From the Department of Physiology, Botterell Hall, Queen's University, Kingston, Ontario K7L 3N6, Canada
K+ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K+ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K+ or Cs+, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K+ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs+ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at ECs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K+ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.
Key words: potassium channel; Kv1.5; gating currentK+ channels are an extremely diverse group of proteins
that are responsible for controlling excitation and membrane potential in many different cell types. Kv channels form part of a superfamily of ion channel proteins
that encode voltage-gated Na+, K+, and Ca2+ channels.
Voltage sensing in these channels is mediated by regions of repeating positively charged residues primarily
in the fourth transmembrane domain (Papazian et al.,
1991; Schoppa et al., 1992
; Perozo et al., 1994
; Goldstein, 1996
; Larsson et al., 1996
; Seoh et al., 1996
) of
each subunit. The opening and closing of voltage-gated channels is characterized by gating currents (Armstrong and Bezanilla, 1973
; Keynes and Rojas, 1974
),
predicted by Hodgkin and Huxley (1952)
, that reflect
displacement of the charged domains (Larsson et al.,
1996
; Mannuzzu et al., 1996
; Yang et al., 1996
) sensing the transmembrane electric field (Sigworth, 1993
; Goldstein, 1996
). The cloning of voltage-gated ion channels
has facilitated the study of Kv gating currents, initially
in Xenopus oocytes using high levels of channel expression and signal averaging (Bezanilla et al., 1991
; Stühmer et al., 1991
), in whole oocytes or using cut-open
oocyte methods (Taglialatela and Stefani, 1993
; Stefani et al., 1994
), or in membrane patches (McCormack et
al., 1994
), and expression in small mammalian cells
(Bouchard and Fedida, 1995
; Fedida et al., 1996
).
In almost every case, ion permeation has been abolished through the replacement of permeant ions with
nonpermeant cations like NMG (Zagotta et al., 1994b;
Fedida et al., 1996
), the use of blockers like intracellular TEA (Bezanilla et al., 1991
; Stühmer et al., 1991
) or
charybdotoxin (Schoppa et al., 1992
), or the engineering of nonconducting mutants (Perozo et al., 1993
; Stefani et al., 1994
). In the absence of permeating ions, a
fundamental feature of Kv channel gating is that the return of gating charge after channels have opened is delayed (Taglialatela and Stefani, 1993
; Stefani et al.,
1994
). On repolarization there is often a detectable rising phase of off-gating current and slow subsequent decay (Perozo et al., 1993
; Stefani et al., 1994
). Some
slowing is expected due to the relative voltage independence of the last closed-open transition (Zagotta and
Aldrich, 1990
), as shown by single channel and whole
cell studies (Zagotta and Aldrich, 1990
; Hoshi et al.,
1994
), but longer depolarizations, even those beyond the time required to move most channels into the open
state, continue to slow charge return on repolarization
(Bezanilla et al., 1994
; Zagotta et al., 1994
a; Fedida et
al., 1996
). To account for the delayed return, gating
models for K+ channels have been proposed for depolarizations to positive potentials which include additional transitions, carrying little charge, around the
open state in order to produce slowed off-gating currents on repolarization (Taglialatela and Stefani, 1993
;
Bezanilla et al., 1994
; Zagotta et al., 1994
b). It is suggested that there is a concerted rearrangement of subunits before the open state (Bezanilla et al., 1994
; McCormack et al., 1994
) and that reversal of this process
accounts for the subsequent very slow charge return from opening potentials (McCormack et al., 1994
; Stefani et al., 1994
). This observation is not universal across
other voltage-gated channels (Neely et al., 1993
; Jones
et al., 1997
), and even in K+ channels, the Shaker mutant V2 lacks such off-charge slowing at depolarizations
that move channels into the open state (Schoppa et al.,
1992
; McCormack et al., 1994
). Small depolarizations
in this channel above the activation threshold give ionic
currents with fast off-gating currents upon repolarization (Schoppa et al., 1992
). Thus, it is a particularly important issue to understand why off-gating currents are
slow in most Shaker channels, and whether K+ channel
gating functions in the same manner in the presence of permeating ions as in their absence. Here we have investigated the problem by comparing off-gating currents in the absence and presence of permeating cations. We demonstrate that when ions are allowed to
permeate through the K+ channel, Kv1.5, in a physiological manner, slowing of off-gating currents on repolarization is largely prevented.
Cells and Solutions
HEK-293 cells were transiently transfected with Kv1.5 cDNA in
pRC/CMV, using LipofectACE reagent (Canadian Life Technologies, Bramalea, Canada) in a 1:10 (wt:vol) ratio. Transfectants
were detected using the phOx system (Invitrogen Corp., San Diego, CA) as described previously (Fedida, 1997). Kv1.5 in the
plasmid expression vector, pRC/CMV was mutagenized using the
Stratagene Chameleon Kit (Stratagene Inc., La Jolla, CA) such
that tryptophan 472 was converted to phenylalanine (W472F).
