Individual Subunits Contribute Independently to Slow Gating of
Bovine EAG Potassium Channels*
Roland
Schönherr
,
Solveig
Hehl
,
Heinrich
Terlau§,
Arnd
Baumann¶, and
Stefan H.
Heinemann
From the
Max-Planck-Gesellschaft, Arbeitsgruppe
Molekulare und zelluläre Biophysik, Drackendorfer Strasse 1,
D-07747 Jena, § Max-Planck-Institut für experimentelle
Medizin, Abteilung Molekulare Biologie neuronaler Signale,
Hermann-Rein-Strasse 3, D-37075 Göttingen, and ¶ Institut
für Biologische Informationsverarbeitung, Forschungszentrum
Jülich, D-52425 Jülich, Germany
 |
ABSTRACT |
The bovine ether à go-go gene
encodes a delayed rectifier potassium channel. In contrast to other
delayed rectifiers, its activation kinetics is largely determined by
the holding potential and the concentration of extracellular
Mg2+, giving rise to slowly activating currents with a
characteristic sigmoidal rising phase. Replacement of a single amino
acid in the extracellular linker between transmembrane segments S3 and S4 (L322H) strongly reduced the prepulse dependence and accelerated activation by 1 order of magnitude. In addition, compared with the wild
type, the half-activation voltage of this mutant was shifted by more
than 30 mV to more negative potentials. We used dimeric and tetrameric
constructs of the bovine eag1 gene to analyze channels with
defined stoichiometry of mutated and wild-type subunits within the
tetrameric channel complexes. With increasing numbers of mutated
subunits, the channel activation was progressively accelerated, and the
sigmoidicity of the current traces was reduced. Based on a quantitative
analysis, we show that the slow gating, typical for EAG channels, is
mediated by independent conformational transitions of individual
subunits, which gain their voltage dependence from the S4 segment. At a
given voltage, external Mg2+ increases the probability of a
channel subunit to be in the slowly activating conformation, whereas
mutation L322H strongly reduces this probability.
 |
INTRODUCTION |
Voltage-gated potassium channels (K+ channels) play
major roles in the control of cellular resting potentials, and they are involved in the regulation of firing rate and shaping of action potentials in excitable cells (1). The progress of molecular cloning
over the past 10 years revealed an astonishing diversity within this
group of membrane proteins, which comprises several related gene
families (2). The ether à go-go gene, which has originally been cloned from Drosophila, provided the name
for a growing sub-family of K+ channels that contain six
transmembrane segments (S1-S6) per subunit and a binding motif for
cyclic nucleotides in the cytoplasmic carboxyl terminus of each
subunit. At present this subfamily consists of three members, the
eag gene, the eag-like K+ channel
gene (elk), and the eag-related gene
(erg) (3). Further diversity is caused by splice variants or
isoforms that have been described for all three genes (4-7). Whereas
the ERG channel is responsible for the cardiac
IKr current (8), the physiological role of EAG
channels still remains to be identified. Recently the first examples of
native EAG currents have been described. A human neuroblastoma cell
line was found to express human eag in a cell
cycle-dependent manner (9). In human myoblasts, the expression of human eag is correlated with a
non-inactivating delayed rectifier K+ current, which is
involved in myoblast fusion (10).
A characteristic feature of all EAG channels that have been examined so
far is the unusual kinetics of the activation process. It displays a
strong dependence on the prepulse potential (11), reminiscent of the
shift originally described by Cole and Moore (12). Test pulses from
potentials just below the activation threshold (e.g.
70
mV) lead to rapid activation, whereas hyperpolarizing prepulses result
in very slow activation kinetics. Terlau et al. (13) showed
that the slow activation of rat EAG not only occurs at
unphysiologically low holding potentials but also under physiological conditions in the presence of extracellular Mg2+ at
mM concentrations. The unusual gating of EAG channels was described using a sequential gating model that considered two voltage-dependent transitions for each single subunit (13). The underlying molecular mechanism, however, has not yet been elucidated.
