From the Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6602
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ABSTRACT |
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Human ether-à-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 ± 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.
Key words: human ether-à-go-go-related gene; potassium ion channel; gating kinetics; membrane surface charge; divalent cation ![]() |
INTRODUCTION |
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Human ether-à-go-go-related gene (HERG)1 encoded K+
channel isoforms exist in a number of cell types including neurons, glia, cardiac myocytes, and tumor cells
(Warmke and Ganetzky, 1994; Trudeau et al., 1995
;
Faravelli et al., 1996
; Arcangeli et al., 1997
; Shi et al.,
1997
; Bianchi et al., 1998
). HERG channels may be involved in neuronal spike frequency adaptation and tumor cell growth (Warmke and Ganetzky, 1994
; Chiesa
et al., 1997
; Bianchi et al., 1998
). In the heart, HERG
K+ channel subunits mediate a delayed rectifier K+ current (IKr) that aids cellular repolarization (Sanguinetti and Jurkiewicz, 1991
; Sanguinetti et al., 1995
). Mutations of the HERG gene or drugs that suppress cardiac
IKr cause the congenital and acquired forms of human
long QT syndrome, respectively (Sanguinetti et al.,
1995
; Sanguinetti et al., 1996
; Spector et al., 1996a
).
Despite homology to the Shaker family of voltage
gated K+ channels, HERG K+ channels exhibit distinctive gating behavior. Voltage-dependent channel activation is much slower than channel inactivation, giving rise to the characteristic rectification-like behavior of
this channel (Trudeau et al., 1995; Smith et al., 1996
;
Spector et al., 1996b
). There is also evidence that
HERG channel inactivation has intrinsic voltage dependence (Wang et al., 1996
, 1997), unlike N- and C-type
inactivation in the Shaker family K+ channels (Hoshi et
al., 1991
; Zagotta and Aldrich, 1990
).
Divalent cations have often been used to probe ion
channel function, and Ca2+ has been implicated as a
modulator of K+ channel gating (Frankenhaeuser and
Hodgkin, 1957; Gilly and Armstrong, 1982
; Armstrong
and Matteson, 1986
; Armstrong and López-Barneo, 1987
; Armstrong and Miller, 1990
; Mazzanti and DeFelice, 1990
; Clay, 1995
). Interaction between Ca2+ and a
distinct region of the channel protein could cause a
specific effect on its function, or Ca2+ could nonspecifically associate with diffuse charged moieties on the
membrane surface, effectively neutralizing charges and leading to a global change in the transmembrane potential perceived by all membrane molecules. Both
mechanisms of modulation have been proposed to explain the effects of divalent cations on ion channels
(Frankenhaeuser and Hodgkin, 1957
; Gilly and Armstrong, 1982
; Green and Andersen, 1991
).
In the squid axon, elevation of extracellular Ca2+
leads to a decrease in the rate of K+ channel activation
(Gilly and Armstrong, 1982; Armstrong and Matteson,
1986
; Armstrong and López-Barneo, 1987
). Complete
removal of extracellular divalent cations and K+ causes
loss of selectivity and gating in the squid axon K+ channel, resulting in channels that are constitutively open. Recombinant K+ channels from Drosophila melanogaster,
including Shaker and EAG, are affected by divalent cations in much the same way as the squid K+ channel
(Armstrong and Miller, 1990
; Spires and Begenisich,
1994
; Ludwig et al., 1994
).
Unlike invertebrate K+ channels, guinea pig ventricular IKr channels appear to retain gating and selectivity
in the absence of extracellular Ca2+ and K+ (Sanguinetti and Jurkiewicz, 1990, 1992
). However, Ca2+
and Mg2+ have been suggested to serve critical roles in
the gating of K+ channels from both rabbit sinoatrial
node and neuronal cell lines, and these channels are
functionally similar to HERG K+ channels (Faravelli et al.,
1996
; Ho et al., 1996
). To gain further insight into the
gating behavior of HERG K+ channels, we have examined the effect of external Ca2+ on HERG channels under conditions in which the extracellular K+ concentration was in the normal physiological range (4 mM).
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MATERIALS AND METHODS |
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Plasmid cDNA Constructs
The HERG cDNA was obtained from Dr. Mark Keating (University of Utah, Salt Lake City, UT). HERG was ligated into the pSI mammalian expression plasmid (Promega Corp.). The CD8 antigen gene in the EBO-pcD Leu2 vector was kindly provided by Dr. Richard Horn (Thomas Jefferson Univ. Medical College, Philadelphia, PA). CD8 is a human T-lymphocyte surface antigen and was used to visually identify transfected cells using CD8 antibody-coated polystyrene microbeads.
Cells
Chinese hamster ovary K1 (CHO-K1) cells were obtained from the American Type Culture Collection and maintained in HAMS F-12 media (Gibco Laboratories) supplemented with 1 mM L-glutamine, and 10% heat-inactivated fetal bovine serum (Gibco Laboratories) in a humidified, 5% CO2 incubator at 37°C.
Transfection
CHO-K1 cells were cotransfected with the HERG and CD8 plasmids in a ratio of 4:1. Transfection was accomplished using the Lipofectamine transfection reagents and method (Gibco Laboratories). Immediately before patch clamping, cells were labeled
with commercially prepared microbeads conjugated to CD8 antibodies (DynaBeads; Dynal) to identify transfected cells. Cells that
displayed CD8 on their surface bound DynaBeads, indicating
successful transfection (Jurman et al., 1994).