This mutation is analogous to the ShH4-IR W434F (Perozo et al.,
1993
). Patch pipettes contained 140 mM N-methyl-D-glucamine
(NMG), 1 mM MgCl2, 10 mM HEPES, 10 mM EGTA, adjusted to
pH 7.2 with HCl. The bath solution contained 140 mM NMG, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM dextrose, adjusted to pH 7.4 with HCl. For Cs+ experiments, pipettes contained 130 mM CsCl, 4 mM Na2ATP, 1 mM MgCl2, 0.1 mM GTP,
5 mM HEPES, 10 mM EGTA, adjusted to pH 7.2 with CsOH. The
bath solution contained 140 mM NMG, 2.5 mM CsCl, 1 mM
CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM dextrose, adjusted to
pH 7.4 with HCl. For the nonconducting mutant, the bath and pipette solutions are as described for the Cs+ experiments. All
chemicals were from Sigma Chemical Co. (St. Louis, MO).
Electrophysiology
Current recording and data analysis were done using an Axopatch 200A amplifier and pClamp 6 software (Axon Instruments,
Foster City, CA). Patch electrodes were fabricated using thin-walled borosilicate glass (World Precision Instruments, Sarasota,
FL). After sylgarding and fire polishing, pipettes used to measure current had resistances of 1.0-2.5 M when filled with control filling solution. Mean series resistance was 3.6 ± 0.4 M
(n = 39), and cell capacitance was 19.9 ± 1.4 pF. Leakage and capacitative currents were subtracted on-line using a P/6 protocol (Zagotta et al., 1994
b). The absence of ionic current at negative
membrane potentials in HEK cells allowed faithful leak subtraction of data. An uncorrected capacity transient is illustrated in
Fig. 1 A for a 10-mV voltage step. A monoexponential fit to data
gave a decay time constant of 35 µs with a mean value of 55.0 ± 5.2 µs (n = 10). When superimposed on typical off-gating current traces in Fig. 1 B, it is obvious that the capacity transient has
settled well before the peak of off-gating currents and does not
limit the
decay measurements of off-gating currents which are
shown by the monoexponential fits to data in Fig. 1 B. These capacity measurements are in keeping with those made by others
from mouse L-cells for hKv1.5 (Snyders et al., 1993
) and our own
previous reports where the mean capacity transient decay rates
were between 46 and 62 µs (Fedida et al., 1996
; Fedida, 1997
).
During the present study we often report
decay of off-gating currents (see Figs. 3-8) where the fastest decay was 0.27 ms, but most
measurements were in the range of 0.4 ms and slower. Given the
properties of the recording system that we describe, our recording bandwidth does not appear to be a limiting factor in our measurements. Capacity compensation and leak subtraction were
routinely used, but series resistance compensation was only rarely
used. No difference between results with and without Rs compensation was observed. Data were sampled at 100-330 kHz (except
for data in Fig. 5, D and E, where sample rate was reduced to 20 kHz to allow for long recording times) and filtered at 5-10 kHz.
All experiments were performed at 22°C, and cells were superfused continually at a flow rate of 1-2 ml/min. All Qoff measurements were obtained by integrating the off-gating currents until
current waveforms decayed to the baseline. This was usually complete by 25 ms. Due to the high level of expression of Kv1.5 channels in HEK cells, there was no need for signal averaging. For a
gating charge of 1 pC, assuming each channel moved 12.3 e0
(Schoppa et al., 1992
) we calculate approximately 508,000 channels per cell, or a density of 254 channels µm
1. This is comparable with the Na channel density of squid giant axon, but much
less than that at the node of Ranvier (Hille, 1992
).
Measurement of Off-gating Currents, with and without Permeating K+
Gating currents from Shaker channels that lack N-type
inactivation are of the general form illustrated in Fig.
2 A with data obtained from Kv1.5 overexpressed in
HEK-293 cells using intracellular and extracellular NMG
to prevent ionic flux. In oocytes where permeant ions
were replaced by NMG in conducting channels, the gating current properties are similar to those described
here (Perozo et al., 1993; Stefani et al., 1994
). However,
when tetraethylammonium or Tris were used as substitute cations, there was an enhancement of gating charge
immobilization which is similar to that seen during the
onset of N-type inactivation (Bezanilla et al., 1991
; Stühmer et al., 1991
). On-gating currents appear during depolarizations positive to
70 mV and increase in amplitude with larger depolarizations, then begin to decay
more rapidly at positive potentials (Fig. 2 A). When on-gating currents are integrated, the waveforms represent the time- and voltage-dependent movement of gating
charge as channels progress towards the open state.
