Báo cáo khoa học: ` Inhibition of human ether a go-go potassium channels by 2+ Ca ⁄calmodulin binding to the cytosolic N- and C-termini potx

Inhibition of human ether a
`
go-go potassium channels by
Ca
2+
⁄ calmodulin binding to the cytosolic N- and C-termini
Ulrike Ziechner
1
, Roland Scho
¨
nherr
1
, Anne-Kathrin Born
1
, Oxana Gavrilova-Ruch
1
, Ralf W. Glaser
2
,
Miroslav Malesevic
3
, Gerhard Ku
¨
llertz
3
and Stefan H. Heinemann
1
1 Institute of Molecular Cell Biology, Research Unit Molecular and Cellular Biophysics, Friedrich Schiller University Jena, Germany
2 Department of Biochemistry and Biophysics, Friedrich Schiller University Jena, Germany
3 Max Planck Research Unit Enzymology of Protein Folding, Halle, Germany
Ether a

`
go-go (EAG) potassium channels are activated
by membrane depolarization. They are widely
expressed in neuronal tissue suggesting a role in neur-
onal signaling. In addition, they are expressed in var-
ious cancer cell lines and primary tumor material
where they are postulated to play a role in regulating
cell growth [1–3]. One of the most remarkable features
of EAG channels is their regulation by intracellular
Ca
2+
. Unlike Ca
2+
-activated potassium channels of
the big-conductance (BK) or intermediate- and small-
conductance (IK ⁄ SK) family, EAG channels are inhib-
ited when the intracellular Ca
2+
concentration rises
above about 100 nm [4,5]. In a previous study we have
shown that this unique inhibition of the channel by
Ca
2+
is mediated by the Ca
2+
-binding protein
calmodulin (CaM) [6]. In excised inside-out membrane
patches the Ca
2+
sensitivity of human EAG1

channels disappeared, but could be reconstituted by
administration of recombinant CaM. Assaying CaM
binding to GST-fusion proteins of the C-terminal
domain of hEAG1 channels, a binding site was locali-
zed within amino-acids 673–770. This stretch of resi-
dues, harboring a putative amphiphilic CaM-binding
helix as mutations in the CaM-binding motif
(F714SÆF717S or R711QÆR712QÆR716QÆR718Q), pre-
vented Ca
2+
⁄ CaM binding to this C-terminal domain
and produced channels that were insensitive to
Ca
2+
⁄ CaM [6].
Keywords
Calmodulin; calcium; potassium channel;
fluorescence correlation spectroscopy;
patch clamp
Correspondence
S. H. Heinemann, Institute of Molecular Cell
Biology, Molecular and Cellular Biophysics,
Friedrich Schiller University Jena,
Drackendorfer Str. 1, D-07747 Jena,
Germany
Fax: + 49 3641 9 32 56 82
Tel.: + 49 3641 9 32 56 80
E-mail:
(Received 17 October 2005, revised 2
December 2005, accepted 10 January 2006)

doi:10.1111/j.1742-4658.2006.05134.x
Human ether a
`
go-go potassium channels (hEAG1) open in response to
membrane depolarization and they are inhibited by Ca
2+
⁄ calmodulin
(CaM), presumably binding to the C-terminal domain of the channel sub-
units. Deletion of the cytosolic N-terminal domain resulted in complete
abolition of Ca
2+
⁄ CaM sensitivity suggesting the existence of further CaM
binding sites. A peptide array-based screen of the entire cytosolic protein
of hEAG1 identified three putative CaM-binding domains, two in the
C-terminus (BD-C1: 674–683, BD-C2: 711–721) and one in the N-terminus
(BD-N: 151–165). Binding of GST-fusion proteins to Ca
2+
⁄ CaM was
assayed with fluorescence correlation spectroscopy, surface plasmon reson-
ance spectroscopy and precipitation assays. In the presence of Ca
2+
, BD-N
and BD-C2 provided dissociation constants in the nanomolar range,
BD-C1 bound with lower affinity. Mutations in the binding domains
reduced inhibition of the functional channels by Ca
2+
⁄ CaM. Employment
of CaM-EF-hand mutants showed that CaM binding to the N- and C-ter-
minus are primarily dependent on EF-hand motifs 3 and 4. Hence, closure
of EAG channels presumably requires the binding of multiple CaM mole-

cules in a manner more complex than previously assumed.
Abbreviations
apoCaM, Apocalmodulin; BD, binding domain; CaM, calmodulin; EAG, ether a
`
go-go; FCS, fluorescence correlation spectroscopy;
hCaM, human calmodulin; hEAG, human ether a
`
go-go; MBP, maltose-binding protein; SPR, surface plasmon resonance; TMR,
tetramethylrhodamine.
1074 FEBS Journal 273 (2006) 1074–1086 ª 2006 The Authors Journal compilation ª 2006 FEBS
The binding of CaM to the C-terminal domain of
hEAG1 channels is dependent on [Ca
2+
]. In this
respect hEAG channels behave very differently to the
Ca
2+
-activated potassium channel SK2. SK2 a-sub-
units form a very stable heteromeric association com-
plex with the C-terminal domain of Ca
2+
-free CaM,
i.e. apoCaM. The activation of these channels is medi-
ated by conformational changes in the CaM ⁄ SK2 com-
plex induced by binding of at least one Ca
2+
ion to
the EF-hand motifs in the N-terminal domain of CaM
[7,8]. In the deactivated state of rSK2 channels the
rSK2-CaMBD ⁄ CaM complex is a monomer. However,

Ca
2+
binding to the CaM-N-lobe caused by increasing
the intracellular Ca
2+
concentration leads to exposure
of regions in the CaM ⁄ SK2 complex that induce an
interaction between neighboring SK2-CaM binding
domains mediated by Ca
2+
⁄ CaM. This interaction
then results in opening of these channels [9,10].
The mechanism of CaM-mediated closure of hEAG1
channels is far from understood mainly because of the
lack of structural information on the cytosolic channel
domains in complex with CaM. Initiated by the finding
that hEAG1 channels lacking the entire N-terminal
cytosolic domain are insensitive to Ca
2+
⁄ CaM we per-
formed a systematic screen for additional putative
CaM binding domains in the channel protein. Indeed,
we found two more sites, one in the C-terminus neigh-
boring to the previously identified site and one domain
in the N-terminus of hEAG1. The latter one binds
CaM only in the presence of Ca
2+
and is functional in
a sense as point mutations in this domain abolished
Ca