In this paper we describe the electrical properties of a point mutation
introduced into the bovine eag1 gene. The mutation in the
extracellular linker of transmembrane segments S3 and S4 strongly
decreased both, the Cole-Moore shift of
b-EAG1 activation and the
apparent regulation by external Mg2+. We used this mutation
as a tool to separate slow and fast gating processes. To control the
stoichiometry within the channel tetramers, we constructed dimeric and
tetrameric b-eag1 genes with different numbers and orders of
the mutated subunits. Using this approach, we could dissect the complex
conformational changes in the multimeric channel protein and assign
them to gating transitions of individual subunits. The activation
kinetics of the linked constructs could be described by a fully
independent model, assuming a binomial distribution of the individual
subunits between two closed states and a voltage-dependent
slow transition between these conformations. The wild-type subunits,
participating in a heterotetrameric channel, are decisive for the slow
gating kinetics.
 |
EXPERIMENTAL PROCEDURES |
Construction of Channel Mutants and mRNA Synthesis--
The
bovine eag1 gene (7) was used to generate the mutants and
chimeric constructs. Constructs were subcloned into the oocyte expression vector pGEMHE (14) using the restriction sites
BamHI and HindIII. The point mutation L322H was
created by site-directed mutagenesis using the polymerase chain
reaction. The polymerase chain reaction product was transferred into
the wild-type plasmid as a BstBI-MluI fragment,
and the sequence of the complete fragment was verified by cycle
sequencing of the plasmid DNA. For construction of dimeric and
tetrameric eag genes, we introduced EcoRV and
HindIII restriction sites at the 3' end of the
eag gene. The modified 3' end reads
GGCGCAAGCggatatcaatgataagctt (lowercase letters are for added
nucleotides). This construct was opened with EcoRV and HindIII, and a second gene copy was coupled to the 3' end as
an SmaI-HindIII fragment. The SmaI
site is part of the upstream region of b-eag in pGEMHE
(cccggggatccaccATG, start codon is in capitals). The junction of the
two gene copies encodes five additional amino acids (GWGST). Two dimer
constructs were then linked using the same restriction sites to obtain
a tetrameric gene. The T7 mMessage mMachine Kit (Ambion, Austin, TX)
was used for mRNA synthesis after linearization with
NheI.
Electrophysiological Measurements--
Stage V oocytes were
prepared from Xenopus laevis as described previously (15),
and 50 nl of in vitro transcribed mRNA (5-500 ng/µl)
were injected. Currents were recorded at 20-23 °C in the period
ranging from 1 to 7 days after injection. A two-electrode voltage clamp
amplifier (Turbo-TEC 10CD, NPI electronic, Tamm, Germany) was used, and
electrodes filled with 2 M KCl had resistances between 0.5 and 0.9 megaohms. The bath solution was normal frog Ringer's (NFR),
containing 115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, pH 7.2 (NaOH). MgCl2 or NiCl2 was added to this
solution at concentrations indicated for individual experiments. Thus, the external solution always contained 1.8 mM
Ca2+ ions, as complete removal of divalent ions resulted in
the emergence of a leak current without strong reduction of the
Cole-Moore shift of b-EAG1 (not shown). Experiment control including
pulse generation and data recording was performed with the Pulse + PulseFit software package (HEKA elektronik, Lambrecht, Germany). A P/n
method, supported by Pulse + PulseFit, was used in all experiments for
correction of leak and capacitive currents.
Data Analysis and Modeling--
Data were analyzed using
PulseFit, PulseTools (HEKA elektronik) and IgorPro (WaveMetrics, Lake
Oswego, Oregon) running on Macintosh and PC-compatible computers.
Analyzed parameters are presented as mean ±S.E., with
n = number of independent experiments.
Normalized conductances as shown in Fig. 1C were obtained by
fitting the following equation to the current-voltage data:
|
(Eq. 1)
|
where
is the maximal conductance, and
Erev is the estimated reversal potential. The
first term accounts for the single-channel conductance according to the
Goldman-Hodgkin-Katz equation. The last term describes the open
probability (popen) of the channels, characterized by the voltage of half-maximal activation,
Vh, and a slope factor, k.
Time course of activation of wild-type b-EAG channels was described
using the following equation:
|
(Eq. 2)
|
In this equation, the symbols have the following meaning:
a = amplitude, kf = rate constant of
channel activation when all slow gates are activated,
ks = rate constant of activation per slow subunit,
n = number of slow subunits, p = probability for a subunit being in a state from which activation is
slow. In the figures, the speed of activation is shown as time constant, i.e. the inverse of the rate constants
(e.g.
s =
1/ks). To account for the limited clamp speed of
the two-electrode voltage clamp, a time delay of 1 ms was assumed. This
model implies a binomial distribution of subunits between "fast"
and "slow" states. Activation of channel constructs with only two
or one wild-type subunit were described by this equation, assuming
n = 2 or 1, respectively. In this case p
denotes the probability of being in a "slow" state for wild-type
subunits only. As shown in the results section, this equation gives a
very reliable description of the data obtained in elevated
concentrations of divalent cations (e.g. Mg2+)
and using a negative holding potential. In such cases
(p > 0.5), the first term, describing fast activation,
basically vanishes ((1
0.5)4 = 0.0625). Thus, the
activation is described by only three free parameters: a,
p, and ks. In cases where p is
small, the fast component adds another free parameter,
kf. We chose to describe fast activation with a
single-exponential component rather than with the fourth power, because
the limited speed of the two-electrode voltage clamp and the rapid
activation rate, kf, hampered a faithful resolution.