The time course of on-charge movement (Qon) is
shown in Fig. 2 C. For larger depolarizations, total gating charge moved upon depolarization increases more
rapidly (Fig. 2 C) and eventually saturates (Fig. 2 C and
3 D,
). When repolarized to
100 mV, off-gating currents represent the return of gating charge as channels
deactivate. In Fig. 2, A and B, these are shown as the
downward current deflections after 15-ms depolarizations to between
80 and +60 mV. In Fig. 2, D and E,
the integrated off-gating currents depict the charge return with time. For small depolarizations to <
10 mV,
off-gating currents reach a peak very rapidly and decay
rapidly and monoexponentially. After depolarizations to more positive potentials, the peak off-gating current
is reduced (Fig. 2 A and 3 A,
) and the time constant
of relaxation of off-current (
decay), while still monoexponential, slows 5-6-fold (Fig. 2 A, inset and Fig. 3 C,
). Depolarizations to +60 mV compared with
10
mV resulted in a decrease in the peak off-gating current to 40.8% of maximum (n = 5), and a slowing of
the decay time constant from 0.59 ± 0.06 to 2.24 ± 0.09 ms. A clear threshold for off-gating current slowing occurs at ~
10 mV, where the pore would normally be open for the channel to conduct ions (Fedida
et al., 1993
). The slow return of charge in this situation
is clearly seen in the integrals (Fig. 2 D), where the rise
of Qoff is progressively delayed for stronger depolarizations. This slow return of charge thus reflects delayed
return from the open state, as described in recent detailed studies on Shaker channels (Perozo et al., 1993
;
Stefani et al., 1994
; Zagotta et al., 1994
a). There is no immobilization of charge as integration of on- and off-gating currents show conservation of charge moved
and returned. It can clearly be seen that in the example
shown that Qon saturated at around 1.4 pC (Fig. 2 C)
and Qoff saturated at exactly the same charge (Fig. 2 D).
For 12-ms depolarizations from
100 to +50 mV, the
mean gating charge ratio (Qoff/Qon) was 1.0 ± 0.04 (mean ± SEM, n = 12). Integration of off-gating currents was carried out for sufficient time to allow relaxation to the zero current level (usually <25 ms). Off-gating charge showed saturation (mean Qoff = 1.4 ± 0.3 pC after depolarization to +40 mV and 1.38 ± 0.3 pC after +60 mV) and a smooth off-gating charge voltage dependence (Q-V curve, Fig. 3 B,
). These results
indicate our ability to accurately measure gating currents from Kv1.5 and that our results in the absence of
permeating ions are comparable with those of others
(Stefani et al., 1994
; Zagotta et al., 1994
a).
The measurements of gating currents in the absence
of ionic currents depend upon the dialysis of K+ from
cells on the attainment of the whole cell recording configuration. I-V voltage protocols performed immediately on patch rupture allowed measurement of gating
currents in the presence of residual cell K+, as shown
by outward ionic currents on depolarization (Fig. 2 B).
The usual problem with the measurement of ionic and
gating currents together is that extremely large ionic
K+ currents dwarf and prevent resolution of gating currents. Here, due to the dialysis of K+ from the cells,
only small outward K+ currents were present, and in
the absence of bath permeant ions, off-gating currents
are uncontaminated by ionic current deactivation (which
would be absent at 100 mV, or extremely small due to
Goldman-Hodgkin-Katz rectification). This was confirmed by integration of records from the same cell before (Fig. 2 E) and after K+ dialysis (Fig. 2 D). Qoff waveforms show a different time course but show clear identity of charge returned before and after K+ dialysis, and
exactly the same charge as that moved during depolarizations (Qon, Fig. 2 C). For the data in Fig. 2, A and B,
charge returned at
100 mV after prepulses to >10
mV was almost identical (i.e., after a prepulse to +40
mV, Qoff was 1.4 pC in both cases; after +60 mV, 1.3 pC
and 1.4 pC). The identity of charge returned during off-gating currents from the same cell in Fig. 2, A and
B, confirms that off-gating currents in Fig. 2 B accurately represent pure Kv1.5 off-gating currents, and
that no significant contamination from ionic tail currents has occurred. In the presence of K+, off-gating
currents showed little decrease in peak or slowing of
decay. The essential finding is illustrated by the arrow
(Fig. 2 B) showing rapid relaxation of off-gating current to the zero line. Here, no slow component of gating current is present, unlike off-gating currents recorded in the absence of permeant K+ (Fig. 2 A).
There is no large decrease in peak off-gating current after prepulses to positive potentials (mean data,
; n = 5 in Fig. 3 A), and much less slowing of
decay (Fig. 3 C,
;
= 0.53 ± 0.1 ms at
10 mV and 0.98 ± 0.1 ms at
+60 mV) which are still monoexponential (Fig. 2 B, inset). We believe that the lack of residual inward gating
current (indicated by the arrow) in Fig. 2 B must indicate that at
100 mV the gating current has decayed more rapidly and completely than off-gating currents
in Fig. 2 A. Any ionic tail, even if it was present, would
have deactivated by this time given ionic tail decay time
constants of ~1 ms at this potential. Similarly, if ionic
currents were contributing significantly to the off-gating currents, there should be a shift in the charge relation (Qoff, Fig. 3 B,
) to the right along the voltage
axis reflecting a contribution from ion flux activating at
more positive potentials than movement of gating
charge (Fig. 3 D,
). This does not occur as shown by
the overlay of the symbols in Fig. 3 B (
,
), and the
smooth Qoff relation in the presence of permeant K+
(Fig. 3 B,
) which does not show any change of slope
or discontinuity positive to
20 mV (where conductance activates, Fig. 3 D), is further evidence for the
lack of contamination of off-gating currents in Fig. 2 B
by ionic current tails at potentials at which channels open.