2+
-sensitivity of the channel. Experiments with
CaM-EF-hand mutants testing for interaction with the
functional C- and N-terminal binding sites of hEAG1
channels highlighted the importance of Ca
2+
-binding
ability of EF-hand motifs 3 and 4 in CaM for its inter-
action with the channel.
Results
Relevance of the N-terminus for Ca
2+
⁄ CaM
regulation of hEAG1 channels
As shown previously [6], EAG channels are inhibited
by Ca
2+
⁄ CaM binding to the C-terminal domain.
However, there is no insight into the molecular mech-
anism by which bound Ca
2+
⁄ CaM makes the EAG
channels close. An obvious possibility is that the
bound complex directly occludes the ion pore or
affects the cytosolic gate formed by the S6 segments.
Alternatively, bound Ca
2+
⁄ CaM might allosterically
alter the gating mode of EAG channels such that the
respective subunits are inhibited from being activated

by voltage. To test for such a scenario, channel
mutants with strongly altered gating properties might
be insightful. As it is known that N-terminal deletions
of the EAG protein have strong effects on the voltage
dependent gating steps [11], we constructed a mutant
of hEAG1 in which the entire N-terminal domain was
deleted (hEAG1D2-190 ¼ hEAG1DN).
As shown in Fig. 1B, such channels do form func-
tional channels as for EAG channels the assembly
domain resides in the C-termini [12] and not in the
N-termini as for K
V
channels. However, the gating of
these hEAG1DN channels is strongly altered with
respect to the wild-type channels. The prominent fea-
ture of hEAG1DN channels is their activation at low
voltages and their slow deactivation kinetics. Fitting
a Boltzmann distribution to the normalized current-
voltage relationships yielded a half-maximal activation
voltage (V
m
) for the wild-type subunits of
AC
B
D
E
Fig. 1. Ca
2+
⁄ CaM sensitivity of wild-type hEAG1 channels and
mutant hEAG1D N (A,B) Recordings from inside-out patches con-

taining wild-type hEAG1 (A) and hEAG1DN channels (B) in response
to depolarizing steps from )140 mV to +20 mV in steps of 10 mV,
from a holding potential of )160 mV (C) Current-voltage relation-
ships of the data shown in (A) and (B); currents were measured at
the end of the test pulses. The continuous curves describe the volt-
age-dependent activation of the channels according to Eqn 4. (D,E)
Current recordings of hEAG1 (D) and hEAG1DN channels (E) in
response to test depolarizations to +50 mV (D), and to )20 and
)80 mV (E) under control conditions, i.e. with Ca
2+
-free bath solu-
tion, upon application of the indicated concentrations of hCaM in
the presence of 1 l
M free Ca
2+
solution, and after washing off the
CaM with Ca
2+
-free solutions.
U. Ziechner et al. Calmodulin binding to hEAG1 channels
FEBS Journal 273 (2006) 1074–1086 ª 2006 The Authors Journal compilation ª 2006 FEBS 1075
)55.4 ± 2.9 mV and for hEAG1DNof)129.6 ±
3.3 mV (n ¼ 7; P ¼ 1.2 10
)9
), while the slope factors
characterizing the voltage sensitivities of the gating
steps (k
m
) did not change significantly (30.8 ± 1.6 mV
versus 28.9 ± 2.2 mV, n ¼ 7, P ¼ 0.48). hEAG1DN

channel subunits activate at about 75 mV lower vol-
tages than the wild-type subunits (also see Fig. 1C)
and, hence, have a stronger tendency of opening that
needs to be overcome by Ca
2+
⁄ CaM.
As a control, we exposed inside-out membrane pat-
ches with wild-type hEAG1 channels to 100 nm hCaM
in 1 lm Ca
2+
solutions (Fig. 1D). This resulted in
complete block of EAG-mediated K
+
currents and this
effect was readily reversible when washing the patches
with Ca
2+
-free solutions. The same procedure applied
to hEAG1DN channels had no effect (not shown).
Even 100-fold higher CaM concentrations (Fig. 1E) did
not result in closure of hEAG1DN channels, neither at
low ()80 mV) nor at high ()20 mV) voltages.
Screening for additional CaM-binding sites in
hEAG1 cytosolic domains
The results shown above may indicate that the strongly
altered gating of hEAG1DN channels is responsible for
the abolition of CaM regulation. However, it may also
be that the deleted N-terminal domain takes part in
CaM binding. Thus, we subsequently searched for
additional CaM-binding sites in the cytosolic domains

of the EAG channel protein.
For that purpose we generated peptide arrays on a
cellulose membrane containing peptides of 15 residues
length scanning the entire N-terminal (between amino-
acids 1–206 in steps of 3) and C-terminal (amino-acids
480–962 in steps of 3 and amino-acids 645–895 in steps
of 2) cytosolic domains of hEAG1. Binding of
BODIPY FL-labeled hCaM in the presence of 25 lm
free Ca
2+
was assayed. As shown in Fig. 2A, dots
with different fluorescence intensity indicate the
amount of bound CaM. The fluorescence change (DFI)
with respect to the array prior to CaM treatment was
analyzed for all spots and is plotted in Fig. 2D nor-
malized to the maximum fluorescence increase as a
function of the residue number (corresponding to the
center of the peptides).
There are three sequence regions with a very strong
fluorescence intensity increase over an extensive
sequence range caused by specific binding of labeled
hCaM to these peptides (Figs 2C,D). The most distal
of these regions encompasses the previously identified
A
D
BC
Fig. 2. Peptide array-based screen for putative CaM interaction sites. (A) Fluorescence images of a cellulose peptide array before (left) and
after (right) exposure to BODIPY FL-hCaM. Dark spots indicate CaM binding. The top frame marks the spots encompassing residues 1–205,
the bottom frame the C-terminal residues 480–962. (B) Topology of the hEAG1 channel subunit consisting of six transmembrane segments
and extended cytosolic N- and C-terminal domains. Functional channels are formed by 4 of such subunits involving the C-terminal assembly

domain. (C) Magnified sections of the peptide array: The numbers indicate the first residue of the 15-mer peptides. In the first row (showing
BD-N), the peptide sequences are advanced by 3 residues, in the other rows (showing BD-C1 and BD-C2) sequences are advanced by 2
residues. (D) Increase in fluorescence intensity (DFI in percentage) of peptide spots after BODIPY FL-hCaM binding relative to the intensity
before CaM incubation plotted against the residue number corresponding to the center of the 15-mer peptides (residues scanned: 1–205
and 480–962). The sequence regions with the highest increase in fluorescence intensity (> 90% of maximum DFI, dashed line) were consid-
ered strong CaM-binding domains, here termed BD-N, BD-C1, and BD-C2.
Calmodulin binding to hEAG1 channels U. Ziechner et al.
1076 FEBS Journal 273 (2006) 1074–1086 ª 2006 The Authors Journal compilation ª 2006 FEBS
Ca
2+
⁄ hCaM binding site, here called calmodulin-bind-
ing domain C2 (BD-C2, Figs 2 and 3C). The other
two regions contain a potential binding site in the
N-terminus (BD-N; Figs 2 and 3A) and in the C-termi-
nus (BD-C1; Figs 2 and 3B).
There are additional peaks detected on this peptide
array membrane. However, relative to the already
mentioned potential binding domains they show fluor-
escence intensity changes not greater than 90% and
the peaks do not exhibit extended plateaus.
In Fig. 3, the sequences of the three potential bind-
ing domains are highlighted. The boxes indicate the
sequence regions in which the strongest fluorescence
was detected. The hydrophobic and basic residues,
both expected to be relevant for CaM binding, are
marked in bold and gray, respectively.
For the N-terminal peptides, the fluorescence inten-
sity increases until R162 is inside the peptide sequence,
stays at a high level (DFI > 90%) as long as the pep-
tides contain the amino acids 151–162, and decreases