The results obtained for the slow
Mg2+-dependent gating of b-EAG channels,
however, were not affected by this limitation.
Individual data traces were fitted according to Equation 2 using a
Levenberg-Marquardt algorithm as implemented in IgorPro. Whole sets of
data traces similar to those shown in Fig. 5 were simultaneously fitted
according to Equation 2 using a Simplex algorithm, programmed after
Caceci and Cacheris (16). In this case, identical kf
and ks values were used for all traces, whereas
individual p values were assigned to each trace as
p is a function of prepulse potential (see Fig. 5).
 |
RESULTS |
Mutation L322H Shifts the Voltage Dependence of Activation to
Hyperpolarized Voltages--
We were interested in identifying amino
acid residues that influence the characteristic gating of EAG channels.
As shown previously, deletions in the cytoplasmic amino terminus of rat
EAG affect both voltage dependence and activation kinetics of the
channels (17). A point mutation at the cytoplasmic face of the S4
segment (H343R) was found to largely compensate for the effect of such deletions (17), and the same phenotype was found for the analogous mutations introduced into the bovine EAG channel (not shown). Recently,
Tang and Papazian (18) described mutations close to the extracellular
part of the S4 segment of the Drosophila EAG channel. A
point mutation that replaced a leucine residue in position 342 by
histidine led to a shift of the current-voltage relation by 16 mV to
depolarized voltages. We introduced the equivalent mutation at the
corresponding position of b-EAG1 (L322H) (Fig. 1A). Under a two-electrode
voltage clamp, this mutant gave rise to delayed rectifier currents,
very similar to wild-type currents when expressed in Xenopus
oocytes (Fig. 1B). A comparison of
popen over the tested voltage range shows that
channels carrying the mutation L322H open at more negative voltages
than wild-type channels (Fig. 1C). The voltage required for
half-maximal activation (Vh) of the mutant channels
was shifted by more than 30 mV to hyperpolarized voltages (wild type,
11.8 ± 2.2 mV, n = 8; L322H,
45.1 ± 1.8 mV, n = 12), whereas the slope factors (k)
were not significantly different (Fig. 1D).

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Fig. 1.
Mutation L322H induces a shift in the
voltage-dependent activation of b-EAG1. A, amino acid
alignment of the putative S3 and S4 transmembrane segments of b-EAG1
and d-EAG channels. In the d-EAG sequence, only, deviations from b-EAG1
are shown, and identical residues are indicated by hyphens.
The arrow indicates the leucine residue that has been
replaced by histidine in b-EAG1 (L322H) in analogy to the d-EAG
mutation (L342H) described by Tang and Papazian (18). B,
currents were measured with a two-electrode voltage clamp from oocytes
injected with mRNA encoding wild type b-EAG1 (top) or
the mutant L322H (bottom). C, the current
magnitude was measured in the last 10% of the depolarization segments
and plotted as a function of voltage. The current was described by
Equation 1. The open probability is given by a single-component
Boltzmann function characterized by the half-maximal activation
voltage, Vh, and the slope factor, k. The
single-channel current was accounted for by assuming a single-channel
conductance according to the Goldman-Hodgkin-Katz formalism with a
reversal potential of 90 mV (Equation 1). Data were fitted to voltage
values ranging between 80 and 20 mV (L322H, filled
circles) or +10 mV (wild type, open circles) to avoid
the influence of current rectification at higher potentials; data
points that were not used for the fit are shown as small
squares. Data sets from individual oocytes were then transformed
to popen values using the data fit results.
These popen values were compiled and are plotted
in C as mean ±S.E. for the wild type (n = 8) and the mutant L322H (n = 12). Data points are
connected by straight lines. In D, the resulting
Vh and k values are compared using
box plots. Bath solution: NFR.
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|
Mutation L322H Accelerates Activation Kinetics--
To
characterize the activation kinetics of b-EAG1 and the effect of the
L322H mutation in more detail, we applied repeated test pulses to +40
mV and varied the prepulse potential between
130 and
70 mV with
increments of 10 mV. The resulting current traces, recorded under a
two-electrode voltage clamp are shown in Fig.