Off-gating Currents in the Presence of Permeant Cs+ Decay Rapidly
We suggest that both ionic and gating currents can be
measured together from the same cell and propose
that permeant K+ in some way speeds the return of gating charge on repolarization. To reproduce a low ionic
permeability which allowed simultaneous measurements
of off-gating currents, we replaced NMG in the pipette solution with Cs+ and added low [Cs+] to the bath to
make continuous measurements of gating current in
the presence of permeating Cs+ without the possibility
of any transient effects during cell dialysis. Cs+ has a
low permeability through K+ channels, so that it is usually thought of as a channel blocker (Hille, 1992). However, at high levels of K+ channel expression, and with
a permeability ratio of 0.11 ± 0.01 obtained from biionic reversal potential measurements (Zhang and Fedida, unpublished data), Cs+ currents are recordable
(De Biasi et al., 1993
) at the same time as on- and off-gating currents (see Figs. 4, 5, and 7).
In an extension of our K+ data, in the presence of
permeating Cs+, little slowing of decay of the off-gating
current was observed on repolarization to 100 mV
(Fig. 4 A, arrow), with a mean change in
decay of the off-gating current from 0.48 ± 0.06 at
10 mV to 0.65 ± 0.06 ms at +60 mV (Fig. 4 B,
). These may be compared with the
decay of off-gating currents in the absence of permeating ions (Fig. 4 B,
). The off-gating
currents in the inset to Fig. 4 A illustrate such monoexponential fits to off-gating current decay in the presence of Cs+, at +10 and +60 mV. Clearly, little off-gating
current slowing has occurred over this potential range
(compare with Fig. 2 A, inset). A dramatic demonstration of the lack of slowing of off-gating current in the
presence of permeant ions is provided by calculating the development of Qoff with time of repolarization.
These data are shown in Fig. 4 C and can be compared
with data obtained with only NMG present (Fig. 4 D).
Off-gating transients rise smoothly and rapidly to a
peak when Cs+ is present (C), but for depolarizations
positive to
10 mV in the absence of ions (D), the rise
time is increasingly slowed.
Validation of Off-gating Currents in the Presence of Permeant Cs+
To validate the measurement of off-gating current in
the presence of permeating Cs+, we used a double pulse
voltage protocol, which allowed comparison of inward
currents at 100 mV before and after ionic current deactivation. Results from the protocol are illustrated in
Fig. 5 A. Here, the calculated ECs was
100 mV (2.5 mM [Cs+]o). Cells were first pulsed to +40 mV to move
channels into the open state, and then repolarized to a
range of potentials between +10 and
100 mV. Outward ionic current tails were visible, at repolarization
potentials from +10 to
20 mV, but then all tails were
inwardly directed (although still 80 mV positive to ECs) and reflected off-gating current as the predominant
component of the observed current. At potentials positive to ECs (between
30 and
100 mV) ionic current
tails should be outwardly decaying, so the inward current tails must represent the dominance of rapidly decaying off-gating current
the question is, how much
are the amplitude and time course of inward off-gating
currents distorted by ionic tail currents? At ECs itself
(
100 mV in our experiments), the tail should represent only off-gating currents as the ionic flux should be
at equilibrium. From the repolarization data in Fig. 5 A
we can calculate the magnitude of ionic tail currents at
the different repolarizing potentials. The slope conductance of the channel conducting Cs+ can be calculated
from the peak current at +40 mV, or instantaneous and steady tail amplitudes at +10 or 0 mV, given the G-V
curve in Fig. 5 D. The mean value for GCs from the cell
in Fig. 5 A was 2.8 ± 0.3 nS, which means that the peak
outward tail current amplitude during repolarization
to
80 mV would be 56 pA, out of a peak tail amplitude of 343 pA (16%). As ECs is approached, for pulses
to
90 or
100 mV, any ionic component to the peak tail current measured diminished to 7% and zero, respectively. This calculation suggests that close to the reversal potential, the large gating currents relative to
Cs+ conductance allow measurement of a peak value
for off-gating current contaminated by less than 10% of
ionic tail current. The data also indicate that accumulation of Cs+ close to the external membrane surface during outward currents is unlikely to affect ECs enough to
give rise to significant ionic tail currents. The clear monoexponential nature of the decay of off-gating currents
observed at ECs (Fig. 5 A, inset) supports this conclusion
that off-gating currents are uncontaminated by a component of ionic current decay.
After 10 ms of the first repolarizing pulse, tail decay
reached a steady state, and the membrane was subsequently pulsed to ECs (100 mV). Here, at ECs no ionic
current should be present, and tails should represent
pure off-gating current which show rapid decay to the
baseline (Fig. 5 A). We have compared off-current amplitude (Igoff), charge (Qoff), and the
decay of the tails at
ECs before and after repolarization to +10, and
20
mV (Fig. 5 A, inset and C). The data displayed in the bar
graph (except
decay) have been normalized to the values obtained from the initial tail at
100 mV in Fig. 5
A. After a prepulse to +10 mV, on return to
100 mV,
there was no change in peak off-gating current, and less than a 10% reduction in charge returned, compared with no prepulse (Fig. 5 C, solid bars). From the
G-V and Q-V relations (Fig. 5 D), there would be a predicted ~45% decrease in the current if it was predominantly an ionic tail (Fig. 5 C), but only a predicted 12%
reduction of charge returned if it represented off-gating current. The data are thus much more consistent with
the tail comprising off-gating rather than ionic current.
