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(Received for publication, September 6, 1994; and in revised form, December
1, 1994) From the
Slowly activating I
Expression of the I Oligonucleotide-directed mutagenesis(7) , in
vitro RNA synthesis, oocyte handling, and injection have been
described previously(8) . Xenopus oocytes were
injected with 1 ng of cRNA/oocyte. The two-microelectrode voltage clamp
configuration was used to record currents from Xenopus laevis oocytes 2-10 days after cRNA injection. Recordings were
performed at 22 °C using a Geneclamp amplifier, pClamp software for
data acquisition and analysis (Axon Instruments, Foster City, CA), and
a Kipp & Zonen chart recorder. If not otherwise stated, I
Figure 1:
Inhibition of I
The membrane-permeable, thiol group
oxidizing reagent DTNP inhibited I In the
intracellular domain of the I
Figure 5:
Determinants for I
Figure 2:
Effects of Hg
The described
positive regulatory effects of Hg
Figure 3:
Use-dependent and use-independent effects
of Hg
Because the predicted chelation
with Hg
Figure 4:
Effects of Hg
Redox
reactions at a Cys have also been shown to regulate the inactivation of
neuronal I The results presented here cannot
exclude the possibility that the I
Volume 270,
Number 8,
Issue of February 24, 1995 pp. 3638-3641
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Regulation by Oxidation or Chelation (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
channels were expressed in Xenopus oocytes and exposed to oxidative agents. Oxidative
treatment reduced the resulting current I, while no
inhibition was observed for I protein mutants carrying a
Ser mutation instead of a highly conserved Cys residue in the
intracellular domain. In contrast, Hg
, which may not
only oxidize thiol groups but also form chelates with dibasic amino
acids, caused a use-dependent, positive regulation of I.
This effect was reversed in an I protein mutant with a
deletion in the extracellular domain. These data suggest opposite
effects of peroxides and Hg on I, a
peroxide-mediated I inhibition by intracellular oxidation
and a Hg-mediated I increase, caused by
extracellular Hg chelation of the I protein.
protein in Xenopus oocytes (1) or HEK 293 cells (2) induces slowly
activating potassium currents, although it is structurally and
functionally distinct from other potassium channel proteins. The
quaternary structure of I channels as well as the
mechanism of I activation are unknown. In contrast, much
is known about regulation of I by a number of second
messengers and kinases, such as
[Ca]
and kinases A and
C (for review, see (3) ). The physiological role of I proteins is best defined in heart, where they mediate the action
potential repolarizing conductance I
(4) .
Interestingly, novel class III antiarrhythmics have been shown to
potently inhibit I expressed in oocytes as well as
I in cardiac myocytes(5) . Inhibition of I may therefore be involved in the mechanism of their
antiarrhythmic action. Since localized inhibition of I by
peroxides may be involved in the genesis of reperfusion-induced
arrhythmias(6) , the influence of oxidation on I channels was investigated.
was evoked with 15-s voltage steps to -10 mV from a holding
potential of -80 mV every 45 s. This protocol was sufficient to
evoke significant I currents without activating
significant endogenous Ca-activated Cl
currents. The I amplitudes were measured at the end
of the depolarizing voltage steps. The superfusing solution contained
(mM): 96 NaCl, 2 KCl, 1.8 CaCl
, 1
MgCl, 5 HEPES (titrated with NaOH to pH 7.4). The
microelectrodes were filled with 3 M KCl solution and had
resistances between 0.6 and 1.3 megohms. Chemicals were added from
stock solutions into the superfusion solution. Chemicals used were:
NE-10064 (
)(1-[[[5-(4-chlorophenyl)-2-furanyl]methylene]amino]-3-[4-(4-methyl-1piperazinyl)butyl]-2,4-imidazolidinedione
dihydrochloride; gift from Procter & Gamble Pharmaceuticals, Inc.).
Dithioerythritol (DTE), 2,2`-dithiobis(5-nitropyridine) (DTNP),
5,5`-dithiobis(2-nitrobenzoic acid), and tert-butylhydroperoxide were purchased from Sigma. Data are
presented as means with standard errors (S.E.), where n represents the number of experiments
performed. The paired Student's t test was used to
calculate statistical significance.
Effects of Peroxides and Oxidative Agents on
I
Human (9) (h-) or rat (1) (r-)
I proteins expressed in Xenopus oocytes induced
the characteristic slowly activating potassium current I during depolarizations to potentials more positive than -50
mV. Superfusion with HO (3 mM) caused
an inhibition of -50.1 ± 2.9% (n = 7; Fig. 1, B and C) and -62.2 ± 2.7% (n = 8) of h- and r-I within 15 min,
respectively. Inhibition of I with HO was enhanced by prolonged superfusion periods (>30 min).