2A. As extracellular bath
solution, we used NFR, containing different concentrations of either
Mg2+ or Ni2+ ions. b-EAG1 channels responded to
changes in the holding potential in a very similar fashion as described
previously for r-EAG channels (13). Hyperpolarizing prepulses caused a
marked slowing of current activation, even in 0 mM
[Mg2+]o. Increasing the concentration of
extracellular Mg2+ to 10 mM resulted in
additional slowing of the activation kinetics. Further slowing of the
activation was observed in solutions containing 1 mM
Ni2+. For a model-independent description of the time
course of activation, we plotted the time to reach 80% of the maximum
current amplitude as a function of the prepulse potential (Fig.
2B). In 0 mM [Mg2+]o, the
slowing effect of the prepulses on b-EAG1 saturates at about
130 mV
(open circles), whereas in 2 mM
[Mg2+]o saturation already occurs at potentials
more negative than
90 mV (open triangles). In 10 mM [Mg2+]o, the channels always
activated slowly, almost independent of the prepulse voltages
(open squares). For comparison, the mutant channels,
analyzed under the same ionic conditions, showed that the point
mutation L322H accelerated the activation kinetics by about 1 order of
magnitude (closed circles). In addition, the prepulse
dependence in Mg2+-free NFR solution was almost completely
abolished. The mutant channels still responded to variations in the
extracellular cation concentration. In 10 mM
[Mg2+]o, the time to reach 80% activation at
different holding potentials is very similar to the values found for
the wild type in the absence of Mg2+ (compare open
circles and filled squares in Fig. 2B).

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Fig. 2.
Mutant L322H strongly reduces the prepulse
dependence of activation. A, activation of wild-type
(left) and L322H (right) channels was assessed by
application of depolarizations to +40 mV from various holding
potentials. The bath solution (NFR) contained, in addition to 1.8 mM Ca2+, the indicated divalent cations.
Although activation of the wild-type channels shows the typical
dependence on both, holding potential and divalent cations, activation
of L322H channels is generally faster. The speed of activation was
quantified by measuring the time required for the current to reach the
80% level. This is plotted in B as a function of voltage
(n = 4-6). The use of the symbols is indicated in A. The data points were connected by straight lines. Current
traces in A were normalized to the current level
at the end of the depolarizations (depolarizations of 5 s were
applied for wild-type channels in Ni2+ to obtain a
steady-state level for normalization (not shown)).
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|
Quantitative Description of b-EAG1 Gating and Mg2+
Regulation--
For a quantitative comparison of the gating kinetics
of mutant and wild-type channels, we built on a gating model introduced by Terlau et al. (13). According to that model, EAG channels undergo two qualitatively different classes of conformational transitions upon depolarization. Rapid activation steps follow a slow
conformational change, which is dependent on extracellular divalent
cations. The latter steps are similar to what has been described for
other voltage-gated potassium channels. Therefore, we assumed at the
holding potential, a binomial distribution of all four individual
subunits between the slow gates being turned on or off. The probability
of being in a "slow" state (p) will be a function of
both the holding potential and the concentration of extracellular
divalent cations. Starting from such a steady-state distribution among
slow and fast states, the activation time course was calculated by
assuming single-exponential relaxation of all "slow" subunits with
a slow rate constant, ks. Fast subunits contribute
to the lumped rapid activation according to kf (Equation 2). Fits according to this simple model (Equation 2 with
n = 4) matched perfectly to experimental data from
b-EAG1, obtained at various concentrations of extracellular
Mg2+ (Fig. 3A,
left, fits in black), regardless of whether prepulses to
130 mV or to
70 mV were applied. The current traces shown represent
the mean currents of six independent experiments. Individual traces
from different experiments were first normalized to the maximum current
level at the end of the test pulse. These traces were then used to
calculate mean current traces. The employed model is obviously well
suited to describe both the hyperpolarization-induced shift and its
regulation by Mg2+. The composition of the mean current
from contributions of fast and slow gating channels is well
demonstrated in 10 mM Mg2+ using a prepulse
voltage of
70 mV (Fig. 3A, lower left panel, lowest trace). A rapidly activating current component is
followed by a slowly activating component. The relative amplitude of
the fast component with about 15% of the total current is in good accordance with a probability of about 38% for individual subunits to
go through the slow gating transition. The two free fit parameters, namely the probability of starting from a slow state, p, and
the time constant for a slow transition, 1/ks, are
plotted as a function of [Mg2+] in Fig. 3, B
and C, respectively (open symbols). Although
p is a function of both, the prepulse potential and
[Mg2+], 1/ks is only a function of
[Mg2+], thus providing a test for the validity of this
simple approach.

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Fig. 3.