During the prepulse to
20 mV, the G-V relation in
Fig. 5 D (
) predicts that complete ionic current deactivation should occur, but from the Q-V relation (
)
only 60% of the gating charge should have returned.
The normalized tail amplitude was still 50% of that
without the
20 mV prepulse (Fig. 5 C), and the normalized charge in the tail after the
20-mV prepulse
(Fig. 5 C, empty bars) was 37% (mean, n = 3) of the total charge moved on depolarization. These values were almost exactly what was expected if the tail comprised
off-gating current rather than ionic current.
It appears then, that the peak off-gating current and
charge content of inward currents at the ionic reversal
potential will not be much affected by permeating Cs+.
A second issue is whether the time course of decay of
the off-gating current will be affected by a decaying inward or outward ionic tail current close to ECs. Representative ionic tail currents for Kv1.5 in K+-containing
pipette and extracellular solutions are as shown in Fig.
5 B for repolarization potentials between 40 and
110
mV (EK in this case was
70 mV). The time constants of
decay (shown by the monoexponential fits) decreased
from 1.48 ms at
40 mV to 1.07 ms at
100 mV. Values
of ~1 ms for the ionic tail decay around
100 mV are
~2× slower than measured gating current
decay (Fig. 5
C, Fig. 4 B). Using the double pulse protocol illustrated in Fig. 5 A, tail decay at
100 mV accelerated from
0.52 ± 0.04 ms (repolarization from +40 mV) to 0.37 ±
0.03 ms (repolarization from
20 mV after complete
ionic current deactivation). Data obtained over the entire depolarization voltage range (Fig. 4 B,
) suggested a mild voltage dependence to off-gating current
decay,
in the presence of permeating ions. We believe that this
reflects the minor voltage dependence to the last
closed-open transition that has been described before
(Zagotta and Aldrich, 1990
). The measurements of
decay
support the idea that off-gating currents at
100 mV measured in the presence of permeating Cs+ are free
of contamination by ionic tail currents. In any case, the
unequivocal action of the presence of permeant ions is
to cause an acceleration of the return of off-gating current and the disappearance of the very slowly decaying
off-gating currents on repolarization from positive potentials (Figs. 2, 4, and 5). This slow decay phase takes
10-15 ms to reach completion and, therefore, is too
slow to be concealed by overlap from rapidly decaying
ionic tail currents.
Time-dependent Slowing of Off-gating Currents Is Prevented by the Presence of Permeant Ions
An important feature of off-gating currents in the
Shaker H4-IR construct (Bezanilla et al., 1994) measured in the absence of permeant ions is a slowed decay
and decreased peak current with longer prior depolarizations, up to about 8 ms. This property is also shown
by Kv1.5 and is illustrated in Fig. 6 A with data from an
envelope test of on- and off-gating currents. From
100 mV, cells were depolarized to +60 mV for varying
lengths of time. The first pulse was 0.25 ms long, and
each subsequent pulse was incremented by 0.50 ms. A
cumulative waveform of on-gating current was built up
which provided a control for cell stability and the constant availability of on-gating current. Off-gating current transients after brief depolarizations rose rapidly
to a peak and initially decayed quickly. After ~2 ms depolarizations, off-gating transients were smaller and
slower as summarized by the graph of
decay against
pulse duration (Fig. 6 B,
). This represents the time
dependence of the slowing of off-gating current described earlier (Fig. 2 A) and known to occur in various
K+ channels after large depolarizations (Stefani et al.,
1994
; Fedida et al., 1996
). The slowing shows a wide
voltage dependence, becoming apparent for depolarizations to potentials greater than those required to
open channels and persisting up to large depolarizations (Fedida et al., 1996
). Despite the slowing, charge
was conserved for short depolarizations allowing about
25 ms to integrate returning charge as reported previously (Bezanilla et al., 1994
; Fedida et al., 1996
). However, for longer depolarizations, up to 358 ms (Fig. 6 C)
the off-gating transients became progressively smaller, and the off-gating current immobilized such that off-gating charge was not conserved when integrated over
20 ms (Fig. 6 D,
). Most of the changes described
above in the off-gating current and charge were prevented when Cs+ was present in the pipette and bath.
There was little decrease in the peak off-gating current
for depolarizations of up to 8 ms duration (Fig. 7 A)
and little slowing of
decay (Fig. 7 B,
), although there
was some slowing at very long pulse durations (Fig. 7
C). When integrated, mean data showed that off-gating charge was extremely well conserved when Cs+ was
present as a permeant ion (Fig. 7 D,
).