Solutions
The intracellular recording solution for all experiments was
(mM): 110 KCl, 5 K2ATP, 5 K4BAPTA, 2 MgCl2, 10 HEPES, pH
7.2. The control extracellular recording solution was (mM): 145 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.35. Solutions with elevated divalent cation concentrations were
made by adding an appropriate amount of 1 M aqueous chloride
salt solution to the control extracellular solution. The 2 mM
EGTA extracellular solutions in Fig. 4 A was the same as control
except that CaCl2 and MgCl2 were omitted and 2 mM EGTA was
added. For the Ca2+ dose response in Fig. 4 B, contaminant Ca2+
was assumed to be 25 µM. MgCl2 was omitted and, for Ca2+ concentrations 100 µM, the appropriate concentration of Ca2+ was
added to the solution without a Ca2+ buffering agent. At concentrations <100 µM, 1 mM EGTA was used to buffer the Ca2+. The
amounts of Ca2+ and chelator needed to obtain a desired concentration of free Ca2+ were determined using Sliders MaxChelator software with the BERS.CCM constants (Chris Patton, Stanford University, Stanford, CA).
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Electrophysiology
The whole-cell patch clamp technique was used to assay HERG
channel function (Hamill et al., 1981). Cells were patch clamped 36-60 h after transfection at a temperature of 20-25°C. HERG
currents in transiently transfected CHO-K1 cells are very stable.
We observe no rundown during whole cell voltage clamp over
the lifetime of the cell, which can be well over 1 h. All recordings
were made using an Axopatch 200A patch clamp amplifier in
conjunction with a Digidata 1200 interface (Axon Instruments).
Patch pipettes were fabricated from capillary glass (1.1 mm outside diameter, #3-00-2-3-G/X; Drummond Scientific Co.; or 1.2 mM outside diameter starbore, #001812; Radnotti) using a Flaming/Brown micropipette puller (P-97; Sutter Instruments, Co.).
Patch pipette resistances were 1-2 M
. Cell and pipette capacitances were nulled and series resistance was compensated (85-
95%) before recording. Data were acquired using pCLAMP programs (6.03; Axon Instruments). Data were analyzed and plotted
using a combination of pCLAMP, Origin (Microcal Software), and SigmaPlot 4.0 (Jandel Scientific). In all figures featuring raw
current recordings, the bottom of the current scale bar indicates the zero current level.
A HERG Model with Voltage-dependent Activation and Inactivation
We adopted a simple multistate Markov kinetic model from the
literature (Scheme I; Wang et al., 1997). Our purpose was not to
uniquely identify a model of HERG K+ currents, but to test
whether a simple voltage shift in one or more parameters of the
published model could account for the data. Multistate Markov
gating models have been successfully used for modeling voltage-gated ion channels and interpreting experimental observations
(Hille, 1992; Sigworth, 1994
; Zagotta et al., 1994
).
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In the model, only the O state conducts ionic current. C states
are closed states primarily occupied at negative membrane potentials. The I state is a closed state primarily occupied during membrane depolarization. The transitions along the activation pathway (see Terminology) are governed by voltage-dependent rate constants (a1, a1, a3, and a
3) and voltage-independent rate constants (a2 and a
2). Transitions to and from the inactivated states
are governed by two additional voltage-dependent rate constants
(a4 and a
4) (as used by Wang et al., 1997). The rate constants
were limited to a maximum value at extreme membrane potentials by applying the following: an = [(1/an) + (1/amax)]
1, where
amax (s
1) is the limiting value of the rate constant (a1max = 100, a3max = 25, and a4max = 400). The simulations were carried out
using ModelMaker (Cherwell Scientific). The rate constants for
the forward and reverse transitions were calculated according to
Eqs. 1 and 2 (Stevens, 1984
; Patlak, 1991
).
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(1) |
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(2) |
where an and an represent the forward and reverse transition
rate constants between the nth state and the n + 1 state in units
of s
1, kB = 1.3807 × 10
23 V C K
1 (Boltzmann constant), T = 293.15°K, e = 1.6022 × 10
19 C (electronic charge in Coulomb).
Each rate constant is defined by two terms. One term is the rate
constant in the absence of a membrane potential, [an(0) and
a
n(0)], and the other term is membrane potential dependent
[z
eV/(kBT)]. The effective charge on the "gate" is z, the fraction
of the electrical field sensed is
, and the membrane potential is
V. The same equations with changes in an(0) and z were used for
the other rate constants. The parameters for each of the transition
rate constant pairs were as follows: a1, a
1:
1 = 0.40, z1 = 1.80, a1(0) = 6.963 s
1, a
1(0) = 11.481 s
1; a2, a
2:
2 = 0.00, z2 = 0.00, a2(0) = 8.172 s
1, a
2(0) = 42.977 s
1; a3, a
3:
3 = 0.40, z3 = 1.98, a3(0) = 11.481 s
1, a
3(0) = 0.094 s
1; a4, a
4:
4 = 0.5, z4 = 0.82, a4(0) = 116.824 s
1, a
4(0) = 14.023 s
1; V = Vclamp
Vshift.
Vclamp is the voltage clamp potential applied. Vshift is the apparent change in membrane potential caused by Ca2+ binding and
was set to zero except to simulate elevation of external Ca2+. We
found that applying the same Vshift to all rate constants could not
simulate the observed data. A shift in a4 and a4 also altered simulated inactivation, inconsistent with our data. Therefore when
simulating elevated Ca2+ (10 mM) Vshift for a1, a
1, a3, and a
3 was
set to +23 mV so that only the voltage dependence of the activation rate constants were affected, and Vshift for a4 and a
4 was set to 0 mV. Rate constants a2 and a
2 are voltage independent.
HERG K+ currents measured with the voltage clamp protocols
described in the RESULTS were used for adjusting the model parameters to approximate the data obtained in 1.8 mM Ca2+. The
model K+ currents were scaled to fit the data sets using a linear
open channel current-voltage relationship and a K+ channel reversal potential of 90 mV to correspond with the experimentally determined value. The HERG open channel current-voltage relationship has been described as linear in several studies
(Sanguinetti et al., 1995
; Smith et al., 1996
).
