From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expressed in Xenopus oocytes, KvLQT1 channel subunits yield a small, rapidly activating, voltage- dependent potassium conductance. When coexpressed with the minK gene product, a slowly activating and much larger potassium current results. Using fluctuation analysis and single-channel recordings, we have studied the currents formed by human KvLQT1 subunits alone and in conjunction with human or rat minK subunits. With low external K+, the single-channel conductances of these three channel types are estimated to be 0.7, 4.5, and 6.5 pS, respectively, based on noise analysis at 20 kHz bandwidth of currents at +50 mV. Power spectra computed over the range 0.1 Hz-20 kHz show a weak frequency dependence, consistent with current interruptions occurring on a broad range of time scales. The broad spectrum causes the apparent single-channel current value to depend on the bandwidth of the recording, and is mirrored in very "flickery" single-channel events of the channels from coexpressed KvLQT1 and human minK subunits. The increase in macroscopic current due to the presence of the minK subunit is accounted for by the increased apparent single-channel conductance it confers on the expressed channels. The rat minK subunit also confers the property that the outward single-channel current is increased by external potassium ions.
Key words: KvLQT1; long QT syndrome; fluctuation analysis; minK ![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of the minK protein is associated with potassium channel activity in a variety of tissues (Busch and
Suessbrich, 1997). The minK (also called ISK) protein
underlies a slowly activating current in uterine smooth
muscle (Boyle et al., 1987
) that is developmentally regulated; it also underlies the slow delayed rectifier current IKs in cardiac tissue (Freeman and Kass, 1993
; Varnum et al., 1993
) and a potassium current in epithelial
cells of the ear (Sakagami et al., 1991
; Marcus and
Shen, 1994
). Expression of this small (129-130 amino
acids) protein in heterologous systems yields at most a
small potassium current whose magnitude saturates at
low expression levels (Lesage et al., 1993
; Blumenthal
and Kaczmarek, 1994
), suggesting that it must combine
with other subunit types to form functional IKs channels. In the channel complex minK appears to be
present in multiple copies (Tzounopoulos et al., 1995
),
quite possibly as few as two (Wang and Goldstein, 1995
).
The other partner in the IKs channel is the product of
the LQT1 gene. Long QT syndrome (LQTS)1 is a genetically heterogeneous disorder that causes cardiac arrhythmias and leads to sudden death. One of several
loci for this disorder, LQT1 is located on chromosome
11 (Keating et al., 1991) and is the gene for a potassium channel subunit named KvLQT1 (Wang et al.,
1996b
). Although KvLQT1 subunits produce a potassium current when expressed alone, much larger currents having the slow kinetic characteristics of IKs are
obtained from the coexpression of KvLQT1 and minK
subunits (Barhanin et al., 1996
; Sanguinetti et al., 1996
;
Yang et al., 1997
). The LQTS-associated mutations in
the KvLQT1 gene appear to reduce the expressed IKs
current in a dominant-negative fashion (Shalaby et al.,
1997
; van den Berg et al., 1997
).
Because KvLQT1 subunits give rise to functional potassium channels when expressed alone, it is interesting
to consider the nature of the interaction between minK
and KvLQT1 that produces larger and more slowly activating currents when these genes are coexpressed. It
has been argued that minK serves as a regulator of
channel activity (Attali et al., 1993; Ben-Efraim et al.,
1996
), but evidence is accumulating that minK residues
form part of the pore of the IKs channel complex
(Wang et al., 1996a
; Sesti and Goldstein, 1998
; Tai and
Goldstein, 1998
). In a recent study using COS cells
(Romey et al., 1997
), it was concluded that the effect of
minK coexpression was greatly to increase channel
number while decreasing the single-channel conductance of the channels expressed from KvLQT1 subunits. In the present study, we revisit the single-channel properties of the KvLQT1 and coexpressed channels,
making use of fluctuation analysis and single-channel
recordings from Xenopus oocytes. A companion study
considers the single-channel properties of coexpressed
channels containing mutant minK subunits as well (Sesti and Goldstein, 1998
).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA and RNA Synthesis
Human and synthetic rat minK cDNAs (Hausdorff et al., 1991;
Goldstein and Miller, 1991
) were obtained from Dr. S. Goldstein (Yale University) and propagated in pGEM-A and pBF2 vectors,
respectively (Swanson et al., 1990
; Tai and Goldstein, 1998
).
Point mutations in the minK constructs were made by PCR and
verified by sequencing. Plasmids of rat and human minK were
linearized with NotI and MluI, respectively. cRNAs were transcribed with the MEGAscript T7 and SP6 RNA polymerase kits
(Ambion Inc., Austin, TX). Two human KvLQT1 constructs were
obtained from Drs. M. Sanguinetti and M. Keating (University of
Utah), which we call s-KvLQT1 and l-KvLQT1. The s-KvLQT1
(Sanguinetti et al., 1996
) has a truncated NH2 terminus, while
l-KvLQT1 is full-length, having 95 additional residues at the NH2
terminus (Yang et al., 1997
). Each KvLQT1 gene was subcloned into a modified Bluescript vector (Bluescript KSM; gift from W. Joiner, Yale University) that incorporates
-globin untranslated sequences and a poly-A tail for increased protein translation in
oocytes. Plasmids of s-KvLQT1 and l-KvLQT1 were linearized with NotI and XbaI, respectively, and transcribed with the
MEGAscript T3 RNA polymerase kit (Ambion Inc.). Sizes of transcribed cRNAs were verified by gel electrophoresis.