Off-gating Current Slowing in a Nonconducting Mutant of Kv1.5
The decreased peak and slowing of off-gating currents
with depolarization, and the progressive slowing of gating charge return are not reproduced when permeant
cations such as K+ and Cs+ are present in the pipette
filling solution and/or bath. Permeant ions passing
through or within the pore of the K+ channels appear
to facilitate a more rapid off-gating of the channel. We
have tested this requirement for permeating ions with a
nonconducting mutant of Kv1.5. The mutation W472F,
analogous to the ShH4-IR W434F (Perozo et al., 1993),
when incorporated into Kv1.5, prevented measurable
ion conduction when channels were transiently expressed in HEK cells, despite high transfection levels
determined by adherence of multiple antigen coated
beads. In this situation we measured only gating currents from Kv1.5 W472F channels using Cs+ in the pipette and Cs+ plus NMG in the bath solution (Fig. 8).
However, gating currents were similar to those observed with NMG present in bath and pipette filling solutions (Fig. 2 A), and quite unlike those recorded from conducting channels in the presence of Cs+ or K+
(Figs. 4, 5, and 2 B). During depolarizations, on-gating
currents appeared identical to those observed before
(Fig. 2), and off-gating currents on repolarization were
slow to peak and to decay for depolarizations positive
to
10 mV (Fig. 8 A, inset and C). Despite the slowing,
the Q-V relation appeared unchanged from that observed in the wild-type channel with a V0.5 of about
19
mV and z = 2.5 (Fig. 8 B). The slowing of off-gating
currents, even in the presence of Cs+ was seen more
dramatically with the envelope test protocol (Fig. 8 D).
Here, increased duration of depolarization did not decrease the amount of charge returned (Fig. 8 E) but reduced the peak and slowed off-gating current decay
(Fig. 8 F ). These data are very similar to those observed
by others (Bezanilla et al., 1994
) for nonconducting
Shaker mutants but fundamentally different from data
obtained when permeant ions are present within the
pore of the channel (Figs. 2, 4, 5, and 7).
The data indicate a central role for the K+ channel pore in the physiological gating properties of K+ channels as they close. These direct measurements of K+ channel gating currents along with ionic currents suggest that permeating K+ or Cs+ can accelerate the return of gating charge on repolarization.
Measurement of Off-gating Currents in the Presence of K+ or Cs+
The major methodological hurdle in this study was to
make measurements of off-gating currents without contamination from deactivating ionic tail currents. This
goal was realized in two ways. In the first, simultaneous
measurements of off-gating currents before and after
K+ dialysis allowed a direct comparison of charge return in the same cells (Figs. 2 and 3). The data showed
no extra charge return (and Qoff/Qon remained at 1.0)
or change in voltage dependence when permeating K+
was present. This experiment virtually excluded a contribution of ionic K+ tail currents to the charge return,
and the data clearly demonstrated a rapid charge return in the presence of intracellular K+. The second
method was to utilize Cs+, an ion of low permeability,
to measure off-gating currents in a controlled manner
on repolarization to ECs to reduce contamination of off-gating charge by ionic charge at measurement potentials (Figs. 4 and 5). It was clearly demonstrated that on
repolarization after a depolarizing prepulse, off-gating
currents predominated over deactivating Cs+ ionic tail
currents, at repolarizing potentials as positive as 30
mV (Fig. 5 A). As repolarizing potentials were made
more negative, reducing Cs+ driving force nearer ECs,
we calculated that off-currents were contaminated by
<7% ionic current at ±10 mV either side of the test
voltage of
100 mV (ECs, where no contamination was
expected). We believe that this gave us sufficient margin for safety, and on repolarization to ECs, the amplitudes of off-gating currents and charge returned were
entirely consistent with gating current rather than ionic
current tail decay, from a comparison of tail amplitudes and consideration of the steady-state Q-V and G-V
curves (Fig. 5, C and D). Consistently, in the presence of
Cs+ (Fig. 4) the
decay of off-gating currents at
100 mV
remained fast, despite time constants for decay of ionic
tail currents that were ~2× slower (Fig. 5 B). Our data
suggest that when permeating ions are present at physiological concentrations within a conducting pore, off-gating currents maintain peak values and decay rapidly, as indicated by the arrows in Fig. 2 B and Fig. 4 A. Below we consider mechanisms for this acceleration of
charge return compared with data obtained in the absence of permeating ions.
Acceleration of a Rate-limiting Step Near the Open State
Two other interventions accelerate off-gating currents;
4-aminopyridine (McCormack et al., 1994; Bouchard
and Fedida, 1995
) and Ba2+ (Hurst et al., 1996
) by different mechanisms in each case. 4-aminopyridine prevents slowing (and blocks the channel) by preventing a
final transition to channel opening (McCormack et al.,
1994
), whereas it is suggested that Ba2+ acts by destabilizing the open state (Hurst et al., 1997
). These data
have been interpreted in terms of existing models of
Shaker K+ channel gating (Bezanilla et al., 1994
; Zagotta et al., 1994
a) and allosterism (McCormack et al.,
1994
). In all cases it is a rate-limiting concerted transition carrying little charge, near the open state that is affected (Bezanilla et al., 1994
; Zagotta et al., 1994
a).