Terminology
The simple model of HERG gating described above can be further simplified to facilitate the discussion of the experimental current measurements. There are three main classes of kinetic states needed for the discussion: closed, open, and inactivated states (Scheme II).
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C (closed) and I (inactivated) are both nonconducting states. Only the O (open) state conducts ionic current. Channels are primarily in closed states at negative voltages. Membrane depolarization predisposes the channels to move toward the open and inactivated states. Repolarization allows channels to return to the closed state. It is important to note that in this model, as in the more complicated model described above, inactivated channels may close only after recovering through the open state.
HERG channel gating has been described as a depolarization-activated channel with slow activation, and fast inactivation gating (Scheme I), in accordance with the previous description of
IKr gating (Shibasaki, 1987; Sanguinetti et al., 1995
; Smith et al., 1996
; Spector et al., 1996b
; Wang et al., 1997). However, it is also
possible to describe this channel as a hyperpolarization-activated channel where channel activation is more rapid than inactivation (Pennefather et al., 1998
; Zhou et al., 1998a
). This distinction is
semantic and we will discuss our data using the terminology indicated in the scheme above.
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RESULTS |
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Ca2+ Decreases HERG Current Amplitude and Shifts the Isochronal Activation Curve
Fig. 1 shows the effects of elevated external Ca2+ on
HERG channel currents. Increasing the external Ca2+
concentration from 1.8 to 10 mM decreased the current amplitude and enhanced the rate of decay of tail
currents at 50 mV (compare Fig. 1, A and B). Plotting
the maximal current during the test step versus the test
potential results in the bell-shaped current-voltage relationship characteristic of HERG channels seen in Fig.
1 C (Trudeau et al., 1995
; Sanguinetti et al., 1995
). The
decrease in the current-voltage relationship at positive
potentials is due to voltage-dependent channel inactivation (Smith et al., 1996
; Spector et al., 1996b
). Elevating
the external Ca2+ concentration increased the degree of
depolarization required to activate HERG currents (Fig.
1, C and D). However, at membrane potentials greater
than +40 mV, current amplitudes during the test pulse
were nearly the same in 1.8 or 10 mM Ca2+ (Fig. 1 C).
Plotting the peak tail current amplitude measured at a
constant potential (
50 mV) as a function of the preceding test potential resulted in a sigmoidal curve that
described the voltage dependence of channel opening
(Fig. 1 D). Elevation of external Ca2+ from 1.8 to 10 mM
shifted the midpoint of this tail current curve by +22.3 ± 2.5 mV (n = 3), in agreement with the shift in voltage dependence of currents measured during the test step
(Fig. 1 C). Ca2+ also decreased the maximum tail current amplitude, but the slope factors of the tail current
curves were not significantly different between 1.8 (8.8 ± 0.2 mV, n = 3) and 10 (9.0 ± 0.4 mV, n = 3) mM Ca2+.
To understand the nature of these Ca2+ effects, the
changes in activation and deactivation kinetics were
characterized in greater detail.
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Ca2+ Slows the Rate of HERG Channel Activation
Because HERG inactivates very rapidly (relative to activation) at most membrane potentials, the rate of activation cannot be observed accurately during a single voltage clamp step (Smith et al., 1996; Spector et al., 1996b
;
Wang et al., 1996
). The time course of the K+ current
observed during a test pulse reflects the rates of both activation and inactivation (Sanguinetti et al., 1995
; Schönherr and Heinemann, 1996
; Smith et al., 1996
; Wang et
al., 1997). To estimate the rate of channel opening at
positive potentials, we took advantage of the channel's
rapid recovery from inactivation (compared with deactivation). This allowed determination of the rate of activation using the method shown in Fig. 2 A (Wang et al.,
1997). A variable duration prepulse is used to drive
channels into the open and/or inactivated state before
stepping to a negative potential (
120 mV), which allows rapid recovery from inactivation (see Scheme II,
the current measured at
120 mV is inward since the K+
reversal potential was
90 mV). Measuring the time
course at different prepulse potentials allowed determination of the voltage dependence of the rate of activation. Comparison of these rates in 1.8 mM extracellular
Ca2+ (Fig. 2 C) and 10 mM Ca2+ (D) shows that activation was slower in the presence of 10 mM Ca2+. Elevated
Ca2+ decreased the rate of HERG activation at all potentials measured (+60 to 0 mV). This can be seen in Fig. 2
B, which shows activation time constants determined by
fitting a second order exponential equation to the data
(see legend for details). The slow time constant of activation was significantly increased (P < 0.05) by elevating extracellular Ca2+ from 1.8 to 10 mM for all test potentials between +50 and 0 mV. The fast time constant of
activation was significantly increased (P < 0.05) for test
potentials +50, +40, +30, and 0 mV.
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Ca2+ Speeds HERG Channel Deactivation
Deactivation kinetics were examined by measuring the voltage dependence of the time course of the K+ current decay upon stepping to different membrane potentials as shown in Fig. 3. Time constants of deactivation were determined by fitting a monoexponential equation to the initial current decay. The initial phase of deactivation is well fit by a monoexponential allowing a simple quantitation of the change in deactivation rate upon elevation of extracellular Ca2+. Time constants determined in this manner were plotted as a function of the test potential to obtain the curves in Fig. 3 D. Increasing external Ca2+ from 1.8 to 10 mM increased the rate of channel deactivation at every potential measured (P < 0.05), resulting in smaller measured time constants (Fig. 3 D).
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Neither External Ca2+ nor Mg2+ Are Required for Channel Closing
Under physiological conditions, Ca2+ is always present
in the extracellular environment, and it is conceivable
that extracellular Ca2+ is required for gating (Armstrong and López-Barneo, 1987; Armstrong and Miller,
1990
; Ho et al., 1996
). Voltage-dependent block of the
open channel could be a mechanism of channel deactivation, as has been suggested for IK in rabbit sinoatrial
node (Ho et al., 1996
). Fig. 4 A shows that HERG channels continued to gate when bathed in a solution with
no added Ca2+ or Mg2+ and buffered with 2 mM EGTA.