when the peptides lack F151 (box in Fig. 3A). Thus,
we can notice that BODIPY FL-labeled hCaM binds
to peptides with a 1-8-14 motif, but the hydrophobic
residues V164 and L165 of this motif do not seem to
be important for a strong interaction of hCaM. At
most, these residues should have a stabilizing effect
on the interaction between hCaM and the
hEAG1 N-terminus.
Among the C-terminal peptide spots there are fluor-
escence signals (DFI > 90%) in positions where pep-
tides contain the amino acids R675–I683 (Fig. 3B) and
R711–K721 (Fig. 3C). We previously showed [6] that
replacements of either the basic arginines or both
phenylalanines (underlined in Fig. 3C) in BD-C2 led to
Ca
2+
⁄ CaM insensitivity of hEAG1 channels.
Ca
2+
-dependent binding of hCaM to the
N-terminal binding site
Because the peptides were spotted onto the cellulose
membrane at high concentration, the array assay may
yield some false-positive hits. Therefore, we confirmed
in GST-pull–down assays the association between the
N-terminus of hEAG1 channel and hCaM. Fusion
proteins of GST linked to either hEAG1(1–206) (GST-
N) or hEAG1(674–867) (GST-C34) were bound to
glutathione-sepharose beads and exposed to hCaM
either in absence or presence of 25 lm free Ca

2+
.As
shown in Fig. 4, both GST-N and GST-C34 bound
hCaM, but only in the presence of Ca
2+
(seen as weak
bands at % 16.8 kDa). As CaM bands were absent in
GST controls, a nonspecific binding of CaM to the
sepharose beads or to GST can be excluded. Hence,
also the N-terminal domain of hEAG1 channels har-
bors a functional binding site for CaM that binds
CaM in a Ca
2+
-dependent manner as for the site in
the C-terminus.
CaM affinity to fusion proteins and peptides
of hEAG1 channels
To quantify the interaction strength between Ca
2+
⁄
hCaM and the N-terminal binding site in hEAG1
A
B
C
D
Fig. 3. Comparison of hEAG1 sequence domains that were identi-
fied as CaM binding sites. (A-C) hEAG1 channel sequences, which
provided peptides with the highest binding of BODIPY FL-labeled
hCaM and which were then synthesized as peptides for FCS meas-
urements. 15-mer peptides on the array that contained the

sequence inside of boxes provided highest increase in fluorescence
intensity (DFI > 90%) after assay with BODIPY FL-labeled hCaM on
the peptide array membrane. Hydrophobic amino acids are bold
basic amino acids gray letters. (A) hEAG1(145–165) covers the
potential 1-8-14 motif in the N-terminus. Residue 145 is a cysteine
that was used for labeling of this synthetic peptide for FCS meas-
urements. (B) hEAG1(671–688) contains the potential 1-5-10 motif
in the C-terminus. The boxed cysteine in the beginning does not
belong to the hEAG1 sequence; it was introduced in the synthetic
peptide for labeling. (C) hEAG1(707–725) is around the already
established binding site in the C-terminus. The boxed cysteine has
the same meaning as in B. Replacement of the underlined residues
in hEAG1 channels were previously shown to reduce Ca
2+
⁄ hCaM
sensitivity. (D) Nomenclature of the GST-fusion proteins used in
this study. The fragments marked in black bound CaM with high
affinity in the FCS interaction assay; gray fragments did not show
significant binding.
U. Ziechner et al. Calmodulin binding to hEAG1 channels
FEBS Journal 273 (2006) 1074–1086 ª 2006 The Authors Journal compilation ª 2006 FEBS 1077
channels we performed FCS measurements. MCGS-
(H)
6
-hCaM was labeled with either TMR or BODIPY
FL and its interaction with GST-N was measured. In
the absence of Ca
2+
, no binding between apoCaM
and GST-N was detected (dissociation constant,

K
D
>5 lm). With 25 lm free Ca
2+
in the buffer,
there was a strong interaction between Ca
2+
⁄ hCaM
and GST-N showing dissociation constants of
184 ± 36 nm for TMR-MCGS-(H)
6
-hCaM (Table 1)
and 194 ± 31 nm for BODIPY FL-MCGS-(H)
6
-
hCaM. FCS measurements using (H)
6
-MBP-N instead
of GST-N provided similar results for both, Ca
2+
-free
(K
D
>5lm) and Ca
2+
-containing solutions (K
D
¼
173 ± 22 nm). Nonspecific binding could be excluded
as TMR- or BODIPY FL-labeled Ca

2+
⁄ hCaM did
not bind to GST alone (K
D
>5lm).
Mutations in the potential N-terminal Ca
2+
⁄ CaM
binding site (BD-N), which replaced hydrophobic resi-
dues by asparagines, reduced the interaction strength.
Both GST-N_LN and GST-N_FNÆLN (for mutant
nomenclature see Table 1) showed a weaker binding to
TMR-MCGS-(H)
6
-hCaM in the presence of 25 lm free
Fig. 4. A GST fusion protein containing the hEAG1 N-terminus binds hCaM in a Ca
2+
-dependent manner. The GST-fusion proteins GST-N
and GST-C34 as well as GST alone was bound to glutathione-sepharose and subsequently incubated with purified hCaM, either in presence
or absence of 25 l
M free Ca
2+
ions. After washing, the retained proteins were eluted with SDS ⁄ PAGE loading buffer. The figure shows the
SDS ⁄ PAGE separation gel of the retained proteins and a protein molecular weight marker. Protein bands were stained with Coomassie blue.
For both, GST-N and GST-C34, an hCaM band is only seen in the presence of Ca
2+
(lanes 5, 6 and 9, 10). In the absence of Ca
2+
(lanes 3, 4
and 7, 8) and for the GST control (lanes 1 and 2) there was no retention of hCaM detectable.