Electrophysiology
Human KvLQT1 cRNA (5.8 ng) was injected into Xenopus oocytes alone or in conjunction with 1 ng minK cRNA. We use the notation hIKs to denote channels resulting from coexpression of human minK and human KvLQT1, rhIKs to denote channels from the combination of rat minK and hKvLQT1, and ILQT to denote channels expressed from hKvLQT1 alone. In this study, only the full-length l-KvLQT1 variant was used for expressing hIKs and ILQT channels; most rhIKs recordings were made with this variant as well. The rhIKs channels formed with the truncated s-KvLQT1 construct had identical behavior in terms of voltage dependence and single channel unitary current.
Patch- and voltage-clamp recordings were done at room temperature, 7-12 d after RNA injection. Patch clamp recordings were obtained using EPC-9 (HEKA Electronic, Lambrecht, Germany) or Axopatch 200B (Axon Instruments, Foster City, CA) amplifiers. Pipettes were pulled from 7052 glass (Corning Glass Works, Corning, NY) to a tip size of 2-5 µm. Pipettes with tip diameters of ~30 µm were used for recording ILQT channel currents. These pipettes were pulled from thin-walled borosilicate capillaries (PG165T; Warner Instruments, Hamden, CT). The standard bath solution for patch clamp recordings was (mM) 7 KCl, 93 K-aspartate, 1 EGTA, and 10 HEPES. The standard pipette solution was (mM) 0.2 KCl, 100 NaCl, 1 MgCl2, 1.8 CaCl2, and 10 HEPES. All solutions were titrated to pH 7.4.
For two-microelectrode voltage clamp recordings, an OC-725
amplifier (Warner Instruments) was driven by the Pulse software (HEKA Electronic) and an analogue interface (ITC-16; Instrutech Corp., Mineola, NY). Microelectrodes were filled with
1 M KCl and had 0.1-0.3 M resistance. The standard bath solution for voltage clamp recordings, denoted ND96, contained
(mM) 96 NaCl, 2 KCl, 0.1 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4.
Half-amplitude threshold analysis (Colquhoun and Sigworth,
1995) was used to idealize single-channel recordings for kinetic analysis and the reconstruction of ensemble time courses. For noise analysis, the macroscopic currents induced by a series of depolarizing pulses were recorded on video tape using a VR-10 Digital Data Recorder (Instrutech Corp.). Data were then transferred digitally from tape through the VR-10 Digital Recorder using the program VCatch developed in our laboratory. The raw
data (94 kHz sampling rate) were filtered and decimated using a
digital Gaussian filter. Power spectra were calculated from data
decimated and filtered to 10 Hz, 100 Hz, 1 kHz, and 10 kHz
bandwidths. A power spectrum covering the frequency range 0.1 Hz-20 kHz was obtained by combining the four individual spectra after correcting for filter responses.
Statistical quantities are expressed as mean ± SEM with the
number of determinations n 3 unless otherwise stated.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activation of IKs and ILQT Channels
In oocytes coinjected with minK and KvLQT1 cRNAs, both cell-attached and inside-out patch recordings showed slowly activating outward currents with monotonically increasing noise during 5-s depolarizations (Fig. 1, A and B). As characterized by Boltzmann fits to isochronal conductance-voltage curves, the voltage dependence of activation of these hIKs and rhIKs channels is quite shallow, with an effective charge of 1-1.2 e0 and a midpoint voltage near +55 mV. The main difference in behavior between the two channel types is the more rapid time course of activation in the hIKs channels. Both channel types show gradually increasing current even at the end of 60-s depolarizations to +50 mV (Fig. 1, D and E).
|
For comparison, the activation of currents resulting
from the injection of hKvLQT1 cRNA alone is shown
from a cell-attached giant patch recording in Fig. 1 C.
The fragility of the giant patches precluded recordings
at large positive voltages, but a Boltzmann fit over the accessible voltage range yields a half-activation voltage of
6 mV, considerably more negative than that of IKs channels and consistent with previous observations (Sanguinetti et al., 1996
). The tail currents show a "hook" in
the time course, characteristic of KvLQT1 currents (Sanguinetti et al., 1996
; Pusch et al., 1998
). The patch recordings shown in Fig. 1 all have similar kinetics and
voltage dependence to the corresponding whole cell-currents obtained by two-electrode voltage clamp.
Reversal potentials of rhIKs currents were measured
from macroscopic tail currents obtained in inside-out or
cell-attached patch recordings with 100 mM K+, Na+,
Rb+, or Cs+ in the pipette; in each case, the bath solution
contained 100 mM K+. Table I (top) shows the reversal
potentials of the rhIKs channels. The table also gives the
computed permeability ratios. The permeability ratios
are very similar to those obtained from voltage clamp recordings of oocytes injected with rat minK RNA (Hausdorff et al., 1991). Like many other potassium channels,
the permeability sequence is K+ > Rb+ > Cs+ > Na+.
|
It was more difficult to obtain patch recordings with macroscopic hIKs currents. Therefore, the ion selectivity of these channels was characterized from whole cell currents with 100 mM K+, Na+, Rb+, or Cs+ in the bath solution. For comparison, the reversal potentials of rhIKs channels were also measured in this way, using the same batch of oocytes. There was no significant difference in reversal potentials between these two channel types (Table I).