Removal of extracellular divalent cations increased the
K+ current immediately upon solution exchange.
Channels opened more rapidly upon membrane depolarization leading to a transient current peak that rapidly decays with the onset of inactivation. Channels also
closed more slowly upon repolarization, but channels
continued to gate. These effects were rapidly and completely reversible upon the return of control extracellular solutions. These results are consistent with the effects of raising extracellular Ca2+ seen in Figs. 1-3,
where opposite effects are seen. Fig. 4 B shows the Ca2+
concentration dependence of the time constant of deactivation at
120 mV. If channel closing results from
block by external divalent cations, then gating should
cease in the absence of divalent cations. This is not the
case. Fig. 4 B shows that gating persists at extremely low
extracellular Ca2+ concentrations. In fact, the rate of
deactivation approaches a limiting value (time constant
~50 ms) for Ca2+ concentrations below 100 µM. Thus,
divalent cation pore block cannot be the mechanism of
deactivation gating for HERG channels, nor are these
ions absolutely required for gating.
Ca2+ Is Not a Fast Voltage-dependent Blocker of HERG Channels
Fig. 1 indicates that raising extracellular Ca2+ results in
decreased outward K+ currents. Fig. 4 shows that time-dependent block of the channel by divalent cations is
not the mechanism of the channel deactivation since
deactivation occurs even in the absence of Ca2+ and
Mg2+. To determine whether the Ca2+ effect is due to a
rapid voltage-dependent block of open HERG channels, the method of Gilly and Armstrong (1982) was
modified to accommodate HERG inactivation behavior. The instantaneous current-voltage relationships
were measured in 1.8 and 10 mM external Ca2+ (Fig. 5,
A and B). Increasing external Ca2+ from 1.8 to 10 mM
did not alter the shape of the open channel current-
voltage relationship. The K+ current was decreased in
10 mM Ca2+ by ~20% at all potentials measured (Fig. 5
C), indicating that the Ca2+ effect was independent of
membrane potential. This finding is not consistent with
the idea that the positively charged Ca2+ is acting as a
rapid voltage-dependent blocker of the channel (Gilly
and Armstrong, 1982
). Therefore, neither slow nor fast voltage-dependent block is the mechanism of the Ca2+
effect. We next investigated the possibility that the apparent Ca2+-induced decrease in conductance was due
to the observed changes in channel gating.
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Increasing extracellular Ca2+ slowed the rate of
HERG activation and accelerated deactivation (Figs. 1,
3, and 4). We therefore examined whether these kinetic changes could account for the Ca2+-induced reduction of HERG current seen in Fig. 5 C. The protocol for measurement of this instantaneous current-voltage relationship contains a 12.5-ms step to 100 mV
that was required to allow the channels to recover from
inactivation. Thus, in elevated Ca2+, accelerated deactivation (Fig. 3) might result in more channels deactivating during the 12.5-ms recovery step, before the measurement of current. This would result in fewer open
channels at the time of the test step and hence would
reduce the instantaneous K+ current measured upon
stepping to +30 mV.
Therefore, we measured the rate of deactivation at
100 mV and used this information to estimate the deactivation that occurred before measurement. This estimation was used to scale instantaneous current-voltage
curves for the deactivation that occurs during the 12.5-ms recovery step at
100 mV (see Fig. 5 legend for details). Exact determination of the degree of deactivation that occurs during the recovery step is not possible
if channels must recover from inactivation before deactivating (Schemes I and II). Nonetheless, because deactivation at
100 mV is more than an order of magnitude slower than recovery from inactivation (see Figs. 2
and 7), it is possible to estimate the fraction of channels that have deactivated during the 12.5-ms recovery
step using the method in Fig. 5 (adapted from Smith
et al., 1996
). When the enhanced rate of deactivation induced by 10 mM Ca2+ was accounted for, the apparent decrease in current was eliminated (Fig. 5 D). This
resulted in instantaneous current-voltage relationships in 1.8 and 10 mM Ca2+ that superimpose. This analysis
illustrates that the reduction of HERG K+ current observed in elevated extracellular Ca2+ concentrations results from the kinetic changes caused by Ca2+.
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Ca2+ Acceleration of Deactivation Alters the Apparent Voltage Dependence of Activation
With this information on the influence of deactivation
on the instantaneous current-voltage curves in Fig. 5
C, the decrease in outward current seen in the isochronal activation curve with 10 mM Ca2+ in Fig. 1 D was
reexamined. To test whether this decrease in current
might also be due to the observed changes in gating kinetics, a three-pulse protocol (Fig. 6) was used to measure the voltage dependence of isochronal activation
(Smith et al., 1996). Deactivation at
100 mV was measured in each test solution immediately after measurement of the voltage dependence of isochronal activation in Fig. 6 B. The activation curves observed with the
three-pulse protocol and the curves scaled to account
for deactivation are shown in Fig. 6, B and C, respectively. This scaling for deactivation removed the observed reduction in HERG peak tail currents, leaving only a parallel shift of the curve to more positive membrane potentials.
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If the observed reduction of K+ current results from enhanced deactivation of the channels before measurement, then the decrease should become greater if a longer recovery period allows the degree of deactivation to increase. We analyzed data from protocols using 12.5-, 25-, and 50-ms recovery periods as an internal control for this method of analysis (data not shown). Increasing recovery duration results in progressive decreases in measured current (due to increasing contamination from deactivation). Likewise, increasing the length of recovery increases the amount of current suppression by addition of 10 mM Ca2+. In each case, this decrease could be removed by accounting for deactivation. This analysis illustrates that the reduction in observed outward current in 10 mM Ca2+ (Figs. 1 D and 6 B) is not due to channel block by Ca2+. The data in Figs. 4 and 5 also demonstrate that Ca2+ does not block HERG directly. Instead, Ca2+ appears to interact with or influence the channel voltage sensor(s) and alter the apparent membrane potential sensed by the channel gating machinery.