Table 1. Binding of MCGS-(H)
6
-hCaM to fusion proteins of cytosolic hEAG1 domains. Dissociation constants K
D
for binding of TMR-MCGS-
(H)
6
-hCaM to the indicated constructs in the absence and presence of 25 lM free Ca
2+
determined by confocal FCS. ND ¼ not determined.
Constructs Short name
K
D
(nM
+
)
+Ca
2+
–Ca
2
GST control GST > 5000 > 5000
GST-hEAG(1–206) GST-N 184 ± 36 > 5000
(H)
6
-MBP-hEAG(1–206) (H)
6
-MBP-N 173 ± 22 > 5000
GST-hEAG(1–206)_L154N GST-N_LN 577 ± 75 ND
GST-hEAG(1–206)_F151NLÆ154N GST-N_FNÆLN 4560 ± 593 ND
GST-hEAG(1–206)_F151SÆL154SÆL158S GST-N_FSÆLSÆLS > 5000 > 5000

GST-hEAG(674–867) GST-C34 127 ± 15 1283 ± 176
GST-hEAG(674–867)_F714SFÆ717S GST-C34_ FSÆFS 735 ± 114 > 5000
GST-hEAG(649–867) GST-C2B34 116 ± 44 1343 ± 213
GST-hEAG(649–867)_F714SÆF717S GST-C2B34_FSÆFS 661 ± 117 > 5000
GST-hEAG(649–867)_F714SÆF717SÆL674NÆI678N GST-C2B34_FSÆFSÆLNÆIN 714 ± 52 > 5000
GST-hEAG(649–867)_F714SÆF717SÆR677NÆR681N GST-C2B34_FSÆFSÆRNÆRN 1340 ± 241 ND
GST-hEAG(649–867)_F714SÆF717SÆR675NÆR677NÆR681NÆK682N GST-C2B34_FSÆFSÆRNÆRNÆRNÆKN 1960 ± 335 > 5000
GST-hEAG(649–867)_R675NÆR677NÆR681NÆK682N GST-C2B34_RNÆRNÆRNÆKN 146 ± 11 1291 ± 139
GST-hEAG(649–700) GST-BDC1 > 5000 > 5000
GST-hEAG(684–867) GST-BDC2 136 ± 19 1239 ± 167
GST-hEAG(479–673) GST-C12 > 5000 > 5000
GST-hEAG(771–962) GST-C45 > 5000 > 5000
Calmodulin binding to hEAG1 channels U. Ziechner et al.
1078 FEBS Journal 273 (2006) 1074–1086 ª 2006 The Authors Journal compilation ª 2006 FEBS
Ca
2+
: The K
D
was increased threefold (577 ± 75 nm)
and 25-fold (4560 ± 593 nm), respectively. Replace-
ment of three hydrophobic amino acids in this putative
1-8-14-Ca
2+
⁄ CaM binding motif by the hydrophilic
serine led to a complete loss of binding (K
D
>5lm,
Table 1).
As the peptide screen identified a putative new CaM
binding site in the C-terminus (BD–C1), the interaction

between TMR-labeled MCGS-(H)
6
-hCaM and GST-
linked fusion proteins of hEAG1 C-terminal fragments
was also quantified (Table 1). The wild-type fragments
GST-C34, GST-C2B34, and GST-BDC2 all bound
CaM in the presence of 25 lm Ca
2+
with K
D
on the
order of 100–150 nm. These fractions have in common
the previously identified CaM-binding domain BD-C2.
In the absence of Ca
2+
these BD-C2 fragments bound
CaM about 10-fold less efficiently. However, compared
to the N-terminal CaM binding site, the C-terminal
site clearly has some finite affinity to apoCaM.
The fragment encompassing only BD-C1 (GST-
BDC1) did not bind CaM with high affinity
(K
D
>5lm), regardless of the Ca
2+
concentration.
The same holds true for the fragments up- and down-
stream of the putative binding regions, i.e. GST-C12
and GST-C45 (Table 1).
Mutations F714SÆ F717S in BD-C2 reduced the K

D
in Ca
2+
-containing solutions to about 660 nm; in the
absence of Ca
2+
no binding could be measured with
FCS. In this background, additional mutations in BD-
C1 only weakly affected CaM binding: L674NÆI678N
had no detectable effect, whereas R677NÆR681N and
R675NÆR677NÆR681NÆK682N increased the K
D
to
about 1.3 and 1.9 lm, respectively. The latter mutation
in the background of GST-C2B34 had no effect. From
these data one can conclude that BD-C1 is not form-
ing a functional CaM-binding site by itself. However,
residues in this domain may interfere with BD-C2 and
may affect CaM binding to the channel protein.
These results could be confirmed by measuring the
association of TMR-labeled synthetic peptides enco-
ding the sequences shown in Fig. 3. The peptide repre-
senting BD-N resulted in a K
D
of 45 ± 9 nm and
peptide BD-C2 in 223 ± 44 nm, while there was no
binding detectable for peptide BD-C1. However, none
of these peptides bound MCGS-(H)
6
-hCaM in the

absence of Ca
2+
including the peptide BD-C2. This
peptide, encompassing amino acids 707–725, may be
too short to bind apoCaM. By contrast, the wild-type
fragments GST-C34, GST-C2B34, and GST-BDC2
contain the complete domain sufficient for a weak
affinity to apoCaM. As BD-C1 seemed to bind CaM
strongly on the peptide array, we reinvestigated the
corresponding peptide using Cy5 as an alternative
fluorescence label. In this configuration the BD-C1
peptide bound CaM in the presence of Ca
2+
with a
K
D
of 327 ± 112 nm; binding of BD-N and BD-C2
were not altered with labeled with Cy5 instead of
TMR.
In FCS measurements fluorescently labeled peptides
of both, the N-terminal or C-terminal binding sites in
hEAG1 channels, could be displaced from binding to
Ca
2+
⁄ hCaM not only by an excess of the same, un-
labeled peptide, but also by an excess of the unlabeled
peptide from the other binding sites (data not shown).
The validity of the results obtained for the binding
of CaM to the N-terminus of hEAG1 channels was
confirmed employing surface plasmon resonance

spectroscopy. Such measurements showed a specific
high-affinity binding of (H)
6
-MBP-hEAG(1–206) to
CaM in the presence of 25 lm Ca
2+
. However, as in
FCS measurements, in the absence of Ca
2+
no specific
binding (K
D
>15lm) was detected. Binding was very
fast such that association and dissociation were mass-
transport controlled and kinetic rate constants could
not be determined. The equilibrium binding response
signal of (H)
6
-MBP-hEAG(1–206) protein with immo-
bilized CaM could be described by Eqn 3 with a disso-
ciation constant of 148 ± 8 nm (see Experimental
procedures). The fit did not show significant systematic
deviations in the range between 0.016 and 10 lm (not
shown).
Functional relevance of CaM binding sites
Mutations inside BD-C2 result in channels insensitive
to Ca
2+
⁄ hCaM (e.g. hEAG_F714SÆF717S [6]). As
mutations in BD-C1 showed some impact on the