Addition of the reducing agent Dithioerythritol (DTE; 5 mM)
completely reversed the HO-mediated inhibition.
Moreover, DTE itself increased r- and h-I by 14.3 ±
3.5% (n = 4) and 31.0 ± 5.7% (n = 4), respectively, suggesting that a part of the I protein population may normally be oxidized. Analysis of r- and
h-I revealed no alterations in the complex activation and
deactivation kinetics of the currents by HO (data not shown; n = 6 and 4, respectively). tert-Butylhydroperoxide (1 mM), another peroxide,
reduced h-I within 10 min by -52.8 ± 5.7% (n = 4).
by DTNP
and HO. A and B, DTNP (100
µM) and HO (3 mM) were
added to the control solution for 15 minutes as indicated by the horizontalbars. The upward deflections reflect rat
(r)-I wild-type (WT), which was evoked with 15 s
depolarizing steps to -10 mV every 45 s from a holding potential
of -80 mV. C, HO inhibits wild-type (WT) human (h)-I, but not h-I C106S (D), an I protein mutant, in which an
intracellular Cys residue was mutated to a Ser. E, relative
change (means ± S.E.) of outward current amplitudes after 15 min
HO superfusion for wild-type h- and r-I and for the mutants h-I C106S, r-I C107S, and r-I(del 10-39)/A94-. The latter
mutant lacks the extracellular amino acids 10-39 and the
intracellular tail from amino acid 94 to the protein end (including
C107). The bars represent the arithmetic means (±
S.E.).
similar to
HO (Fig. 1A), while the
impermeable analog 5,5`-dithiobis(2-nitrobenzoic acid) had no effect.
At a concentration of 100 µM, DTNP caused within 15 min a
decrease of h- and r-I amplitude of -83.0 ± 1.0% (n = 5) and -76.4 ± 2.0% (n = 7), respectively, and almost completely suppressed
h-I after an extended superfusion period (>30 min; n = 5). This inhibition was partially reversible during
washout (to 47 and 61% of control r- and h-I) and was
completely reversed by DTE (5 mM) (108.0 ± 7.5% of h-
and 97 ± 3% of r-I; n = 5 and 7,
respectively). These results suggest that I can be
inhibited by intracellularly acting thiol-modifying agents. protein resides a highly
conserved Cys (1, 4, 9, 10) in all
species. Mutation of this Cys to a Ser in the r- and h-I protein (r-I C107S and h-I C106S)
resulted in proteins, which induced upon expression in Xenopus oocytes potassium currents with similar general properties to the
wild-type I under control conditions. However, as shown in Fig. 1D, mutation of Cys to Ser in the I protein abolished the inhibitory effect of
HO. Under HO (3 mM for 15 min) h-I C106S was 113.5 ± 5% of
control (n = 7) and r-I C107S was 100.4
± 2.1% of control (Fig. 1E; n =
6). Protein mutants, which lacked the intracellular part Ala-94 to
Ser-130, induced I with the same general properties as the
wild-type proteins(7) , but the resulting current could also
not be inhibited by HO (n = 4;
see Fig. 1E). These data suggest that I is
negatively modulated by oxidation of a highly conserved intracellular
Cys residue of the I protein. The inhibition may be caused
by sequestration of I proteins and/or an impairment of
mobility of the intracellular protein tail (see Fig. 5). There
were no obvious changes in the rate of I activation after
peroxide-mediated inhibition, which supports the hypothesis that after
oxidation simply less I proteins can be recruited for
channel formation. When the highly conserved Cys residue is substituted
by an amino acid which cannot form disulfide-bonds or when it is simply
deleted, the mutant protein cannot be sequestered by oxidation.
activation
and Hg-mediated positive regulation. A,
depolarizations induce I, when the intracellular domain is
not sequestered (i.e. when the Cys residue is reduced in the
wild-type protein or is mutated or deleted for the described I mutants). Hg can only form I protein chelates after I activation, which implies
that conformational changes occur in the extracellular domain during
channel activation. B, oxidation of the intracellular Cys
residue results in the formation of disulfide bonds with another
protein, thereby sequestering the number of I proteins,
which can be recruited for the formation/induction of a functional
channel.
Effects of Hg
In contrast to H on
IO, the
heavy metal Hg not only oxidizes thiol groups but
also causes the formation of chelates with dibasic amino acids.