A common way in which multivalent cations have
been found to influence ion channel gating is through
screening of diffuse negative surface charges (Frankenhaeuser and Hodgkin, 1957; Cole, 1969
; Gilbert and
Ehrenstein, 1969
; McLaughlin et al., 1971
; Århem, 1980
; Green and Andersen, 1991
; Hille, 1992
; Elinder
et al., 1996
). This can cause shifts in the membrane potential sensed by a membrane protein, such as a voltage-gated K+ channel. This type of nonspecific surface
charge screening should affect all channel parameters
that depend on membrane potential (Gilly and Armstrong, 1982
). To determine whether all voltage-dependent channel parameters are equally affected by external Ca2+, the inactivation of HERG currents in the presence of 1.8 and 10 mM Ca2+ was examined.
Ca2+ Does Not Affect the Rate of Inactivation
The rate of channel inactivation was measured in 1.8 and 10 mM external Ca2+ over a range of membrane
potentials and under conditions designed to isolate the
inactivation process. Two voltage-clamp protocols were
used to determine the voltage dependence of inactivation and recovery from inactivation (Figs. 5 B and 7 A).
A three-pulse protocol (see Fig. 5 B) was used to measure the rate of inactivation at membrane potentials
more positive than 60 mV (Smith et al., 1996
). Monoexponential curves were fit to the rapidly declining current during the step to the test potential, and the
time constants obtained from these fits are plotted versus membrane potential in Fig. 7 C (circles, right side).
Fig. 7 C shows that, surprisingly, the voltage-dependent
rate of inactivation was almost unaffected by elevation
of extracellular Ca2+ from 1.8 to 10 mM. The large
shift in voltage-dependent gating behavior observed
for activation and deactivation was not present. At
membrane potentials more negative than
60 mV, it
was difficult to accurately measure inactivation by the
three step method described above. Therefore, a two-pulse protocol, described in Fig. 7 A, was used to measure the rate of recovery from inactivation between
130 and
50 mV. In this case, the channels were activated and inactivated by a 2-s pulse to +50 mV. Then,
stepping to the test potential results in a rising phase
of the tail current that is largely due to recovery from
inactivation (Smith et al., 1996
). The rate of recovery
was estimated by fitting a monoexponential function
to the rising recovery phase, and the time constant
obtained was plotted as a function of the test potential
in Fig. 7 C (squares, left side of curve) along with the
time constants obtained for inactivation with the three-pulse protocol (Fig. 7 C, circles, right side). As observed for the rate of inactivation, the rate of recovery
from the inactivated to the open state was largely unaffected by increasing the Ca2+ concentration to 10 mM. These findings support the hypothesis that there
are separate voltage sensors for channel activation and inactivation gating (Wang et al., 1997; Zou et al.,
1998
) and indicate that external Ca2+ affects them differently.
Voltage Dependence of Availability Is Ca2+ Independent
The very slow activating and fast inactivating phenotype
of HERG currents prevents traditional determination
of a steady state inactivation (h) curve (Hodgkin and
Huxley, 1952
; Hille, 1992
; Smith et al., 1996
; Schönherr
and Heinemann, 1996
; Spector et al., 1996b
; Wang et
al., 1997). The voltage dependence of the distribution
of channels between the open and inactivated states can be tested by the method illustrated in Fig. 8 (Smith
et al., 1996
; Zou et al., 1998
).
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A three-pulse protocol was used and the instantaneous current measured in the third step to +30 mV
was plotted as a function of the test membrane potential (Fig. 8 B). Note that these curves (Fig. 8 B) largely
superimpose except at potentials less than 80 mV, indicating that 10 mM Ca2+ had no effect on the voltage
dependence of this relationship. The decrease in K+
current observed for test steps more negative than
100
mV is again due to channel deactivation at these membrane potentials. Accounting for the enhanced rate of
deactivation eliminates this decrease (Fig. 8 C). Correction was accomplished in the same manner as was described for Figs. 5 and 6, with the exception that the fraction of current lost due to deactivation must be determined for each test potential. Thus, both the corrected
and uncorrected data indicate that Ca2+ affects only deactivation and does not affect the voltage dependence of
inactivation. A decrease in current measured with this
voltage clamp protocol due to accelerated deactivation is
also predicted by the model discussed below.
Small Changes in External Ca2+ Alter HERG K+ Current During an Action Potential Clamp
To determine whether changes in external Ca2+ near the
physiological range impact HERG K+ currents during the
time course of a cardiac action potential, we used a voltage-clamp protocol with the shape of an action potential (Fig. 9). A similar protocol has been used to study HERG
current stably expressed in HEK-293 cells (Zhou et al.,
1998b). Human total serum Ca2+ concentrations range
between 1.5 and 4.25 mM (average ~2.5 mM), ~50% of
which is in the free ionized form (Guyton, 1991
). Fig. 9 illustrates how Ca2+ modulation of HERG gating results in
changes in current during an action potential clamp. The
action potential clamp protocol was applied to a cell expressing HERG K+ channels in solutions containing 1, 2, 3, or 10 mM external Ca2+. Relatively little outward K+
current was apparent during the peak of the action potential clamp, but as the cell membrane potential decreases
(at the end of the action potential), HERG current rises
sharply. HERG currents during this voltage clamp protocol were highly sensitive to changes in extracellular Ca2+
concentration between 1 and 3 mM. Note that, although
our experiments were at room temperature, the action
potential duration used (~400 ms) is appropriate for a
human ventricular myocyte at 37°C. As a consequence,
these experiments may underestimate the impact of Ca2+
on the currents during an actual action potential. Changing external Ca2+ from 1 to 3 mM resulted in a 50% reduction of the maximum outward K+ current during this
action potential voltage clamp protocol. For comparison
with the concentrations used in the biophysical characterization of the Ca2+ effect, 10 mM Ca2+ was applied to the
cell and caused an 80% reduction of the maximum current under these conditions.