Here we can explain our observations if the rate of this
transition is different in the absence or presence of K+
or Cs+. This is depicted by the left transitions of the
model in Fig. 9. For simplicity, the closed transitions
have been grouped into one closed state (C). The channel can exist in an open state without ions (O), or with
ions permeating through the channel (OK). Any model
will predict fast on-gating and slowed off-gating currents with a rate limiting slow transition, close to the
open state that carries little charge. After voltage-dependent conformational movement of the charged domains, in the absence of permeating cations, the final
transition to the open state carries little charge, but is
slow in both directions (C
O, filled arrow in Fig. 9).
This provides a rate-limiting step on deactivation that visibly slows returning charge (dashed box). Accelerated
return of off-gating charge requires a speeding of this
returning transition. In the presence of physiological
concentrations of intracellular and extracellular ions,
our data suggest that the final transition is fast, and
thus no longer rate-limiting (OK
C, open arrow). In
the absence of permeant ions (Fig. 2) or when a nonconducting mutant was used, which may prevent free
movement of permeating ions within the pore (Fig. 8),
return of gating charge followed a fundamentally different and slower time course than activation, as has
been observed previously (Stefani et al., 1994
).
In the absence of permeant ions, the activation energy barrier that the channel has to overcome to deactivate is greater. We calculate (from data in Fig. 4, C and
D) at +60 mV, a difference in the activation energies of
~1.2 kcal/mol in the absence and presence of permeant ions, respectively. This difference is small but facilitates rapid return of charge when permeant ions are present (Fig. 9, solid box). The relative independence of
rate of charge return on pulse duration or amplitude
that we have observed can then support gating models
which utilize independence of subunit movement during activation (and deactivation) (Zagotta et al., 1994a).
Such an allosteric role for permeating ions in K+ channel gating may then be of fundamental importance in
the maintenance of faster cycling as the channel moves
from depolarization to repolarization, particularly for
channels like Kv1.5 from excitable tissues (heart, brain)
where repetitive activation and deactivation is the norm.
Role of Permeating Ions as Gating Modifiers
Permeating ions within ion channel pores are increasingly thought to be involved as modulators of gating.
Permeating ions can influence the direction of a cyclical gating reaction in NMDA channels (Schneggenburger and Ascher, 1997). In Shaker channels, high external [K+] (in some channels; Matteson and Swenson,
1986
), and [Cs+] or [Rb+] (in most channels; Zagotta
et al., 1994
b; Clay, 1996
) slow deactivating ionic tail currents in a "foot-in-the-door" manner (Matteson and Swenson, 1986
; Demo and Yellen, 1992
) and slows the
return of gating charge (Loboda and Armstrong, 1997
).
Inactivation can also be modified by the presence of
small cations and recent studies have shown that the
rate of C-type inactivation is modulated by different
[K+] at the outer pore mouth (Baukrowitz and Yellen,
1995
, 1996
). In the extreme case, in the absence of external K+ or in the nonconducting Shaker mutant
W434F, it has been suggested that the channel exists in
a permanently C-type inactivated state (Yan et al.,
1996
). These studies all describe situations where the
channel conformational changes of gating can be seen
to be coupled to ion concentrations within the vicinity
of the pore. Here we have shown that the rate of return
of charge after channel opening depends on the presence of permeating ions at physiological concentrations within the pore.
A provocative suggestion from a consideration of the
above literature is that our data reflect a prevention of
C-type inactivation by small cations somewhere in the
permeation pathway. This would provide an explanation of our results that fits neatly with data on the rate
of C-type inactivation being dependent on the cation occupancy of a site in the external mouth of the pore,
when there is an acceleration in the rate of C-type inactivation with a decrease in K+ flux (Baukrowitz and
Yellen, 1995, 1996
). Furthermore, it has been shown that
the time course of C-type inactivation is slowed by increasing external K+, Rb+, and Cs+ (Lopez-Barneo et
al., 1993
) through a foot-in-the-door manner which is
dependent on the residue at site 449 in Shaker channels lacking N-type inactivation. In the model in Fig. 9,
since C-type inactivation depends on the occupancy of
cations in the external mouth of the pore, a single inactivated state (I) is shown as coupled directly to the
open state. We have ignored the possibilities of closed-state inactivation (Marom and Levitan, 1994
) and an inactivated state which has K+ bound to explain faster
recovery from inactivation by elevated [K+o] (Levy and
Deutsch, 1996
). In the case where there is NMG alone, upon activation, the channel would follow the upper
pathway (dashed box) but with a fast C
O transition.
Since there would not be any ions occupying the binding sites within the pore, the O
I transition would be
rapid and preferred. Upon repolarization, the I
O
transition would be the slow, rate-limiting step which would explain the slow return of gating charge. The
O
C transition would remain fast and be rate-limited
by the speed of the I
O transition.
When pulses of increasing duration were given (Fig.