Single hIKs Channel Current
If we assume that the IKs channel only has one conductance level with unitary current i, then for n channels the variance of current fluctuations will depend on the mean current I according to
![]() |
(1) |
(Sigworth, 1980). We shall denote by iv an estimate of i
obtained from fitting Eq. 1 to the variance-mean relationship. For this analysis, a series of current sweeps was
collected by applying repetitive depolarizing pulses to
+50 mV. The mean current and variance from hIKs
channels (Fig. 2 A) were computed using groups of two
sweeps to minimize errors due to slow current drifts
(Heinemann and Conti, 1992
). Shown in Fig. 2, B and
C, are two mean-variance plots computed from data filtered to different extents. Fitting Eq. 1 yielded the estimates iv = 0.28 pA at 100 Hz and 0.51 pA at 10 kHz
bandwidth. The discrepancy between the two estimates of unitary current suggests that a substantial amount of
variance is contained in high-frequency components.
|
To investigate the high-frequency components of the
hIKs current fluctuations, we computed the power spectrum of the macroscopic currents. Pairs of aligned current traces were subtracted as shown in Fig. 2 D. Power
spectra were computed from subtracted traces (Sigworth, 1981) by fast Fourier transform and the resulting
power spectrum after correction for background noise is
shown in Fig. 2 E. It has a remarkably straight 1/f dependence over five decades of frequency. The weak frequency dependence of the spectral density implies that
the observed noise variance will be heavily dependent on
filter cutoff frequency. From Parseval's theorem, we have
![]() |
(2) |
where S(f) is the power spectral density of the current fluctuations and H(f) is the filter transfer function. To give an idea of the effect of filter bandwidth, the spectral density in Fig. 2 E was integrated numerically and converted into unitary current amplitude according to the expression
![]() |
(3) |
where I is the time-averaged mean current and is(f) is the apparent unitary current at bandwidth f. As can be seen in Fig. 2 F, is increases strongly with filter bandwidth, and is still increasing at f = 20 kHz. Thus, fluctuation analysis is expected to yield any of a variety of unitary current amplitudes, depending on the bandwidth. At 20 kHz, is is 0.47 pA at +50 mV.
The expression in Eq. 3 is missing a correction term
(Sigworth, 1981) and therefore underestimates the unitary current by a factor of about
where
is the
mean open probability. Thus, the apparent unitary currents from spectral analysis (Fig. 2 F) of 0.2 and 0.47 pA, at 100 Hz and 20 kHz bandwidth, respectively, become ~0.25 and 0.6 pA when
= 0.2 is assumed.
These values agree with those obtained from the mean-
variance analysis (Fig. 2, B and C).
Unitary currents roughly 0.5 pA in size should be visible in single-channel recordings. Obtaining single-channel patches was difficult, however, because the hIKs channels appeared to be highly clustered in the oocyte membrane so that patches typically contained either tens of channels or no channels at all. The distribution of patch current density was very broad, as determined from more than 100 patches (Fig. 3). Fig. 4 A shows one of our best candidates for an hIKs single-channel current. This sweep was recorded from a multiple-channel patch but appears to have only one channel active. As would be expected from the very broad power spectrum of macroscopic current fluctuations, the channel current shows very rapid flickering. From recordings at three voltages, the single-channel conductance is estimated to be 3 pS at 200 Hz bandwidth (Fig. 4 C).
|
|
Conductance of ILQT Channels
Injection of the KvLQT1 cRNA alone results in small K+ currents having more rapid kinetics than IKs channels. Might the smaller current result from smaller single-channel currents? In the case of the hIKs channels, mean-variance and spectral fluctuation analysis yielded reasonable estimates of the unitary current at +50 mV, comparable to what was observed in a patch recording. To determine the ILQT unitary current, we used the same fluctuation-analysis methods and similar experimental protocols. The only difference was that in attempting to record those currents, we encountered a very low channel density. Using pipettes with 2-5-µm tip diameters, we saw no current in 12 patches from oocytes having mean whole-cell currents of 4 µA. Therefore, we used much larger pipettes (30-µm tip diameter) to obtain macroscopic channel currents. Fig. 5 A shows one cell-attached giant patch having a mean current of 240 pA at +50 mV. This recording shows the characteristic "hook" of outward tail current that is seen in ILQT channels. The mean-variance relationship, computed from data filtered at 200 Hz, is poorly fitted by the parabolic function of Eq. 1; however, linear fits are consistent with unitary currents of 0.03-0.04 pA, as is shown in Fig. 5 C.
|
Spectral analysis of the fluctuations was also performed. The spectrum shows several discernible components, and can be well fitted by the sum of three Lorentzian functions (Fig. 5 E). The integral of the spectrum, scaled to show the apparent unitary current, shows is increasing with bandwidth but possibly reaching a limiting value of ~0.09 pA at 20 kHz. The unitary current is therefore about one fifth of that of the hIKs channels. We were not able to obtain any convincing single-channel recordings of this current.