CaM binding to the C-terminal binding complex, we
also tested whether such mutations affect the channel
regulation by CaM. Mutant hEAG_R675NÆR677NÆ
R681NÆK682N, destroying the putative CaM-binding
domain C1, was expressed in Xenopus oocytes. In the
presence of 1 lm free Ca
2+
a preparation of hCaM,
reducing the currents through wild-type channels to
20.6 ± 6.3% (n ¼ 7), only reduced the currents medi-
ated by the mutant channels to 89.7 ± 2.3% (n ¼ 6).
To test for the functional importance of BD-N, the
double mutant hEAG_F151NÆL154N was assayed.
200 nm hCaM in 1 lm Ca
2+
solutions to inside-out
patches with wild-type hEAG1 channels resulted in
strong current reduction (to 13.8 ± 3.4%, n ¼ 11).
The mutant was much less sensitive: Application of
500 nm hCaM only reduced the current to 81.9 ±
5.3% (n ¼ 7).
In Fig. 5 A the direct comparison of wild-type,
hEAG_F151NÆL154N and hEAG_R675NÆR677NÆ
U. Ziechner et al. Calmodulin binding to hEAG1 channels
FEBS Journal 273 (2006) 1074–1086 ª 2006 The Authors Journal compilation ª 2006 FEBS 1079
R681NÆK682N with respect to the effect of CaM in
1 lm Ca
2+
using the same batch of oocytes and the
same batch of CaM is shown. The voltage dependence

of channel activation in the mutants was left-shifted
with respect to the wild type, but had no resemblance of
mutant hEAG1DN. V
m
values were: )21.7 ± 2.1 mV
for hEAG_F151NÆL154N and )22.4 ± 3.5 mV for
hEAG_R675NÆR677NÆR681NÆK682N. As can be seen
in Fig. 5A, activation kinetics of mutant hEAG_
R675NÆR677NÆR681NÆK682N seems to be particularly
slow. We thus analyzed the activation time course with
single exponentials at +40 mV and obtained the
following time constants: wild type, 8.4 ± 1.5 ms (n ¼
7); hEAG_F151NÆL154N, 4.7 ± 1.6 ms (n ¼ 7);
hEAG_R675NÆR677NÆR681NÆK682N, 104 ± 12.7 ms
(n ¼ 8).
Contribution of CaM EF-hand motifs
The results shown above indicate that the N-terminus
of hEAG1 channels only binds CaM in the presence of
Ca
2+
, while the C-terminus has some finite affinity
to CaM even in Ca
2+
-free solutions. The reason for
this apparent difference might reside in a different
contribution of the Ca
2+
-binding sites (EF-hands) in
hCaM. Therefore, we compared wild-type hCaM with
EF-hand mutants, in which the relevant aspartates

were replaced with alanines, with respect to the bind-
ing to synthetic TMR-labeled peptides (see Fig. 3A–C)
coding for the putative binding sites in hEAG1.
In the presence of 25 lm free Ca
2+
, the aspartate-
to-alanine mutations in the N-terminal half of CaM
(hCaM-EF12) increased the dissociation constant for
binding of the N1-peptide by a factor of 9 (K
D
¼
393 ± 81 nm compared to 45 ± 9 nm for the wild-
type peptide); EF-hand mutations 3 and 4 abolished
binding (K
D
>5lm for hCaM-EF34 and hCaM-
EF1234). None of the CaM constructs bound to the
peptide in the absence of Ca
2+
. For the TMR-labeled
peptide C2, EF-hand mutations 1 and 2 increased the
K
D
by about a factor of seven (to 1560 ± 190 nm
compared to 223 ± 44 nm for the wild type); EF-hand
mutations 3 and 4 resulted in a complete loss of bind-
ing (K
D
>5lm for hCaM-EF34 and hCaM-EF1234).
EF-hand mutants of hCaM were also employed for

functional tests on hEAG1 channels expressed in Xen-
opus oocytes. The channel-inhibiting potency of wild-
type hCaM and the EF-hand mutants were assayed in
inside-out patches under repetitive channel activation
at +50 mV in solutions containing 1 lm free Ca
2+
.
While 270 nm hCaM reduced the current to 9 ± 3%
(n ¼ 4), 1080 nm hCaM-EF12 reduced the current to
34 ± 10% (n ¼ 4) and 1080 nm hCaM-EF34 had no
effect: 97 ± 8% (n ¼ 3) (Fig. 5B). hCaM-EF1234 had
no effect either (n ¼ 4, not shown).
Discussion
Use of FCS measurements and peptide arrays for
CaM binding site characterization
For the mapping of CaM binding sites at hEAG1
channels the entire cytosolic sequence of the channel
was presented as an array of short peptides, attached
to a cellulose membrane. Detection of CaM binding
was realized by BODIPY FL labeling of recombinantly
produced MCGS-(H)
6
-hCaM. Analysis of this assay
resulted in unambiguous detection of CaM binding
domains when setting a stringent detection threshold
(Fig. 2) and by assuming that a CaM binding domain
has to have a length of at least around 15 residues.
For quantitative estimates of the binding of CaM
and CaM mutants to peptides and fusion proteins we
performed FCS measurements in the autocorrelation

mode. Using fusion proteins as unlabelled interaction
partner, the molecular weight ratio of the fluorescently
labeled molecule and the formed binding complex was
A
B
Fig. 5. Functional impact of mutations in the N-terminal CaM-bind-
ing site and in the EF-hand lobes of hCaM. (A) Normalized current
traces obtained from inside-out patches with wild-type hEAG1
channels (wt), mutant F151NÆL154N (FL) and mutant hEAG_R675NÆ
R677NÆR681NÆK682N (RRRK). ‘–’ and ‘+’ indicate absence and
application of about 1 l
M CaM in 1 lM Ca
2+
solutions. (B) Similar
experiments as in (A) for a single patch with hEAG1 channels
before (–) and after (+) application of the indicated type of CaM in
the presence of 1 l
M free Ca
2+
. Pulses to +50 mV were given;
traces in (B) are shown in a chronological order, scaled to control
currents in order to eliminate effects of current run-down.
Calmodulin binding to hEAG1 channels U. Ziechner et al.
1080 FEBS Journal 273 (2006) 1074–1086 ª 2006 The Authors Journal compilation ª 2006 FEBS
smaller than 1 : 6 in all cases. Therefore, great caution
had to be taken in validating the results. For that rea-
son we also employed surface plasmon resonance
spectroscopy for measuring the interaction of immobi-
lized CaM with (H)
6