|
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DISCUSSION |
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Mechanisms of Ca2+ Modulation
Our data suggest an intimate association of Ca2+ with
the HERG channel protein that alters the voltage dependence and kinetics of channel activation gating,
while having little effect on inactivation gating. Figs. 1-3
show that increasing extracellular Ca2+ from 1.8 to 10 mM had at least two effects on HERG currents. The
voltage dependence of channel activation was shifted
to more depolarized membrane potentials and the current amplitude was decreased at membrane potentials
less than +50 mV. Several simple mechanisms could
potentially explain these effects of Ca2+. First, voltage-dependent block of the channel might decrease HERG
current at potentials less than +50 mV, producing an
apparent shift in the voltage-dependent activation of
the channels. Relief of block at positive potentials
could then account for the lack of block at potentials
+50 mV. This possibility was ruled out by two interdependent findings. Removing external divalents does not remove deactivation (Fig. 4), eliminating the possibility that slow voltage-dependent block by divalent cations causes HERG deactivation. It is impossible to assure that all divalent cations have been removed from
the external solution, but by omitting them from the
solution and adding the chelator EGTA, we can assure that the concentration is extremely low (~2.5 nM). We
also showed (Fig. 4 B) that the time constant of HERG
deactivation reaches a maximum (~50 ms) in 100 µM
Ca2+ and does not increase for lower concentrations
of Ca2+ over five orders of magnitude. The shape of
the open channel current-voltage relationship is not
changed by elevating external Ca2+ (Fig. 5 C); a voltage-dependent open channel blocker would change the shape of this curve (Gilly and Armstrong, 1982). A
second possible mechanism for Ca2+ modulation is
through charge screening of negative membrane surface charges, resulting in a change in the membrane
potential sensed by the channel (Green and Andersen,
1991
). Such a screening could be nonspecific, resulting
from association with diffuse charged moieties like
membrane phospholipid head groups, or it could occur through specific interaction with the channel protein. Nonspecific surface charge screening would cause
a global change in the membrane potential (V) perceived by the entire channel protein, and therefore all
voltage-dependent parameters of the channel would be
shifted in a parallel manner along the voltage axis by
the change in perceived membrane potential (V = Vclamp
Vshift). The fact that the voltage dependence of
activation gating was shifted by elevated external Ca2+
(Figs. 1-4 and 6) while inactivation gating was not
(Figs. 7 and 8) indicates that nonspecific charge
screening inducing a change in the membrane potential sensed by the entire channel cannot be the mechanism of Ca2+ modulation. Ca2+ association with a specific channel residue(s) could cause more selective effects on channel function since it need not result in a
global change in the potential sensed by the channel
protein. The observation of differential modulation of
the voltage dependence of activation and inactivation
greatly constrains the possibilities for the nature of the
channel voltage sensing machinery. Both HERG activation and inactivation gating appear voltage dependent (Figs. 2, 3, and 7; Sanguinetti et al., 1995
; Trudeau et al., 1995
; Smith et al., 1996
; Schönherr and Heinemann,
1996
; Spector et al., 1996b
; Wang et al., 1996
, 1997; Zou
et al., 1998
). However, it is possible that inactivation derives its apparent voltage dependence from coupling to
voltage-dependent channel activation, as is true for
Shaker potassium channels (Zagotta and Aldrich, 1990
;
Hoshi et al., 1991
). In this case, any effect on the activation voltage sensor would also alter the apparent voltage dependence of inactivation, contrary to our data
(Figs. 1-3, 7, and 8) and previous observations of K+
modulation of HERG (Liu et al., 1996
; Wang et al., 1996
,
1997). Alternatively, both processes could be intrinsically
voltage dependent, but use the same voltage-sensing machinery. This possibility is also excluded by the specificity
of the Ca2+ effect. If the same voltage sensor controlled
both activation and inactivation, Ca2+ shielding of that
sensor should affect both gating processes, contradicting
the data. Our data seem to indicate that there are at least
two voltage-sensing regions involved in HERG channel gating: one governing channel activation and another for
inactivation. Furthermore, these distinct sensors can detect different local values of membrane potential and be
differentially screened by Ca2+.
To test the feasibility of this idea, we used a simplified
HERG channel gating model to determine whether
our observations could be predicted by a voltage shift
in only the voltage dependence of the rate constants
that govern channel activation (see METHODS for details). The model simulates the major features of our
own data and is consistent with data from the literature
(Trudeau et al., 1995; Sanguinetti et al., 1995
; Smith et al.,
1996
; Schönherr and Heinemann, 1996
; Wang et al.,
1997). In the model, both activation and inactivation
are voltage dependent (Wang et al., 1997). Fig. 10 A illustrates simulated currents for the voltage-clamp protocol in Fig. 1. Elevated Ca2+ was simulated (Fig. 10 B)
by including a +23-mV bias in the rate equations for
the activation rate constants (a1, a
1, a3, a
3). As seen in
Fig. 10, this change alone accounts for a decrease in K+
currents and an increase in the rate of deactivation
(compare with Fig. 1, A and B). Fig. 10 C plots the simulated outward current after 2 s at the test potential,
and D shows the peak tail current (compare with Fig. 1,
C and D, respectively). Fig. 10 E indicates that the simple shift of voltage-dependent activation rate constants in the model also results in an apparent decrease in the
open channel current-voltage relationship (compare
with Fig. 5 C). Furthermore, the modeled voltage shift
does not affect the voltage dependence of the equilibrium between the open and inactivated states (Fig. 10
F). The model also predicts the Ca2+-induced decrease
in K+ current measured at negative test potentials with
this protocol (compare Figs. 10 F and 8 B). This occurs
because the rate of deactivation is accelerated in elevated extracellular Ca2+ (Fig. 3). Therefore, at test potentials more negative than the threshold for channel
activation, the faster deactivation in 10 mM extracellular Ca2+ allows more channels to make the transition
from the open to the closed state. As a result, 10 mM
Ca2+ exaggerates the decrease in the channel availability curve measured at negative potentials in Fig. 8 B, as
is simulated in the modeled data (Fig. 10 D). Thus, a
simple shift in the membrane potential felt by the activation process, but not the inactivation process, explains the effects experimentally observed upon elevation of extracellular Ca2+. Such an effect can be envisioned if we assume Ca2+ binds to a region of the
HERG protein near the region that controls activation
voltage sensing and alters the membrane potential
sensed by amino acids in the vicinity of the binding site.