Single Channel Properties of rhIKs Channels
We also studied channels formed by coexpression of rat
minK with human KvLQT1 subunits. The currents
from these channels (Fig. 1) show similar noise properties and voltage dependence to those containing human minK subunits. Fig. 6 shows the fluctuation analysis of these channels. The power spectrum (Fig. 6 B)
does not have the simple power-law frequency dependence of the hIKs channels, but can be fitted by one 1/f
component plus several Lorentzian components, where
a minimum of four Lorentzians was required for a
good fit. The presence of discernible Lorentzians suggests that rhIKs channels may have more clearly distinguishable kinetic states than hIKs channels. The unitary
current is was calculated from the power spectrum from
this experiment. Again, is shows a strong dependence
on bandwidth, as shown in Fig. 6 C, with an estimated unitary current of 0.67 pA at 20 kHz. Depending on
the estimated open probability, this value should be
increased somewhat, for example to 0.84 pA assuming
= 0.2.
|
The mean-variance analysis was also applied to same set of data. The fit of Eq. 1 to the mean-variance plot (Fig. 6 E) yields an estimate of the unitary current iv of ~0.28 pA at 100 Hz bandwidth. This is similar to the value obtained from hIKs channels at this bandwidth.
The presence of discernible Lorentzian components
in the power spectrum suggests that the rhIKs channels
should show less flickering than the hIKs channels.
Patch recordings indeed showed single-channel events,
but as was the case with hIKs channels, of >200 trials, we
were unable to obtain a one-channel recording of sufficient duration to allow kinetic analysis. Shown in Fig. 7
is a recording from a patch containing three channels
using pulses to +50 mV. Channels open after a latency
of a few seconds, often first to a subconductance level
before reaching the full single-channel current (Fig.
7 B). To verify that these channel events correspond to
the macroscopic currents, we computed the channel
open probability from the idealizations of 60 sweeps. It
has a slowly activating time course that reaches an open
probability of 0.45 at the end of the 5-s depolarization.
This time course superimposes well on the time course
of current in a multichannel patch (Fig. 7 C). The time
course of activation can be described by the distribution of first latencies to channel opening. Ignoring the
subconductance levels, we measured the first latency to
the fully open state and corrected it for the presence of
three channels (Aldrich et al., 1983). When scaled by
the factor 0.8, it matches very well the time course of
the open probability. This correspondence is consistent
with the idea that, once a channel opens, it remains open with a substantial probability (apparently 0.8 at
this time resolution).
|
Slow variations in single-channel activity were observed in this patch, with occasional null sweeps (Fig. 7 B, middle) occurring throughout the recording (Fig. 7 F). A subsequent recording at +20 mV from the same patch (Fig. 8) shows similar kinetic behavior of the single channels. Again, subconductance levels are sometimes seen to precede the full opening of the channel (Fig. 8 A) and the slow time course of activation is explained by long first latencies (Fig. 8 B). At this smaller depolarization, a higher frequency of null sweeps was seen (Fig. 8 C).
|
External Potassium Dependence of IKs Channels
The conductance and gating of some types of K+ channels depend on external K+ concentration. We tested
the external potassium dependence of whole-cell currents using continuous bath perfusion. Fig. 9 shows currents at +30 mV as the bath solution was switched
between 0.2 and 10 mM K+. The hIKs and rhIKs channels have opposite responses to the change in external
potassium. Switching from 0.2 to 10 mM K+ reduces
the hIKs current by 20%, an effect that can be explained by the decrease in driving force; however, there is a
20% current increase under the same conditions with
rhIKs channels. That higher external potassium increases outward current was also observed in the IKr
current through HERG channels (Sanguinetti et al., 1995). The effect of external K+ was also tested for ILQT
channels (Fig. 9 C). In this case, the currents were
smaller when external K+ was increased. These experiments show that coexpression with the rat minK gene
product changes the sensitivity the ILQT channels to external potassium.
|
A comparison of the human and rat minK sequences
(Murai et al., 1989; Fig. 9 D) shows many differences in the
extracellular (NH2-terminal) and intracellular (COOH-terminal) regions, but only one nonconserved residue
in the putative transmembrane domain. As a first attempt to locate the region responsible for the differences in K+ sensitivity, we made complementary mutations at this position. The resulting constructs, human
(V47I) and rat (I48V) minK were coexpressed with human KvLQT1 and the external potassium sensitivity was
assayed by the ratio of peak current in 10 mM external
K+ to that in 0.2 mM K+ (Table II). The mutation in
human minK had no significant effect on the K+ sensitivity, but, with the rat mutation increased, K+ significantly decreased the current (P < 0.01), which is the opposite effect to that seen with the wild-type rat minK
subunit. Thus, this residue in the membrane-spanning
region appears to contribute to the external potassium
sensitivity.
|
Because the single-channel rhIKs currents can be resolved in patch-clamp recordings, it should be possible
to examine the origin of the increase in outward current
in these channels when extracellular potassium concentration is increased. Fig. 10 A shows three representative
single-channel currents from inside-out patches at +50
mV and filtered at 100 Hz. The currents were estimated to be 0.56, 0.44, and 0.37 pA with [K]o equal to 10, 0.2, and 0 mM. The single-channel current is seen to increase
when [K]o increases, even as the driving force decreases.