-MBP-N in the presence of Ca
2+
yielding approximately the same dissociation constant
(% 150 nm) as determined with FCS (% 170 nm,
Table 1).
Quantitative estimates of the binding constant
between peptides encoding the minimal CaM-BDs and
fragments of the hEAG1 protein, encompassing more
than just the binding site, resulted in a fourfold stron-
ger binding of the BD-N peptide and a twofold weaker
binding for the BD-C2 peptide. In the first case this
could be due to a better accessibility of CaM to the
site. In the latter case it may indicate that the peptide
does not contain all critical residues necessary for com-
plexing CaM with the channel protein. It cannot be
excluded, however, that these differences are brought
about by the specific fluorescence labels at the pep-
tides. Such an effect was observed for BD-C1: The
TMR-labeled peptide encompassing this domain did
not bind Ca
2+
⁄ CaM, while Cy5 as label did not
impede binding.
Functional properties of N-terminally deleted
hEAG1 channels
Potassium channels of the EAG family assemble via
the C-terminal ends of their a-subunits [12]. Therefore,
complete removal of the N-terminus (hEAG1D2-190)
still results in functional K
+

channels as previously
shown for rat EAG1 [11]. However, the activation
threshold of these channels is shifted by about )75 mV
and deactivation of the channels is slow. As a result,
these channels are constitutively open around the nor-
mal resting potential of an excitable cell. In addition,
hEAG1DN channels inactivate at higher voltages (not
shown here). The inference to be drawn for the gating
of EAG channels is that their N-terminal domains are
essential for the stabilization of the closed channel
state. As pointed out by Terlau et al. [11], a direct
interaction of the N-terminal domain with the intracel-
lular S4-S5 linker may take part in this stabilization.
For the subject studied here one can conclude that
CaM is unable to close the hEAG1 channels in the
absence of the N-terminal domain.
Mutations in the N- and C-terminal CaM binding
sites also affected the gating parameters of the chan-
nels, but to a much smaller extent than the complete
deletion of the N-terminus. Remarkably, mutations in
the N-terminus (hEAG_F151NÆL154N) markedly slo-
wed down channel activation further supporting the
notion that the N-terminal domain takes part in chan-
nel gating.
The N-terminus of EAG channels harbors a
functional CaM binding site
Using a peptide array representing the entire cytosolic
domains of human EAG1 channels we identified three
regions where fluorescently labeled CaM binds. One
binding site in the C-terminus (BD-C2) overlapped

with that previously reported [6]. In its vicinity there is
an additional motif with CaM-binding capacity
(BD-C1). CaM binding to this site seems to be weak,
but functional assays led us to conclude that this puta-
tive binding site is relevant for channel inhibition by
Ca
2+
⁄ CaM under in vivo conditions.
The third site, located in the N-terminus (BD-N),
bound CaM in an in vitro assay but was also necessary
for function. Mutagenesis of this site resulted in a loss
of CaM sensitivity. Likewise, complete deletion of the
N-terminus rendered the channels insensitive to CaM.
Thus, it is very likely that the altered CaM sensitivity
of the hEAG1DN mutant is not due to its strongly
modified gating properties. Instead, binding of CaM to
the N-terminal domain is needed for channel closure.
However, it cannot be excluded that the specific inter-
action of the N-terminus with the voltage sensor ele-
ments of the channels is involved in CaM-mediated
channel closure.
Type of CaM binding motif
Most CaM-BD peptides bind Ca
2+
⁄ CaM with a K
D
of
about 0.1–100 nm [13] and this is also true for BD-N
and BD-C2 of hEAG1 channels. Although quite
diverse in structure, some types of CaM binding motifs

can be specified [14]. The previously identified BD-C2
shows some resemblance to a 1-5-8-14 motif where
position ‘14’ is occupied by an alanine (Fig. 3C). This
may be the reason why this motif separated in the BD-
C2 peptide binds CaM in a strictly Ca
2+
-dependent
manner; in the absence of free Ca
2+
a weak binding
was only detected if this BD-C2 was included in wild-
type hEAG1 protein fragments. The N-terminal bind-
ing domain BD-N is of the 1-8-14 type and BD-C1
shows a 1-5-10 motif. While BD-N clearly falls into this
category by binding CaM in a strictly Ca
2+
-dependent
manner (% 40 nm for the peptide), BD-C1 showed wea-
ker binding to CaM, even in the presence of Ca
2+
, but
mutagenesis of the BD-C1 site in the background of a
fragment containing a mutated BD-C2 site reduced the
binding affinity of CaM. This could mean that the BD-
C1 site contributes to the coordination of CaM that
U. Ziechner et al. Calmodulin binding to hEAG1 channels
FEBS Journal 273 (2006) 1074–1086 ª 2006 The Authors Journal compilation ª 2006 FEBS 1081
primarily binds BD-C2. Both sites together may form a
complex CaM-binding structure.
Importance of EF-hands 3 and 4

As BD-N bound CaM only in the presence of Ca
2+
,
while BD-C2 also showed some weak binding of CaM
in the absence of Ca
2+
, it was feasible that the inter-
action of CaM with the respective binding sites is
dominated by different sets of EF-hand motifs. Assay
of hCaM-EF12 and hCaM-EF34 mutants for binding
to peptides coding for the binding sites showed that in
both cases, i.e. for BD-N as well as for BD-C2, lack
of EF-hand motifs 1 and 2 only resulted in a mild
reduction of binding (factor 7 and 9, respectively),
while EF-hand motifs 3 and 4 were essential. The
same results were obtained for functional assays on
hEAG1 channels expressed in Xenopus oocytes. Thus,
both CaM binding domains seem to require a func-
tional C-lobe of CaM. This result appears quite rea-
sonable as the binding constant of Ca
2+
to the C-lobe
of CaM is up to 10-fold smaller than that for the
N-lobe [13,15]. Upon binding of Ca
2+
to the C-lobe,
CaM undergoes a first conformational change, expos-
ing a hydrophobic binding pocket and, hence, promo-
ting binding to the target (e.g. [13,16,17]). Binding to
the target can increase the Ca

2+
-binding affinity to
CaM further [17,18]. In addition, subsequent binding
of Ca
2+
to the N-lobe then induces further conforma-
tional changes of CaM [19,20] that may trigger an
increase of CaM affinity to the target protein. Thus,
the importance of functional EF34 domains of CaM
for inhibiting hEAG1 channels indicates that associ-
ation of Ca
2+
-bound CaM to the channel is a necessary
requirement. Interestingly, Ca
2+
binding to the N-lobe
facilitates channel closure as more CaM-EF12 than
wild-type CaM is needed for an equivalent effect
(Fig. 5). However, binding of Ca
2+
to the N-lobe does
not seem to be a strict requirement, providing some
insight into the underlying molecular mechanism.
Mechanism of channel regulation
Several ion channels are regulated by cytosolic Ca
2+
by means of CaM. Among the channels with a-sub-
units of the 6-TM family similar to hEAG1, Ca
2+
-

activated K
+
channels of the IK ⁄ SK type [7,8,10],
nonselective cation channels gated by cyclic nucleotides
(CNG) [21–25], and voltage-gated channels of the
KCNQ [26] type are known to interact with CaM.
hEAG1 channels studied here share some properties
of CNG channels and of KCNQ channels. On the one
hand they are activated by membrane depolarization
and do not inactivate as do KCNQ channels. On the
other hand EAG channels harbor a C-terminal puta-
tive binding site for cyclic nucleotides; however, thus
far there is no compelling evidence for a functional
impact of this site. In terms of Ca
2+
regulation,
hEAG1 channels are readily inhibited by Ca
2+
⁄ CaM
at low Ca
2+
concentrations [6] and low CaM concen-
trations (Fig. 1). As for CNG channels, hEAG1
harbors CaM-binding motifs in the N- and C-terminal
domain and both of them are functional in a sense
that mutagenesis inside these domains results in a loss
of Ca
2+
sensitivity. Given the similarity to CNG chan-
nels it is tempting to assume that hEAG1 channels are