Voltage shifts applied to both activation and inactivation rate constants in the model failed to qualitatively
reproduce the experimental observations (not shown).
Although we cannot definitively rule out the possibility that Ca2+ causes an allosteric change in HERG channels that mimics a specific association near the activation voltage sensor, our data are consistent with a simple Ca2+-induced change in membrane potential
sensed specifically by the activation process.
|
Comparison with Previous Studies
Ca2+ is critical in the gating of some voltage-gated
channels. HERG belongs to the family of six transmembrane domain voltage-gated K+ channel genes such as
Shaker, EAG, and the mammalian Kv channels. The effects of Ca2+ have been most thoroughly characterized
in the squid delayed rectifier K+ channel and the recombinant Shaker K+ channel; both require divalent
cations to stabilize the closed channel and maintain
channel selectivity (Armstrong and Matteson, 1986;
Armstrong and López-Barneo, 1987
; Armstrong and
Miller, 1990
). Unlike these channels, we find that
HERG channels do not absolutely require Ca2+ for selectivity and gating, though even control extracellular Ca2+ concentrations cause a shift relative to the gating
behavior in their absence (Fig. 4). These results agree
with data for IKr recorded from guinea pig ventricular
myocytes in high K+ (Sanguinetti and Jurkiewicz,
1992
). Our data show that the voltage dependence of
the HERG activation curve was shifted by +22.3 ± 2.5 mV upon raising extracellular Ca2+ from 1.8 to 10 mM.
This shift was accompanied by slowed channel activation, and enhancement of the rate of channel deactivation, even in physiological concentrations of external
permeant cations (4 mM K+). Deactivation of IK in rabbit sinoatrial node myocytes is sensitive to external divalent cations, and block by the extracellular divalent cations Ca2+ and Mg2+ has been proposed to be the primary determinant of voltage-dependent deactivation
gating of that current (Ho et al., 1996
). Our data indicate that HERG K+ channels retain deactivation gating
even in the absence of external divalent cations (Fig.
4). Furthermore, the apparent decrease of current
through open HERG channels was not voltage dependent (Fig. 5). This indicates that Ca2+ does not experience the transmembrane field when interacting with
HERG channels. Thus, neither a time-dependent nor
-independent voltage-dependent block of open HERG
channels can explain the effect of extracellular Ca2+.
The observed decrease in HERG K+ currents upon elevation of external Ca2+ was completely explained by
modulation of gating. Correction of the current amplitudes for these Ca2+-induced changes in gating removed the apparent current suppression (Figs. 5, 6,
and 8). Thus, external divalent cations modulate, but
do not cause, HERG deactivation gating. This is in contrast with the conclusions that divalent cations cause
deactivation gating of rabbit IK (Ho et al., 1996
).
There are several possible explanations for this discrepancy. It is conceivable that native rabbit sinoatrial
node K+ channels interact with divalent cations in a
fundamentally different manner than heterologously
expressed HERG channels due to differences in structure or subunit composition. However, there is considerable similarity in the behavior of rabbit sinoatrial
node IK and HERG (Shibasaki, 1987; Trudeau et al.,
1995
; Sanguinetti et al., 1995
; Ho et al., 1996
). A more
likely explanation involves the differences in the external ionic conditions between the two studies. Our study
was performed in 4 mM external K+, while studies in
rabbit sinoatrial node were performed in the presence
of high extracellular K+ (140 mM). Elevated external
K+ was necessary in part because the native currents
are small in physiological K+ (Ho et al., 1996
). Both
HERG K+ currents and IKr are modulated by external
K+(Sanguinetti and Jurkiewicz, 1992
; Trudeau et al.,
1995
; Sanguinetti et al., 1995
; Schönherr and Heinemann, 1996
; Smith et al., 1996
; Wang et al., 1996
, 1997;
Yang et al., 1997
). Elevated extracellular K+ slows inactivation and deactivation gating of HERG. Slow deactivation kinetics induced by high K+ could make deactivation in the absence of extracellular divalent cations
difficult to observe. This difficulty is exacerbated by the
apparent competition between external K+ and Ca2+
(Ho et al., 1998
). Elevated extracellular K+ (20 mM)
decreases the shift in the HERG channel isochronal
current-voltage curve caused by Ca2+ (Ho et al., 1998
).
This functional interaction was interpreted as a competition for a binding site within the channel pore (Ho et al.,
1998
). Since our data indicate that Ca2+ does not directly occlude the permeation pathway of HERG channels, this competition between external K+ and Ca2+ is
most likely at a site outside the pore, or it may be a
functional result of the two ions exerting antagonistic
effects on channel gating at distinct sites.
The specificity of Ca2+ for HERG activation gating is
further evidenced by the identity of K+ current at membrane potentials +10 mV in Fig. 1 C. This lack of current suppression could be interpreted as relief of Ca2+
block by positive membrane potentials (Ho et al.,
1998). However, Figs. 4 and 5 illustrate that Ca2+ is not
a voltage-dependent blocker of HERG currents. HERG
outward currents are determined by both activation
and inactivation controlling channel open probability.