Recordings from six patches show increased conductance over the voltage range of 30 to +80 mV with
higher [K]o (Fig. 10 B). The [K]o dependence of conductance appears to saturate above 2 mM, and its magnitude accounts for all of the increase in macroscopic current
observed on raising extracellular potassium in the case of
the rhIKs channel.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study has considered the single-channel properties of IKs channels obtained from the coexpression of the human or rat minK protein with human KvLQT1, and has compared these properties with the expression of KvLQT1 subunits alone in Xenopus oocytes. We conclude that the IKs channels have a higher single-channel conductance than channels from KvLQT1 alone, and that the sensitivity to external potassium ions is reflected in the size of single-channel currents in the case of the rhIKs hybrid channel. The IKs single-channel currents are roughly 0.6 pA at +50 mV. The relatively low conductance of these slowly activating channels might be important to reduce membrane potential fluctuations in cells where IKs serves to shape long-duration action potentials.
Size of Single-Channel Currents
The rapid flickering of currents in these channels makes difficult the determination of the single-open-channel current. When fluctuation analysis is used to estimate the single-channel current, the bandwidth of the recording must be sufficient to capture the fastest fluctuations, or else the variance will be underestimated, providing an underestimate of the single-channel current. The direct observation of single-channel currents suffers from a similar limitation: if a channel's current contains many brief interruptions, a single-channel recording at low bandwidth will show a reduced apparent single-channel current and increased apparent open probability. The very noisy appearance of the hIKs recording at 500 Hz (Fig. 4 A) suggests that this bandwidth is not sufficient to resolve the true open-channel current. Fluctuation analysis allows a wider range of frequencies to be explored.
A two-state channel with opening and closing rate
constants and
yields current fluctuations having a
Lorentzian power spectrum with a corner frequency fc = 1/2
(
+
). Above fc, the Lorentzian decays with frequency as f
2; this relatively rapid decay means that the
observed variance of the fluctuations converges rapidly
to the correct value as the bandwidth is increased above
fc. On the other hand, no convergence results in the
case of an f
1 frequency dependence, like that shown in
Fig. 2 E for the hIKs channels. Such a frequency dependence results in an observed variance that increases
without limit as the bandwidth increases. Because bandwidth is related to the time scale of measurement,
one could speak of an effective single-channel current
value that depends on the time scale under which it is
measured. Channels having stable open and closed
states, such that the power spectrum of fluctuations
from these channels decay rapidly at higher frequencies, show a distinct single-channel current value given
sufficient recording bandwidth. For the hIKs channel,
however, the 20-kHz limit of our power spectrum measurements was not sufficient to reach this regime. Thus
we do not know the asymptotic value of the variance;
we also do not know the exact open probability value that is necessary to correct the estimate of the single-channel current. The variance computed from fluctuations up to 20 kHz (Fig. 2), when corrected for an estimated absolute open probability of ~0.2, result in the
estimated single-channel current of 0.6 pA at +50 mV,
or a chord conductance of ~4.5 pS. These values are
consistent with the current extremes observed in single-channel recordings (Fig. 4).
Another way to summarize the problem posed by the hIKs channel is that the very rapid current fluctuations make it difficult experimentally to distinguish, on the basis of time scales, between gating or channel-block phenomena on the one hand and the ion conduction process on the other. The apparent single-channel conductance values are influenced by the very rapid interruptions in the channel current.
The channels formed by hKvLQT1 subunits expressed alone or in combination with rat minK subunits showed less extreme behavior. Although the spectra of the current fluctuations are also very broad, they are not as featureless as those of hIKs currents and can be fitted by multiple Lorentzian components. Limiting single-channel current values at +50 mV of 0.09 and 0.84 pA are obtained from fluctuation analysis. These correspond to conductances of ~0.7 and 6.5 pS. The rhIKs channels resulting from coexpression were also observed directly from single-channel recording at 100 Hz bandwidth. There they appeared to have single-channel currents at +50 mV of ~0.5 pA, depending on extracellular K+ concentration (Fig. 10).
In native tissues, the cardiac IKs current has been seen
to have small fluctuations. Walsh et al. (1991) estimated
unitary conductances of <1 pS in guinea pig myocytes.
Taking into account their recording bandwidth of 200 Hz, we obtain a similar value. At 200 Hz, we would estimate a conductance of ~2 pS, as calculated from the estimated single-channel current at +50 mV of ~0.2 pA in both hIKs and the hrIKs channels (Figs. 2 F and 6 C).
It should be kept in mind that fluctuation analysis depends on several assumptions about the behavior of
channels. We assume homogeneous populations of independently gating channels, and have also used the assumption that there is only one nonzero conductance level. It is likely that one or more of these assumptions is
false. Evidence has been presented by Pusch et al. (1998)
that KvLQT1 channels have two open states, and we see
clear subconductance levels in single-channel recordings of the rhIKs channels (Figs. 7 B and 8 A). If there are
multiple conductance levels, the estimated single-channel current will lie between the largest and smallest single-channel current, and will depend on the probabilities of occupancy of the various conductance states. It
should be kept in mind, however, that the high-conductance states tend to dominate the estimated conductance, because the contribution of a state's current i to
the variance is proportional to i 2. Thus, our single-channel conductance estimates are likely to approximate the
values for the largest conducting states.