gated via CaM by a distortion of an interaction
between the N- and C-termini. We do not think that
this is a likely scenario, because in precipitation assays
with N- and C-terminal protein fragments of hEAG1
we could not detect interaction, neither alone nor
in the presence of CaM (not shown). In addition,
peptides encoding the N- and C-terminal binding
domains competed for CaM binding.
With respect to Ca
2+
regulation the strongest simi-
larity of hEAG1 channels can be found to heteromeric
olfactory CNG channels. In both cases N- and C-ter-
minal CaM-BDs associate with CaM at low Ca
2+
con-
centration resulting in channel closure. However, as
for hEAG1 channels the mechanism is not clear [27].
In summary, the identification of three functional
CaM-binding sites in hEAG1 channels shows that the
mechanism of CaM-induced channel closure is much
more complex than previously anticipated. In partic-
ular, the molecular mechanism of EAG channel regu-
lation via Ca
2+
⁄ CaM markedly differs from those of
other ion channels of the 6TM gene family.
Experimental procedures
Production of calmodulin
The cloning of full-length cDNA encoding hCaM was pre-

viously described [6]. Point mutations were generated by
overlap extension PCR [28]. EF-hand mutants used were:
hCaM-EF12 (D21AÆ D57A), hCaM-EF34 (D94AÆD130A),
and hCaM-EF1234 (D21AÆD57AÆD94AÆD130A).
(H)
6
-hCaM and (H)
6
-hCaM EF-hand mutants were
cloned into pQE30, expressed in E. coli M15 cells, and
purified with Ni-NTA agarose according to the suppliers
protocols (Qiagen, Hilden Germany). Integrity of the pro-
teins was verified with SDS ⁄ PAGE analysis.
For labeling of CaM the arginine residue in the His-tag
(MRGS-(H)
6
) was replaced by cysteine. The resulting
MCGS-(H)
6
-hCaM was produced as described, followed by
hydrophobic interaction chromatography according to a
Calmodulin binding to hEAG1 channels U. Ziechner et al.
1082 FEBS Journal 273 (2006) 1074–1086 ª 2006 The Authors Journal compilation ª 2006 FEBS
protocol by Hayashi et al. [29] using a phenyl-sepharose
6FF (high sub) column from Amersham Biosciences (Frei-
burg, Germany).
For GST precipitation assays and FCS measurements
with labeled peptides wt-hCaM was used. From a pET-14b
vector it was expressed in E. coli BL21 cells and purified by
phenyl-sepharose chromatography [29].

Production of hEAG1 fusion proteins
Cloning of full-length cDNA encoding hEAG1 was pre-
viously described [6] and point mutations were generated by
overlap extension PCR. GST fusion proteins, encoded in the
pGEX-5X vector (Amersham Biosciences), were expressed
in E. coli BL21 cells and purified using GSH sepharose.
(H)
6
-MBP fusion proteins of hEAG1 were produced using
the pETM 40 vector from EMBL (Heidelberg, Germany) in
E. coli BL21 cells and purified on Ni-NTA agarose.
Labeling of MCGS-(H)
6
-hCaM with BODIPY FL
or TMR (tetramethylrhodamine)
BODIPY FL N-(2-aminoethyl)maleimide and TMR-6-
maleimide were purchased from Molecular Probes (Eugene,
OR, USA). A 10-fold molar excess of reactive dye
(BODIPY FL- or TMR-maleimide in dimethyl sulfoxide)
was added to purified MCGS-(H)
6
-hCaM in 20 mm Tris
buffer (pH 7.4). This mixture was incubated under shaking
for 60 min at room temperature. Subsequently, the excess of
dye was inactivated with a fivefold molar excess (relative to
the reactive dye) of glutathione under shaking for 20 min.
The dye-labeled hCaM was separated by size exclusion
chromatography using a HiPrep 16 ⁄ 60 Sephacryl S 200
HR-column (Amersham Biosciences) and 50 mm phosphate
buffer (pH 7.4) with 500 mm NaCl and 2 mm glutathione.

Synthesis and labeling of peptides
Three peptides encompassing putative CaM-binding sites of
hEAG1 (BD-N, BD-C1, and BD-C2) were synthesized
using standard protocols. As illustrated in Fig. 3(A–C), the
BD-N naturally starts with a cysteine residue; in the other
peptides a cysteine was placed at the N-terminus before the
natural hEAG1 sequence. These peptides were labeled at
their N-terminal cysteines with TMR or Cy5 (Amersham
Biosciences) according to the same procedure as applied for
MCGS-(H)
6
-hCaM. Size exclusion chromatography was
performed with a Superdex Peptide PE 7.5 ⁄ 300 column
(Amersham Biosciences).
GST precipitation assay
GST-fusion proteins bound to glutathione-sepharose were
washed three times with Ca
2+
-free or Ca
2+
-containing
buffer and then incubated with purified hCaM either in the
presence or absence of Ca
2+
for 10 min at room tempera-
ture. Following threefold washing of the beads with the
relevant buffer, the resin-bound proteins were eluted with
SDS ⁄ PAGE loading buffer. Subsequently, SDS ⁄ PAGE
analysis was performed and protein bands were stained
with Coomassie blue.

The Ca
2+
-free buffer contained (in mm): 100 K-aspar-
tate, 15 KCl, 10 EGTA, 2 glutathione, 10 Hepes (pH 7.2
with KOH). The Ca
2+
-containing buffer consisted of (in
mm): 115 K-aspartate, 8.9 CaCl
2
, 10 hEDTA, 2 glutathi-
one, 10 Hepes (pH 7.2 with KOH) yielding 25 lm free
Ca
2+
ions.
Peptide array assay
Arrays of immobilized peptides (15-mers) comprising
sequences of the hEAG1 potassium channel with a shift of
2 or 3 amino acids were prepared by automated spot syn-
thesis on Whatman 50 filter paper (Whatman, Maidstone,
England). Peptides were C-terminally attached to cellulose
via a (beta-Ala)
2
spacer. The quality of the synthetic pep-
tides on spots was evaluated by mass spectrometry analysis
and the amount of peptides on spots was determined by
HPLC analysis after releasing the peptides from cellulose
membrane using ammonia vapor [30,31].
Before screening, the dry cellulose membrane was pre-
treated with methanol for 10 min and then equilibrated,
first, five times for 20 min with a Tris-buffer (30 mm