Quantitative description of the activation in
various Mg2+ concentrations. A, activation
elicited by voltage steps to +20 mV were measured for the wild type
(open symbols) and L322H (closed symbols) from
holding potentials of 70 (bottom) and 130 mV
(top). The extracellular Mg2+ concentrations
were: 0.2, 0.5, 1, 2, 5, and 10 mM. Data traces
from 6 oocytes were normalized to the current level after 1-s
depolarization. The mean traces are shown in A. A
data fit was performed according to Equation 2 using n = 4, and the fit results (black) are superimposed to the
data (gray). In the left panels, the fit matches
perfectly such that it cannot be distinguished from the data. The two
free parameters of Equation 2, namely the probability of starting in a
slow state (p-slow) (B) and the time constant of
activating a subunit from a slow state (1/ks)
(C), are plotted as a function of the Mg2+
concentration. Data points are connected by straight lines.
Symbols as indicated in A.
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Data for the mutant L322H (Fig. 3A, right) were
analyzed with the same method, using n = 4 in Equation 2. Because of the more rapid activation, fits were only performed to
the first 300 ms after depolarization. Although the initial part of the
traces was described well, a minor third current component with very slow kinetics could not be accounted for. The fit parameters for the
mutant are shown as closed symbols in Fig. 3, B
and C. p was generally much smaller than for the
wild type, and the time constants for the activation of the slow gates
did not depend on [Mg2+]. In addition, this time constant
was similar to the time constant of wild-type subunits in the absence
of Mg2+ (
50 ms). Thus, these results indicate that data
of the wild-type channel were adequately described by Equation 2.
Moreover, the mutant subunits do not seem to contribute significantly
to the slow gating, and they can be safely discriminated from wild-type subunits in high [Mg2+].
Activation of Linked Channel Constructs--
Potassium channels
are formed as symmetric tetramers of
-subunits assembled around a
central pore (19-21). Thus, the S3-S4 linker region, which is
apparently involved in the process leading to slow activation, is
represented in four copies in the functional channel. This raises the
question, whether the slow activation of wild-type channels after
hyperpolarization is a concerted action of all four subunits (mediated
by the binding of up to four Mg2+ ions) or if it is a
function of single and independent gating events. To assess this
problem, we chose the approach of concatenated genes to control the
stoichiometry of fast (L322H) and slow (wild type) gating subunits in
the tetramer. A dimeric construct containing one mutant and one
wild-type subunit was made by linking the cytoplasmic carboxyl terminus
of a mutant to the amino terminus of a wild-type subunit
(m-w, see "Experimental Procedures"). Expression of this construct should result in channels with a 2 + 2 stoichiometry. The
same stoichiometry was expected for another dimer (w-m) and two tetrameric constructs (m-w-w-m, w-m-w-m),
which resulted from the coupling of two dimers. All constructs were
functionally expressed in oocytes. To obtain similar current
amplitudes, mRNA concentrations 5 times and 20 times higher than
for the wild type had to be injected for dimers and tetramers,
respectively. A channel formed by wild-type dimers (w-w) did
not show significantly different activation than channels formed by
wild-type monomers (not shown). As shown in Fig.
4A, all four constructs with a
2 + 2 stoichiometry resulted in intermediate activation kinetics when
compared with wild-type (w-w) or mutant (m)
channels. An additional tetramer construct containing only one
wild-type subunit (m-m-m-w) activated faster than channels
with a 2 + 2 stoichiometry but slower than the mutant monomers. In the
upper panel of Fig. 4A, currents were elicited from hyperpolarized holding potentials in the presence of 5 mM Mg2+, favoring the slow channel activation.
In the lower panel,we applied test pulses from a holding
potential of
70 mV. All traces shown are mean traces from at least 5 individual experiments. Note that the four tested constructs with
theoretical 2 + 2 stoichiometry show similar but not identical
activation kinetics. This probably reflects a possible deviation of the
actual channel assembly from the expected structures, as depicted in
Fig. 4B (see "Discussion"). Apart from these minor
deviations, the linked constructs clearly show that the activation
kinetics of b-EAG channels is determined by the gating properties of
the individual subunits constituting the channel rather than by a
concerted action that requires all four subunits for slow
activation.

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Fig. 4.
Activation of linked constructs.
A, activation time courses of the indicated channel
constructs were measured in 5 mM Mg2+. Voltage
steps from the indicated holding potentials were elicited to +20 mV.
The data traces are mean traces from 5-10 individual
experiments, normalized to the maximal current. It is clearly seen that
an increasing number of wild-type subunits per channel results in a
decreased speed of activation. B, schematic for the ideal
assembly of the constructs to form functional potassium channels.