6 A), the off-gating currents that resulted initially decayed rapidly (Fedida et al., 1996), but after depolarizations longer than 1-2 ms they began to slow and then
became increasingly slower (Zagotta et al., 1994
a; Fedida et al., 1996
). These effects were prevented by the
presence of K+ (data not shown) or Cs+ (Fig. 7 A).
With permeating K+ or Cs+, there would be an extra
transition which is fast (O
OK, open arrow) that channels prefer to the inactivated state (due to the lower activation energy barrier). The majority of channels would then prefer the sequence outlined in the solid box
which would not result in the conformational changes
at the outer mouth of the channel associated with inactivation (Liu et al., 1996
). Return of gating charge would
remain fast because the OK
C or OK
O
C pathways avoid the rate-limiting I
O step. In the definitive case, inactivation should lead to charge immobilization
similar to N-type inactivation in Shaker channels (Stühmer et al., 1991
; Perozo et al., 1992
). However, charge
return was well-conserved both when return was slow in
NMG alone and when return was rapid in the presence
of K+ or Cs+. There was also no change in Q-V relations
nor the Qoff/Qon ratios (Figs. 2 and 3). A more subtle
initial shift in the Q-V curve to more negative potentials
without reduction in the total charge moved has been
suggested to result from C-type inactivation (Olcese et
al., 1994
). This inactivation becomes more obvious
when the preparation is held for several seconds, and
there is a reduction in charge. In Figs. 6 and 7 we
showed that pulses of increasingly longer duration (up
to 400 ms) did lead to charge immobilization that was
also prevented, or slowed considerably, by permeating cations, so it is possible that the very early slowing of
off-gating charge represents the very earliest steps of inactivation, one end of a continuum that will eventually
lead to measurable charge immobilization. This also
provides a possible explanation of our data from the
nonconducting mutant, W472F. If this channel predominantly exists in a C-type inactivated state, as suggested previously (Yan et al., 1996
), then permeating
cations may not be able to access the pore quickly or
deeply enough to prevent off-gating current slowing.
The channel would show rapid C-type inactivation which would bypass the O
OK transition and follow
the sequence outlined by the dashed box. In the same
way as in NMG solutions alone, the I
O transition
would be the rate-limiting step and return of gating
charge would be slow.
A Site for the Action of Cations
Although it is possible that the K+ or Cs+ binds to an intracellular site outside the pore itself to produce the effects on off-gating currents, data from the nonconducting mutant with high intracellular Cs+ (Fig. 8) do not
show the same acceleration in the return of gating charge seen in conducting Kv1.5 channels with identical Cs+ concentrations (Figs. 5 and 7). This result suggests that the single W472F mutation in the nonconducting mutant might disrupt a binding site for a permeating monovalent cation within the pore of the
channel or prevent access to such a site. A putative
structural explanation would involve sites near the
equivalent of the Shaker 449 site. In addition to its involvement in C-type inactivation (Lopez-Barneo et al.,
1993) and TEA block (MacKinnon and Yellen, 1990
),
this site has been suggested to be an external K+ binding site in DRK1 (Krovetz et al., 1997
). Ba2+ block in
Shaker channels also speeds the return of off-gating charge and an adjacent residue, D447, has been shown
to contribute to Ba2+ binding while T449 is a barrier to
the site from the outside of the pore (Hurst et al.,
1996
). Hurst et al. also found that a D447N substitution
is nonconducting. Residues near the 434 equivalent site in Kv2.1 have recently been shown to affect C-type
inactivation and TEA sensitivity (Kirsch and Shieh,
1997
). It is therefore possible that the 434 and 449 sites
are in close physical proximity in the external mouth of
the pore and together affect TEA binding, C-type inactivation, and are responsible for producing nonconducting mutants. In addition, they may cooperate to
confer a cation binding site which is also affected by
both Cs+ and Ba2+. When there are no permeating ions
(or ions at the binding site), return of gating charge
will be slowed after short depolarizations. However,
having K+, Cs+, or Ba2+ at the binding site prevents off-gating current slowing, either via allosteric actions on
channel closing or via a prevention of C-type inactivation (or, of course, a combination of both). In a situation where the binding site is disrupted by a mutation in the 434 residue, it is possible that K+ cannot pass
through the pore and C-type inactivation is strongly accelerated, causing a slow return of gating charge, which
cannot be prevented by Cs+ (Fig. 8). However, these
suggestions are speculative and in the present study we
cannot distinguish whether the rapid return of gating
charge is due to a slow step near the open state which is
facilitated by permeant ions or due to a rate-limiting
step resulting from C-type inactivation which is prevented (or slowed considerably) by permeant ions.
Original version received 12 March 1997 and accepted version received 9 May 1997.
Address correspondence to Dr. David Fedida, Department of Physiology, Botterell Hall, Queen's University, Kingston, Ontario K7L 3N6, Canada. Fax: 613-545-6880; E-mail: fedidad{at}post.queensu.ca
A preliminary report of this work has appeared in abstract form (Chen, F.S.P., and D. Fedida. 1997. Biophys. J. 72:A27).Supported by grants from the Heart and Stroke Foundation of Ontario and the Medical Research Council of Canada to D. Fedida.