Tris ⁄ HCl, 170 mm NaCl, 6.4 mm KCl, pH 7.6) containing
1% BSA and 1% sucrose to prevent unspecific binding to
the membrane surface during the assay and, second, three
times for 10 min in binding buffer with 25 lm free Ca
2+
.
In order to investigate which peptides on the membrane
are able to interact with hCaM, 10 lm BODIPY FL-labeled
hCaM in the same Ca
2+
-containing buffer was added to
the membrane surface. After 30 min incubation, the
unbound excess of labeled hCaM was removed by washing
the membrane in Ca
2+
-containing buffer. Subsequently, the
fluorescence was measured with a fluorescence scanner,
FLA-5000 (FUJIFILM Europe GmbH, Du
¨
sseldorf,
Germany). Quantification of signal intensity was performed
by analysis of the spot intensity using nih image Software.
Confocal fluorescence correlation spectroscopy
(FCS)
FCS measurements were performed in vitro using Confo-
Cor-2 equipment (Carl Zeiss AG, Jena, Germany) in auto-
correlation mode. The smaller interaction partner was
labeled with either TMR, Cy5 or BODIPY FL. These fluor-
escence dyes were excited with a HeNe laser (543 nm or
633 nm) or by an Ar

+
laser (488 nm), respectively, and the
emitted photons were detected using emission filters LP560,
U. Ziechner et al. Calmodulin binding to hEAG1 channels
FEBS Journal 273 (2006) 1074–1086 ª 2006 The Authors Journal compilation ª 2006 FEBS 1083
LP650, or BP505-550, respectively. Autocorrelation analysis
was performed with the confocor-2 software according to
Eqn 1:
GðsÞ¼
1
N
1 þ
T
1 À T
e
Às=s
T

X
m
i¼1
y
i
1 þ
s
s
Di

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1 þ

s
s
2
s
Di
q
0
B
@
1
C
A
þ 1
ð1Þ
with T, triplet fraction; s
T
, decay time of triplet state; N,
average number of fluorescent molecules in the detection
volume; m, number of fluorescent components; y
i
, contribu-
tion of i-th fluorescent component; s
Di
, diffusion time of
i-th fluorescent component; and S, structure parameter.
We measured the labeled C-hEAG1-CaM-BD-peptide or
labeled MCGS-(H)
6
-hCaM alone and determined the diffu-
sion time s

D,free
and the structure parameter. The diffusion
time of the complex was estimated from the molecular
weight of the complex and free ligand (Eqn 2).
s
D;complex
¼ s
D;free
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
M
complex
M
free
3
s
ð2Þ
The equilibrium dissociation constant K
D
was calculated
based on the assumption that there is only one binding
site.
Surface plasmon resonance (SPR) spectroscopy
The real–time interaction between the (H)
6
-MBP fusion
protein of hEAG(1–206) and immobilized hCaM was ana-
lyzed using the BIAcore 2000 system (Biacore AB, Sweden).
(H)
6
-hCaM was covalently attached to the sensor surface

with a carboxylated dextrane matrix (CM5 sensor chip).
(H)
6
-MBP fusion protein of hEAG1(1–206) was diluted
into a buffer containing (in mm): 10 Hepes (pH 7.2), 100
K-aspartate, 40 KCl, and 0.005% surfactant P20 and either
8.9 CaCl
2
, 10 hEDTA (free Ca
2+
concentration of 25 lm)
or 10 EGTA (Ca
2+
-free buffer). The SPR measurements
were performed with a flow rate of 20 lLÆ min
)1
at 20 °C.
To determine the affinity of (H)
6
-MBP-hEAG(1–206) for
apoCaM or Ca
2+
⁄ CaM in a direct binding assay, variable
concentrations of (H)
6
-MBP-hEAG(1–206) protein were
applied to the immobilized CaM on the sensor surface and
the binding response was detected. A fit of the equilibrium
binding response signal (R
eq

) versus analyte concentration
(C
A
) yielded the dissociation constant (K
D
), where R
max
is
the maximal resonance signal as shown in Eqn 3:
R
eq
¼
C
A
K
D
þ C
A
R
max
ð3Þ
Functional expression of hEAG1 channels
Capped mRNA was synthesized in vitro using the mMes-
sage mMachine kit (Ambion, Austin, TX, USA).
Stage-V oocytes from Xenopus laevis were surgically
obtained under ice water ⁄ tricaine anesthesia according to
the local animal care program. Manually defolliculated
oocytes were microinjected with channel-coding mRNA
(< 50 nL).
Currents were measured 2–4 days after mRNA injection

in the inside-out patch-clamp configuration using aluminum
silicate glass pipettes with resistances of about 1 MW.As
patch-clamp amplifier an EPC-9 operated with PatchMaster
software (both HEKA Elektronik, Lambrecht, Germany)
was employed. The bath solutions contained (in mm): ‘1 lm
Ca
2+
’: 115 K-aspartate, 10 EGTA, 8.73 CaCl
2
, 10 Hepes
(pH 7.2 with KOH) or ‘Ca
2+
-free’: 100 K-aspartate, 10
EGTA, 15 KCl, 10 Hepes (pH 7.2 with KOH). The pipette
solution was composed of 103.6 Na-aspartate, 11.4 KCl,
1.8 CaCl
2
, 10 Hepes (pH 7.2 with NaOH). The concentra-
tion of free Ca
2+
ions in the bath solutions was calculated
according to Tsien and Rink [32]. As EGTA is not a good
buffer for high concentrations, the actual free Ca
2+
concen-
tration is higher than predicted (about 1.5 lm) as verified
with Fura-2 fluorometry. Important for this study, we
assured that addition of 1 lm CaM to the Ca
2+
-containing

buffer did not alter the concentration of free Ca
2+
ions.
For application of His-tagged hCaM, the purified protein
was transferred to the nominally ‘1 lm Ca
2+
’ solution with
1mm dithiothreitol. For solution change around the
excised patches we used a multichannel perfusion system
and placed the excised patch directly in the middle of
streaming solution.
Data were analyzed with FitMaster (HEKA Elektronik)
and IgorPro (WaveMetrics, Lake Oswego, OR, USA).
Unless otherwise stated, data are presented as mean ±
SEM (n ¼ number of independent measurements). Statisti-
cal tests were performed with two-sided Student’s t-tests
with unequal variances and the resulting P-values are given.
Voltage-dependent activation of hEAG1 channels was
estimated by fitting the steady-state current as a function of
test potential according to a Hodgkin & Huxley formalism
with m ¼ 4 activation gates and a single-channel character-
istics according to Goldman-Hodgkin-Katz (Eqn 4):
I
peak
ðVÞ¼
I
max
1 þ e
ÀðVÀV
m

Þ=k
m
ðÞ
4
I
max
¼ CV
1 À e
ÀðVÀE
rev
Þ=25mV
1 À e
ÀV=25mV
ð4Þ
V
m
is the voltage of half-maximal gate activation and k
m
the corresponding slope factor. G is the maximal conduct-
ance of all channels and E
rev
the reversal potential.
Acknowledgements
This work was supported by the Deutsche Fors-
chungsgemeinschaft SFB 604 (TP A4 and B4). We like
Calmodulin binding to hEAG1 channels U. Ziechner et al.
1084 FEBS Journal 273 (2006) 1074–1086 ª 2006 The Authors Journal compilation ª 2006 FEBS
to thank Dr D. Wicher for helpful comments, and Dr
F. Große for sharing the SPR facility.
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