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In contrast to this additive regulation of the gating kinetics, the
changes in the current-voltage dependence of steady-state currents were
much more dominated by wild-type subunits. The
Vh values of the tetramers with 2+2
stoichiometry (m-w-w-m:
11.1 ± 0.7 mV,
n = 11; w-m-w-m:
6.1 ± 2.3 mV,
n = 7) did not significantly differ from the wild type
(-11.8 ± 2.2 mV, n = 8). Only the
m-m-m-w tetramer was shifted to hyperpolarized potential
(-22.9 ± 0.6 mV, n = 12), though much less
pronounced than the completely mutated channel (-45.1 ± 1.8 mV,
n = 12). Obviously the wild-type subunit, which needs
more positive potentials to gate open, dominates the behavior of the channel.
Quantitative Analysis of Linked Channel Constructs--
The data
shown in Fig. 4 already indicate that an increasing number of wild-type
subunits per channel progressively slow down channel activation. As the
mutated subunits give rise to considerable faster activation and small
p values (see Fig. 3), it might be possible to describe the
data for the linked constructs with a 2 + 2 or 1 + 3 stoichiometry by
using Equation 2 with n = 2 or 1, respectively. In this
case, the mutant subunits are only considered to contribute to the
lumped fast activation component. In addition, it should be assumed
that both rate constants for activation, kf and
ks, are only a function of the test potential and
not of the prepulse potential. Mean normalized currents in 5 mM Mg2+, elicited by test pulses to +20 mV, are
shown in Fig. 5 for the indicated
constructs and prepulse voltages. All recorded currents of the
wild-type construct (w-w) were simultaneously fitted with Equation 2 with n = 4, yielding 1/kf = 10 ms and 1/ks = 215 ms. Note that in contrast to
the fits shown in Fig. 3, we now used more constrained fit conditions,
as nine different activation time courses (corresponding to 9 prepulse
potentials) had to be described simultaneously. Fitted curves are
superimposed (black), indicating that even this heavily
constrained fit describes the data (gray) well. The
resulting p values are plotted in Fig.
6 as a function of prepulse potential
(open circles). The data of constructs m-w and
m-m-m-w were then fitted to Equation 2 assuming n = 2 or 1, respectively. The data fits resulted in
1/kf = 9.9 ms and 1/ks = 152 ms
for m-w and 1/kf = 11.9 ms and
1/ks = 171 ms for m-m-m-w. It is clearly shown that the data are described well. Similar fits with
n = 4 gave consistently worse results (not shown). The
p values are also shown in Fig. 6, indicating that,
similarly to the ks and kf
values, they are rather similar for wild-type subunits in a
homotetrameric channel (w-w). Mutant subunits, however, yield much smaller p values, as already shown in Fig. 3 and
smaller time constants: 1/kf = 7.2 ms and
1/ks = 79 ms (mutants (m) were fitted
with Equation 2 using n = 4). The shown fits and the
high similarity of 1/ks, 1/kf, and p-slow values for the wild-type subunits in all analyzed
constructs strongly support our assumption that slow gating of EAG
channels can be quantitatively described by the independent gating of
the participating wild-type subunits.

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Fig. 5.
Superposition of normalized and averaged data
traces (gray) obtained in 5 mM Mg2+ for voltage steps to +20 mV from the
indicated holding potentials. The data were obtained from oocytes
expressing the indicated channel constructs: w-w (wild-type
dimer, n = 4), m-w (L322H, wild type,
n = 9), m-m-m-w (tetramer with three L322H
subunits and one wild-type subunit, n = 5), and
m (L322H, n = 5). Superimposed to the data
are the results of simultaneous data fits (black). The
theory of these fits considers the number of wild-type subunits per
channel (for details see text).
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Fig. 6.
The p-slow values of the
wild-type subunits as a result of the data fits shown in Fig.
5 are shown here as a function of the holding voltage. For
the monomeric mutant channels, activation was described like that of
the wild type, yielding estimates for the p-slow values of
mutant subunits. Because these are much smaller than those of the wild
type, they were neglected for the theoretical description of the
heteromeric constructs. Data points for the mutant (m) were
connected by straight lines, and the other data points are
superimposed to Boltzmann fit functions (see "Discussion").