A similar argument can be made concerning the possible heterogeneity of channel types. When minK and KvLQT1 cRNAs are coinjected, it is possible that hybrid channels are expressed having various stoichiometries, and the fluctuation analysis will give a weighted-average value. Again, it should be kept in mind that larger channel currents make larger contributions to the variance, and therefore predominate in the weighted average. Thus, if our coinjections produced a variety of channel types, the estimated conductance probably reflects the largest conductance value. Further, the good correspondence between the fluctuation analysis of rhIKs currents and direct single-channel recordings argues that heterogeneity in channel conductances is not a serious problem.
How Coexpression of minK Affects KvLQT1 Current
Expression of KvLQT1 subunits produces small, rapidly
activating potassium currents; coexpression of these
with minK results in slowly activating IKs currents that
are several-fold larger. These differences in the expressed currents have been seen in a variety of expression systems, including Xenopus oocytes, Sf9 cells, and
in the mammalian cell lines CHO and COS (Barhanin
et al., 1996; Sanguinetti et al., 1996
; Romey et al.,
1997
). Is the increase in KvLQT1 current on coexpression with minK due to an increase in channel density or
an increase in the single-channel current? Romey et al.
(1997)
addressed this question through single-channel recordings and noise analysis of expressed currents in
COS cells. They concluded that the addition of minK
subunits to KvLQT1 channels caused a reduction of
single-channel conductance from 7.6 to 0.6 pS. To account for the increase in macroscopic current, they
conclude that coexpression with minK causes the channel density to increase by a large factor, some 60-fold.
Our studies of these channels in Xenopus oocytes lead
to the opposite conclusion, that a large part of the observed current increase on coexpression of minK arises
from an increase in single-channel conductance. From
fluctuation analysis, we estimate a single-channel conductance of ~0.7 pS for KvLQT1 channels. We estimate the conductance of human IKs channels to be
~4.5 pS. The discrepancy between our results and
those of Romey et al. (1997) might be explained by the
difference between COS cell and oocyte expression systems. This, however, is unlikely because the behavior of
the channels is similar in the various systems; further,
Romey et al. (1997)
report the same single-channel
conductance value for IKs channels expressed in Xenopus oocytes as in COS cells.
Our results disagree with this previous work in two respects. First, we obtain a larger single-channel conductance for the human IKs channels than reported by
Romey et al. (1997). This can be explained largely by
the frequency dependence of fluctuations in this channel. Our value of 4.5 pS is based on fluctuation analysis at 20 kHz and on single-channel recordings at 500 Hz
bandwidth; their value of 0.6 pS was based on fluctuation analysis at a bandwidth of 150 Hz under similar
ionic conditions. Our conductance estimate of 6.5 pS
for the closely related rhIKs channel (Figs. 6-10), for which openings are more readily resolved, supports the
higher conductance estimate.
The other disagreement concerns the conductance
of channels arising from the expression of KvLQT1
subunits alone. Romey et al. (1997) found well-resolved
single-channel events in COS cells having a conductance of 7.6 pS. In our macropatch recordings, we find
a remarkably noiseless current (Fig. 5). The power
spectrum from the macropatch recording shows a
broad frequency dependence, with a limiting conductance value of ~0.7 pS apparently being reached at 20 kHz bandwidth. There is always the danger that the currents in patch-clamp recordings are not properly
identified, such that unitary events from one channel
type are ascribed to another. Although we have not performed a pharmacological identification of our currents, we note that the kinetics of activation and the tail
currents in our macropatch recordings agree very well
with the currents observed from KvLQT1 channels in
whole oocytes and in other expression systems (Fig.
1 C; Barhanin et al., 1996
; Sanguinetti et al., 1996
;
Romey et al., 1997
), supporting the view that it is these
channels whose fluctuations we have measured.
Our results agree well with those of Sesti and Goldstein (1998), who studied channels expressed from
KvLQT1 subunits alone and with human minK. They
used symmetrical 100 mM potassium solutions and
thereby obtained higher conductance values (4 and 16 pS) compared with ours (0.7 and 4.5 pS). Under the
different ionic conditions, the single-channel outward
currents are expected to be similar at large depolarizations. At +50 mV and 20 kHz bandwidth, our estimate
for the single-channel current of hIKs channels is 0.6 ± 0.2 pA; here the error bounds reflect an estimate of statistical and systematic errors in the fluctuation analysis
used. The corresponding estimate at 25 kHz bandwidth
given by Sesti and Goldstein (1998)
is 0.8 ± 0.2 pA.
Kinetics of IKs Channels
In addition to rapid flickering, the currents through single IKs channels show slow gating processes. At depolarizations to +20 and +50 mV (Figs. 7 and 8), the main determinant of the activation time course is seen to be the latency to first channel opening. In the rhIKs channels where single-channel events could be readily resolved, dwells in a subconductance state were often seen to precede full channel opening. The rhIKs channel activity also waxes and wanes on a time scale of ~30 s, as seen by groups of successive blank sweeps in patch recordings (Figs. 7 and 8).