Outward currents during depolarization to intermediate potentials are suppressed because a shift of the voltage dependence of channel opening to more positive
potentials leads to a decrease in channel open probability (Figs. 1 and 6). At membrane potentials greater
than +10 mV, activation approaches a maximal level,
and therefore channel inactivation is the primary determinant of HERG channel open probability at these
voltages. HERG inactivation is much less sensitive to external Ca2+ than is activation (Figs. 7 and 8). Therefore
at potentials greater than +10 mV, where inactivation
largely determines HERG open probability, there is no
Ca2+-induced decrease in current. Still another illustration of the specificity of the Ca2+ effect on activation
gating is seen in Fig. 4 A. The current recorded in divalent cation-free solution showed a distinct inactivating phase during depolarization to +50 mV. This is because the rate of activation was increased upon removing Ca2+. Increased Ca2+ slowed activation (Fig. 2, B
and C), but complete removal of divalent cations increased the rate of activation, while the rate of inactivation was little affected by changes in external Ca2+
(Figs. 7 and 8). The result is that a significant fraction
of the channels were activated before the onset of inactivation, and a portion of the current inactivation could
therefore be directly observed (Fig. 4 A).
Implications for the Nature of HERG Inactivation
It has been suggested that the inactivation of HERG K+
channels is mechanistically related to the C-type inactivation identified in Shaker K+ channels (Hoshi et al.,
1991; Schönherr and Heinemann, 1996
; Smith et al.,
1996
). Indeed, HERG inactivation shares several characteristics with C-type inactivation. For example, the
rate of HERG inactivation is sensitive to extracellular
K+ and TEA, but insensitive to intracellular TEA (Choi
et al., 1991
; Smith et al., 1996
). The rate and voltage dependence of HERG inactivation are also dependent
upon the identity of the amino acid present at position
631, analogous to Shaker residue 449, which in part determines the rate of C-type inactivation in those channels (López-Barneo et al., 1993
; Schönherr and Heinemann, 1996
; Zou et al., 1998
). Unlike C-type inactivation in Shaker channels, HERG inactivation occurs
more rapidly than activation at some potentials and has been suggested to have its own independent voltage
sensor (Liu et al., 1996
; Wang et al., 1996
, 1997). Our
data support this conclusion. Neither N- nor C-type inactivation of Shaker K+ channels have significant inherent voltage sensitivity. The apparent voltage dependence at positive potentials occurs through coupling to
the voltage dependence of activation gating (Zagotta
and Aldrich, 1990
; Hoshi et al., 1991
). However, it is
now clear that, for HERG channels, mutation of serine
631 to alanine causes a shift in the voltage dependence
of channel inactivation without shifting the voltage dependence of activation (Zou et al., 1998
). The selectivity of the effect of Ca2+ on the activation gating process
provides further support for the idea that HERG-type
inactivation has a distinct voltage sensor and is therefore fundamentally different from C-type inactivation in Shaker-like channels.
Implications for HERG Function In Vivo
Fig. 9 shows that raising external Ca2+ from 1 to 3 mM
reduced the HERG current maximum by 50% during
an action potential voltage clamp. This large effect may
seem surprising, but it is actually quite consistent with
the biophysical characterization of the Ca2+ effect. The
characteristic slow activating, fast inactivating behavior
of HERG channels results in outward currents during
the action potential clamp that are largest near the
midpoint of the voltage-dependent activation curve.
Hence, Ca2+-induced shifts in the midpoint of the activation curve greatly affect the current level during an
action potential clamp. HERG currents recorded with a
voltage clamp protocol of this type at 35°C display more
current during the plateau phase of the action potential waveform due to the temperature dependence of
the activation process (Zhou et al., 1998b). As a result,
calcium effects could be even larger at physiological
temperatures than they are in our study. Our results
demonstrate how even modest changes in external
Ca2+ concentration could significantly change the fraction of HERG channels available to participate in cellular repolarization in vivo.
Conclusions
We conclude that elevation of extracellular Ca2+ interacts specifically with channel activation voltage sensing, and does not act as a direct blocker of the open pore. This interaction is likely mediated by the association of Ca2+ with distinct, possibly negatively charged, channel residues near a voltage sensor. Voltage-dependent inactivation gating was nearly unaffected by elevating external Ca2+. The differential effect of external Ca2+ on the voltage dependence of activation and inactivation indicates that these two processes sense different values of transmembrane potential and are therefore probably physically separated in space. Further studies will be needed to elucidate the specific channel residues that are responsible for this interaction.
![]() |
FOOTNOTES |
---|
Address correspondence to Paul B. Bennett, Ph.D., Department of Pharmacology (WP26-265), Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486. Fax: 215-652-0800; E-mail: paul_bennett{at}merck.com
Original version received 11 August 1998 and accepted version received 3 February 1999.
This project was completed in partial fulfillment of the requirements for the Ph.D. degree in Pharmacology at Vanderbilt University School of Medicine (J.P. Johnson), and supported by National Institutes of Health (NIH) training grants T32 HL07411, and T32 GM07628. The work was also supported by institutional funding of the Medical Scientist Training Program (F.M. Mullins) and NIH grants HL-51197 and HL-46681 (P.B. Bennett).We thank Drs. Louis J. DeFelice and Christoph M. Fahlke for insightful discussions and critical review of the manuscript. We also thank Michelle Choi and Brady Palmer for technical assistance, Dr. Richard Horn for the CD8 plasmid, Dr. Mark Keating for the HERG cDNA, and Dr. Sabina Kupershmidt for the final HERG-pSI construct.
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Abbreviations used in this paper |
---|
CHO, Chinese hamster ovary; HERG, human ether-à-go-go-related gene; IKr, delayed rectifier K+ current.
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