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 |
DISCUSSION |
EAG potassium channels activate in a rather unique way as the
kinetics of channel opening strongly depend on the voltage that the
channel proteins experience before depolarization. Hyperpolarizing prepulses result in a slowed activation and cause a more sigmoidal rising phase of the current waveform (9, 11, 13, 22). Similar to
hyperpolarizing prepulses, extracellular divalent cations, e.g. Mg2+, slow the activation kinetics and
increase the sigmoidicity of the rising phase. Because of this
similarity, a common mechanism has been proposed for Cole-Moore shift
and Mg2+ regulation. Mg2+ ions could shift the
equilibrium voltage between two closed states, leading to either slow
or fast activation (13). Detailed analysis of the prepulse dependence
and the effect of extracellular Mg2+ gave rise to a model
in which up to four slow gating steps precede fast channel activation
(13).
Importance of the S3-S4 Linker for Cole-Moore Shift and
Mg2+ Regulation--
In Drosophila EAG, (d-EAG)
channels, Tang and Papazian (18) found that deletions and point
mutations in the extracellular S3-S4 linker had severe effects on the
gating characteristics. A deletion of five charged residues slowed the
activation in the presence of Mg2+ ions. A direct hint,
pointing to the S3-S4 linker as regulator of the Cole-Moore shift, was
the observation that a naturally occurring splice variant (b-EAG2) of
the recently cloned bovine EAG channel is characterized by a 27-amino
acid insertion in this linker, making this protein more sensitive to
[Mg2+]o compared with b-EAG1 (7). Mutagenesis in
b-eag1 now provided further evidence for the importance of
the S3-S4 linker. The single amino acid exchange in the b-EAG1 channel
(L322H) significantly reduced the prepulse dependence of the channels (Fig. 2). Interestingly, in addition to the faster activation, the
voltage dependence of the steady-state current was shifted toward
hyperpolarizing voltages for this mutant (Fig. 1). This is in striking
contrast to a depolarizing shift observed by Tang and Papazian (18) for
the d-EAG L342H mutant. The opposite effects of analogous mutations may
be because of further differences in the S3-S4 linker regions of both
channels (Fig. 1A).
The Mutation L322H Reduces Cole-Moore Shift in b-EAG1--
The
mutation L322H caused a strong reduction of the EAG-specific slow
activation process. In particular, the prepulse dependence and the
influence of extracellular divalent cations was strongly reduced. It
could be speculated that L322H alters a regulatory Mg2+
binding site on the channel and thereby leads to faster channel activation. However, this interpretation would not explain the accelerated activation kinetics of L322H channels even in the absence
of Mg2+. Furthermore, a leucine is very unlikely to
participate directly in binding divalent cations. Thus, it appears that
the mutation primarily reduces the marked prepulse dependence of the
channels but does not abolish the binding of Mg2+ ions.
An Independent Gating Model Describes the Cole-Moore
Shift--
For a quantitative description of the gating
characteristics of wild-type and mutant channels, we assumed a binomial
distribution of four subunits among slow and fast states. This simple
independent model yielded convincing fits to the experimental data,
suggesting that the main effect of the L322H mutation is a strong
reduction of the probability for a subunit to be in the slowly
activating state (p-slow), both in the absence and presence
of Mg2+ ions. One could speculate that Mg2+
binding is reduced in the fast state, but there is no direct evidence
yet. Interestingly, the time constant of L322H subunit activation does
not depend on the Mg2+ concentration anymore, whereas for
the wild-type channels, it is increased by a factor of 2 in 10 mM [Mg2+] (Fig. 3C). Presently, it
is not clear whether or not this results from limitations of the simple
model employed. It also remains to be investigated to what extent
changes in surface potential with increasing [Mg2+] can
account for such an effect. In this respect it is noteworthy that an
analysis of b-EAG1 activation with double exponentials, thus not taking
into account an independent activation of up to four subunits, resulted
in ks values independent of [Mg2+]
(7). Our previous results with rat EAG (13) and the results with
b-EAG1, strongly suggest that the independent model gives rise to
better fits without introducing more free parameters.
Concatenated Gene Constructs of b-eag1 Show Independent Subunit
Gating--
As our independent model for the slow gating of b-EAG
channels was well suited to fit the ionic currents, the Cole-Moore
shift of the channels should be because of a
voltage-dependent gating step that is independent for all
four subunits. To assess this question experimentally, we engineered
channel constructs of concatenated wild-type and mutant eag1
genes, leading to dimers and tetramers with various combinations of the
different subunits. The aim was to control the number of subunits in
the channel that undergo the slow gating transition. Mutant L322H was
well suited for that purpose as its rapid gating property can be safely
distinguished from wild type. Although all combinations gave rise to
functional channels, the expression level decreased with the increasing
number of linked subunits. Although concatenated genes became a common tool to assess subunit stoichiometry (14, 20, 23-28), the properties of such channels cannot necessarily be explained from the expected assembly of the constructs (23, 24). Using dimeric constructs of
Shaker K+ channels, McCormack and co-workers
(23) described a preferential insertion of the NH2-terminal
subunit into the tetramers. Thus, it may happen that functional
tetramers are formed from three or even four dimers if only one-half of
a dimer contributes to the channel. Therefore, quantitative analysis of
linked constructs should include controls with opposite order of the
linked subunits. We constructed m-w and w-m
dimers and found that activation of w-m dimers was slightly
slower than that of m-w dimers (Fig. 4A), suggesting a smaller probability of functional incorporation of the
carboxyl-terminal subunit. The two tetrameric constructs with 2 + 2 stoichiometry showed almost no deviations in the activation kinetics,
regardless of the order of the subunits (m-w-m-w or m-w-w-m), indicating the assembly of the complete entity.
