium channels.
Neuron
10:
533-541,
1993[ISI][Medline].
5.
Del Camino, D,
Holmgren M,
Liu Y,
and
Yellen G.
Blocker protection in the pore of a voltage-gated K+ channel and its structural implications.
Nature
403:
321-325,
2000[ISI][Medline].
6.
Doyle, DA,
Cabral JM,
Pfuetzner RA,
Kuo A,
Gulbis JM,
Cohen SL,
Chait BT,
and
MacKinnon R.
The structure of the potassium channel: molecular basis of K+ conduction and selectivity.
Science
280:
69-77,
1998[Abstract/Free Full Text].
7.
Glauner, KS,
Mannuzzu LM,
Gandhi CS,
and
Isacoff EY.
Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel.
Nature
402:
813-817,
1999[ISI][Medline].
8.
Guy, HR.
Amino acid side-chain partition energies and distribution of residues in soluble proteins.
Biophys J
47:
61-70,
1985[Abstract].
9.
Liman, ER,
Hess P,
Weaver F,
and
Koren G.
Voltage-sensing residues in the S4 region of a mammalian K+ channel.
Nature
353:
752-756,
1991[ISI][Medline].
10.
Li-Smerin, Y,
Hackos DH,
and
Swartz KJ.
A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel.
Neuron
25:
411-423,
2000[ISI][Medline].
11.
Liu, Y,
Holmgren M,
Jurman ME,
and
Yellen G.
Gated access to the pore of a voltage-dependent K+ channel.
Neuron
19:
175-184,
1997[ISI][Medline].
12.
Liu, Y,
and
Joho JH.
A side chain in S6 influences both open state stability and ion permeation in a voltage-gated K+ channel.
Pflügers Arch
435:
654-661,
1998[ISI][Medline].
13.
Lopez, GA,
Jan YN,
and
Jan LY.
Evidence that the S6 segment of the Shaker voltage-gated K+ channel comprises part of the pore.
Nature
367:
179-182,
1994[ISI][Medline].
14.
Milligan, CJ,
and
Wray D.
Local movement in the S2 region of the voltage-gated potassium channel hKv2.1 studied using cysteine mutagenesis.
Biophys J
78:
1852-1861,
2000[Abstract/Free Full Text].
15.
Neher, E.
Correction for liquid junction potentials in patch clamp experiments.
Methods Enzymol
207:
123-131,
1992[ISI][Medline].
16.
Ogielska, EM,
and
Aldrich RW.
A mutation in S6 of Shaker potassium channels decreases the K+ affinity of an ion binding site revealing ion-ion interactions.
J Gen Physiol
112:
243-257,
1998[Abstract/Free Full Text].
17.
Papazian, DM,
Timpe LC,
Jan YN,
and
Jan LY.
Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence.
Nature
349:
305-310,
1991[ISI][Medline].
18.
Perozo, E,
Cortes DM,
and
Cuello LG.
Structural rearrangements underlying K+-channel activation gating.
Science
285:
73-78,
1999[Abstract/Free Full Text].
19.
Perozo, E,
Santacruz-Toloza L,
Stefani E,
Bezanilla F,
and
Papazian DM.
S4 mutations alter gating currents of Shaker K channels.
Biophys J
66:
345-354,
1994[Abstract].
20.
Rich, TC,
and
Snyders DJ.
Evidence for multiple open and inactivated states of the hKv1.5 delayed rectifier.
Biophys J
75:
183-195,
1998[Abstract/Free Full Text].
21.
Rodriguez, BM,
and
Bezanilla F.
Transitions near the open state in Shaker K+-channel
probing with temperature.
Neuropharmacology
35:
775-785,
1996[ISI][Medline].
22.
Rodriguez, BM,
Sigg D,
and
Bezanilla F.
Voltage gating of Shaker K+ channels the effect of temperature on ionic and gating currents.
J Gen Physiol
112:
223-242,
1998[Abstract/Free Full Text].
23.
Schoppa, NE,
McCormack K,
Tanouye MA,
and
Sigworth FJ.
The size of the gating charge in wild type and mutant Shaker potassium channels.
Science
255:
1712-1715,
1992[ISI][Medline].
24.
Seoh, SA,
Sigg D,
Papazian DM,
and
Bezanilla F.
Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel.
Neuron
16:
1159-1167,
1996[ISI][Medline].
25.
Smith-Maxwell, CJ,
Ledwell JL,
and
Aldrich RW.
Role of the S4 in cooperativity of voltage-dependent potassium channel activation.
J Gen Physiol
111:
399-420,
1998[Abstract/Free Full Text].
26.
Smith-Maxwell, CJ,
Ledwell JL,
and
Aldrich RW.
Uncharged S4 residues and cooperativity in voltage-dependent potassium channel activation.
J Gen Physiol
111:
421-439,
1998[Abstract/Free Full Text].
27.
Snyders, DJ,
Tamkun MM,
and
Bennett PB.
A rapidly activating and slowly inactivating potassium channel cloned from human heart. Functional analysis after stable mammalian cell culture expression.
J Gen Physiol
101:
513-543,
1993[Abstract].
28.
Uebele, VN,
England SK,
Chaudhary AC,
Tamkun MM,
and
Snyders DJ.
Functional differences in Kv1.5 currents expressed in mammalian cell lines are due to the presence of endogenous Kv
2.1 subunits.
J Biol Chem
271:
2406-2412,
1996[Abstract/Free Full Text].
29.
Yeola, SW,
Rich TC,
Uebele VN,
Tamkun MM,
and
Snyders DJ.
Molecular analysis of a binding site for quinidine in a human cardiac delayed rectifier K+ channel. Role of S6 in antiarrhythmic drug binding.
Circ Res
78:
1105-1114,
1996[Abstract/Free Full Text].
30.
Zhou, YY,
Jiang M,
and
Tseng GN.
Stabilization of a channel's open state by a hydrophobic residue in the sixth membrane-spanning segment (S6) of rKv1.4.
Pflügers Arch
437:
114-122,
1998[ISI][Medline].
31.
Zuhlke, RD,
Zhang HJ,
and
Joho RH.
Role of an invariant cysteine in gating and ion permeation of the voltage-sensitive K+ channel Kv2.1.
Receptors Channels
2:
237-248,
1994[ISI][Medline].
Am J Physiol Cell Physiol 282(1):C161-C171
0363-6143/02 $5.00
Copyright © 2002 the American Physiological Society