The Conductance of minK "Channels"
The discovery that KvLQT1 subunits coassemble with
the minK gene product to produce the IKs current (Barhanin et al., 1996; Sanguinetti et al., 1996
) has clarified
some of the puzzling aspects of the "IminK current" that
is seen in Xenopus oocytes when minK is expressed
alone (Busch and Suessbrich, 1997
). It is now clear that
this current results from the combination of minK with
an endogenous, Xenopus KvLQT1 homologue that is expressed at low levels (Sanguinetti et al., 1996
). The difficulty that we and others have had in attempting to define the single-channel characteristics of IminK are now
understandable in view of the difficulties we have encountered in recording from single IKs channels. In a
preliminary communication (Yang and Sigworth, 1995
),
we reported fluctuation analysis of a slowly activating
current seen in patches from Xenopus oocytes, but
many subsequent attempts were unsuccessful to establish this current as the same as the macroscopic, potassium-selective IminK. It is possible that our patch currents, which from fluctuation analysis had a unitary current value below 1 fA, were contaminated with currents
from an endogenous channel or ion transporter having slow kinetics, similar perhaps to the transporter studied
by Schlief and Heinemann (1995)
.
![]() |
FOOTNOTES |
---|
Address correspondence to F.J. Sigworth, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8026. Fax: 203-785-4951; E-mail: fred.sigworth{at}yale.edu
Original version received 30 July 1998 and accepted version received 21 October 1998.
We thank W.N. Joiner for the Bluescript-KSM vector and Y. Yan for cRNA preparation and oocyte injection. We also thank Dr. S.A.N. Goldstein for minK cDNA, and Drs. M. Sanguinetti and M. Keating for the human KvLQT1 cDNA.
![]() |
Abbreviation used in this paper |
---|
LQTS, long QT syndrome.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Aldrich, R.W., D.P. Corey, and C.F. Stevens. 1983. A reinterpretation of mammalian sodium channel gating based on single channel recording. Nature. 306: 436-441 [Medline]. |
2. |
Attali, B.,
E. Guillemare,
F. Lesage,
E. Honore,
G. Romey,
M. Lazdunski, and
J. Barhanin.
1993.
The protein IsK is a dual activator
of K+ and Cl![]() |
3. | Barhanin, J., F. Lesage, E. Guillemare, M. Fink, M. Lazdunski, and G. Romey. 1996. K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature. 384: 78-80 [Medline]. |
4. |
Ben-Efraim, I.,
Y. Shai, and
B. Attali.
1996.
Cytoplasmic and extracellular IsK peptides activate endogenous K+ and Cl![]() |
5. | Blumenthal, E.M., and L.K. Kaczmarek. 1994. The minK potassium channel exists in functional and nonfunctional forms when expressed in the plasma membrane of Xenopus oocytes. J. Neurosci. 14: 3097-3105 [Abstract]. |
6. | Boyle, M.B., N.J. MacLusky, F. Naftolin, and L.K. Kaczmarek. 1987. Hormonal regulation of K+-channel messenger RNA in rat myometrium during oestrus cycle and in pregnancy. Nature. 330: 373-375 [Medline]. |
7. | Busch, A.E., and H. Suessbrich. 1997. Role of the ISK protein in the IminK channel complex. Trends Pharmacol. Sci. 18: 26-29 [Medline]. |
8. | Colquhoun, D., and F.J. Sigworth. 1995. Fitting and statistical analysis of single-channel records. In Single Channel Recording. 2nd ed. Plenum Publishing Corp., New York. 483-587. |
9. | Freeman, L.C., and R.S. Kass. 1993. Expression of a minimal K+ channel protein in mammalian cells and immunolocalization in guinea pig heart. Circ. Res. 73: 968-973 [Abstract]. |
10. | Goldstein, S.A., and C. Miller. 1991. Site-specific mutations in a minimal voltage-dependent K+ channel alter ion selectivity and open-channel block. Neuron 7: 403-408 [Medline]. |
11. | Hausdorff, S.F., S.A. Goldstein, E.E. Rushin, and C. Miller. 1991. Functional characterization of a minimal K+ channel expressed from a synthetic gene. Biochemistry. 30: 3341-3346 [Medline]. |
12. | Heinemann, S.H., and F. Conti. 1992. Nonstationary noise analysis and application to patch clamp recordings. Methods Enzymol. 207: 131-148 [Medline]. |
13. | Keating, M., C. Dunn, D. Atkinson, K. Timothy, G.M. Vincent, and M. Leppert. 1991. Consistent linkage of the long-QT syndrome to the Harvey ras-1 locus on chromosome 11. Am. J. Hum. Genet. 49: 1335-1339 [Medline]. |
14. | Lesage, F., B. Attali, J. Lakey, E. Honore, G. Romey, E. Faurobert, M. Lazdunski, and J. Barhanin. 1993. Are Xenopus oocytes unique in displaying functional IsK channel heterologous expression? Receptors Channels. 1: 143-152 [Medline]. |
15. |
Marcus, D.C., and
Z. Shen.
1994.
Slowly activating voltage-dependent K+ conductance is apical pathway for K+ secretion in vestibular dark cells.
Am. J. Physiol
267:
C857-C864
|
16. | Murai, T., A. Kakizuka, T. Takumi, H. Ohkubo, and S. Nakanishi. 1989. Molecular cloning and sequence analysis of human genomic DNA encoding a novel membrane protein which exhibits a slowly activating potassium channel activity. Biochem. Biophys. Res. Commun 161: 176-181 [Medline]. |
17. |
Pusch, M.,
R. Magrassi,
B. Wollnik, and
F. Conti.
1998.