However, all tetramers lead to lower current amplitudes, probably
because of impaired translation/translocation processes for these large proteins. In summary, we found that linked b-EAG1 channels do form
functional complexes that possess almost the expected configuration as
illustrated in Fig. 4B.
Linked Channel Constructs Support Independence of the Slow Gating
Transitions--
After validating the approach of concatemer
expression of b-EAG1 with L322H mutations, we set out to perform a
quantitative description of channel activation, based on the predicted
subunit stoichiometry. As shown in Fig. 5, simultaneous data fits to
whole Cole-Moore data sets resulted in reasonable fits of the wild type and constructs with a 2 + 2 and 1 + 3 subunit stoichiometry. We chose
the tetramer m-m-m-w; given the slightly smaller probability of functional incorporation of the last subunit, any slow activation will be a result of functional constructs of exactly that
stoichiometry. Thus, these results show that the slow,
Mg2+-dependent activation of b-EAG channels is
mediated by the slow activation of each of the four subunits. At
present, we cannot completely exclude that there is a certain degree of
cooperativity among the slow gating steps. We tried to address this
question by comparing the constructs m-w-m-w and
m-w-w-m. As illustrated in Fig. 4B, upon correct
assembly, these constructs both will have a 2 + 2 stoichiometry. In the
latter case, channels contain two wild-type and mutant subunits as
direct neighbors, whereas in the former case, the subunits are
distributed diametrically. Comparison of the activation kinetics in 5 mM Mg2+ from
130 and
70 mV did not reveal
differences between these isoforms. This suggests that the subunit
position within the channel construct is not important for channel
gating. The number of wild-type subunits per channel, however, may
still give rise to cooperative gating. In the framework of the employed
gating model (Equation 2), the dependence of p for wild-type
subunits on the prepulse voltage should not depend on the construct
type. As shown in Fig. 6, this is qualitatively the case when compared
with mutant subunits. However, it seems that the steepness of the
prepulse dependence depends on the number of wild-type subunits per
channel. Boltzmann fits to the data plotted in Fig. 6 reveal slope
factors of 12.6 mV for the wild type (w-w), 18.5 mV for the
dimeric construct m-w, i.e. a 2 + 2 stoichiometry, and 28.2 mV for the tetrameric construct
m-m-m-w, i.e. a 1 + 3 stoichiometry. This
behavior may indeed be indicative of some subunit cooperativity, which
remains to be investigated in more detail.
What can we learn from b-EAG1 L322H about the mechanism underlying the
Cole-Moore shift of EAG channels? The introduced mutation is located
close to the external part of the S4 segment. As it strongly reduced
the prepulse dependence rather than the Mg2+ regulation, it
is suggested that the S4 segment is involved in the conformational
change underlying the slow gating of EAG channels. Thus, it is very
likely that this conformational change also derives its voltage
dependence from the S4 voltage sensor, although operating at a much
more negative voltage than the fast gating, which leads to channel
opening. This interpretation is consistent with our previous results,
which showed that the effect of the NH2 terminus on the
Cole-Moore shift is based on a close contact between this cytoplasmic
domain and the cytoplasmic part of the S4 segment (17).
 |
ACKNOWLEDGEMENTS |
We thank A. Ro
ner and S. Arend for
excellent technical assistance and J. Tytgat for providing the vector pGEMHE.
 |
FOOTNOTES |
*
This work was partially supported by the DFG (SFB197).The costs of publication of this
article were defrayed in part by the payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Tel.:
++49-3641-304541; Fax: ++49-3641-304542; E-mail:
ite{at}rz.uni-jena.de.
 |
ABBREVIATIONS |
The abbreviations used are:
b-EAG, bovine EAG;
d-EAG, Drosophila EAG;
NFR, normal frog Ringer's
solution.
 |
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