Activation
and inactivation of homomeric KvLQT1 potassium channels.
Biophys. J.
75:
785-792
|
18. |
Romey, G.,
B. Attali,
C. Chouabe,
I. Abitbol,
E. Guillemare,
J. Barhanin, and
M. Lazdunski.
1997.
Molecular mechanism and functional significance of the minK control of the KvLQT1 channel
activity.
J. Biol. Chem.
272:
16713-16716
|
19. | Sakagami, M., K. Fukazawa, T. Matsunaga, H. Fujita, N. Mori, T. Takumi, H. Ohkubo, and S. Nakanishi. 1991. Cellular localization of rat Isk protein in the stria vascularis by immunohistochemical observation. Hear. Res. 56: 168-172 [Medline]. |
20. | Sanguinetti, M.C., M.E. Curran, A. Zou, J. Shen, P.S. Spector, D.L. Atkinson, and M.T. Keating. 1996. Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel. Nature. 384: 80-83 [Medline]. |
21. | Sanguinetti, M.C., C. Jiang, M.E. Curran, and M.T. Keating. 1995. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell. 81: 299-307 [Medline]. |
22. | Schlief, T., and S.H. Heinemann. 1995. H2O2-induced chloride currents are indicative of an endogenous Na+-Ca2+ exchange mechanism in Xenopus oocytes. J. Physiol. (Lond.). 486: 123-130 [Abstract]. |
23. |
Sesti, F., and
S.A.N. Goldstein.
1998.
Single channel characteristics
of wild-type IKs channels and channels formed with two minK mutants that cause long QT syndrome.
J. Gen. Physiol.
112:
651-663
|
24. |
Shalaby, F.Y.,
P.C. Levesque,
W.P. Yang,
W.A. Little,
M.L. Conder,
T. Jenkins-West, and
M.A. Blanar.
1997.
Dominant-negative
KvLQT1 mutations underlie the LQT1 form of long QT syndrome.
Circulation.
96:
1733-1736
|
25. | Sigworth, F.J.. 1980. The variance of sodium current fluctuations at the node of Ranvier. J. Physiol. (Lond.). 307: 97-129 [Medline]. |
26. | Sigworth, F.J.. 1981. Interpreting power spectra from nonstationary membrane current fluctuations. Biophys. J. 35: 289-300 [Abstract]. |
27. | Swanson, R., J. Marshall, J.S. Smith, J.B. Williams, M.B. Boyle, K. Folander, C.J. Luneau, J. Antanavage, C. Oliva, and S.A. Buhrow. 1990. Cloning and expression of cDNA and genomic clones encoding three delayed rectifier potassium channels in rat brain. Neuron 4: 929-939 [Medline]. |
28. | Tai, K.K., and S.A. Goldstein. 1998. The conduction pore of a cardiac potassium channel. Nature. 391: 605-608 [Medline]. |
29. | Tzounopoulos, T., H.R. Guy, S. Durell, J.P. Adelman, and J. Maylie. 1995. minK channels form by assembly of at least 14 subunits. Proc. Natl. Acad. Sci. USA. 92: 9593-9597 [Abstract]. |
30. | van den Berg, M.H., A.A. Wilde, E.O. Robles de Medina, H. Meyer, J.L. Geelen, R.J. Jongbloed, H.J. Wellens, and J.P. Geraedts. 1997. The long QT syndrome: a novel missense mutation in the S6 region of the KVLQT1 gene. Hum. Genet. 100: 356-361 [Medline]. |
31. | Varnum, M.D., A.E. Busch, C.T. Bond, J. Maylie, and J.P. Adelman. 1993. The minK channel underlies the cardiac potassium current IKs and mediates species-specific responses to protein kinase C. Proc. Natl. Acad. Sci. USA. 90: 11528-11532 [Abstract]. |
32. |
Walsh, K.B.,
J.P. Arena,
W.M. Kwok,
L. Freeman, and
R.S. Kass.
1991.
Delayed-rectifier potassium channel activity in isolated
membrane patches of guinea pig ventricular myocytes.
Am. J. Physiol.
260:
H1390-H1393
|
33. | Wang, K.W., and S.A. Goldstein. 1995. Subunit composition of minK potassium channels. Neuron. 14: 1303-1309 [Medline]. |
34. | Wang, K.W., K.K. Tai, and S.A. Goldstein. 1996a. MinK residues line a potassium channel pore. Neuron. 16: 571-577 [Medline]. |
35. | Wang, Q., M.E. Curran, I. Splawski, T.C. Burn, J.M. Millholland, T.J. VanRaay, J. Shen, K.W. Timothy, G.M. Vincent, T. de Jager, et al . 1996b. Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias. Nat. Genet. 12: 17-23 [Medline]. |
36. |
Yang, W.P.,
P.C. Levesque,
W.A. Little,
M.L. Conder,
F.Y. Shalaby, and
M.A. Blanar.
1997.
KvLQT1, a voltage-gated potassium channel responsible for human cardiac arrhythmias.
Proc. Natl. Acad.
Sci. USA.
94:
4017-4021
|
37. | Yang, Y., and F.J. Sigworth. 1995. The conductance of minK `channels' is very small. Biophys. J. 68: A22 . |