Superfusion with Hg (1 µM) resulted in
an use-dependent increase of the outward current amplitude to 221.2
± 21.6% (n = 6) of control within 15 min, which
was mainly the consequence of a retarded deactivation of
I. Under repetitive stimulation the holding current at -80
mV shifted in outward direction and an instantaneous current appeared (Fig. 2, A, C, and F). Both
time-dependent I and the instantaneous current under
Hg could be almost completely inhibited with the
I blocker NE-10064 (5) (Fig. 2, B, D, and G; n = 6), suggesting that the
instantaneous currents were indeed currents through delayed
deactivating I channels. These currents displayed an
almost linear current-voltage relationship with reversal potentials of
-95.9 ± 2.0 and -31.8 ± 3.4 mV (n = 4) under 2 and 20 mM extracellular
K
, respectively, suggesting a high selectivity for
K ions. The deactivation kinetics of r-I (at -80 mV) could be fitted to a single exponential
function resulting in a deactivation time constant
(
) of 1.14 ± 0.17 s (n =
6). Under Hg, the deactivation of I was
changed into two distinct events, the deactivation of time-dependently
activating I ( was 2.18 ± 0.23
s; n = 6), and a deactivation rate that was too slow to
get precisely quantified (estimated > 1 min).
Washout of Hg reversed the I stabilizing
effect and resulted in an inhibition of I compared to
control (Fig. 2, A and C).
on
r-I. In A and B the upward deflections
represent r-I evoked with 15-s depolarizing steps to
-10 mV every 45 s from a holding potential of -80 mV. A, under these conditions Hg increased the
total current amplitude. The increase of r-I mediated by
Hg was mainly the result of an outward shift of the
holding current. The wash-out of Hg resulted in an
inhibition of r-I indicating an additional, irreversible
inhibitory effect of Hg on r-I. B, the I inhibitor NE-10064 (10 µM)
inhibited both time-dependent I and the holding current at
-80 mV. C, panel shows in a higher time resolution the
extremely slow deactivation of r-I under
Hg, the appearance of an instantaneous outward
current and the outward shift of the holding current. D,
NE-10064 (10 µM) inhibits instantaneous outward current,
the positively shifted holding current, and the time-dependent outward
current. E and F, recordings of instantaneous
currents during 200-ms voltage steps to potentials from -120 to 0
mV with 30-mV increments at an interval of 2 s taken 10 s after a 15-s
depolarization to -10 mV. Because of the slow I activation, no significant currents could be activated with 200
ms depolarizations under control conditions (C). D,
Hg produces an instantaneous potassium current with a
linear-I-V relationship, which was almost completely inhibited by 10
µM NE-10064 (E). In the same batches of oocytes
as used for the experiments, in HO-injected oocytes,
Hg did not induce any instantaneous potassium
currents, but it induced in one batch of oocytes (4 out of 8 oocytes) a
small Ca-activated chloride current. In three other
batches of oocytes (n = 30), Hg (1
µM) did not induce any currents. The dashed line in C-E indicates 0
current.
were strongly
dependent on a prior activation of I. In contrast, when
Hg (1 µM for 20 min; see Fig. 3)
was superfused in the absence of repetitive depolarizations (see Fig. 3), no alterations in the holding current (at -80 mV)
were observed. The first depolarization after Hg washout resulted in a reduced r-I (-66.1
± 3.9%; n = 4). However, a subsequent second
Hg superfusion period during repetitive
depolarizations (see Fig. 3) caused an increase of previously
inhibited r-I and an outward shift of the holding current.
The use-independent Hg-mediated I inhibition was also present in I protein mutants
r-I C107S and h-I C106S (data not shown). We
conclude, therefore, that other mechanisms than intracellular thiol
group interaction is the main mechanism for this
Hg-mediated I inhibition. However, the
inhibitory mechanism of Hg-mediated I inhibition remains unclear.
on I. When Hg (1
µM for 20 min) was superfused in the absence of repetitive
depolarizations, the holding current was not significantly affected. In
addition, depolarizations after the washout of Hg resulted in a reduced I. A subsequent second
superfusion of Hg during repetitive depolarizations
resulted in an increase of I and an outward shift of the
holding current, which reversed again upon
washout.
was very similar to the persistent activation
of I previously described for organic,
membrane-impermeable cross-linkers (11) and the Cl channel blocker DIDS and mefenamic acid(12) , binding of
Hg to the extracellular domain of the I protein was predicted. Two r-I protein
mutants(7) , with deletions of amino acids 10-25
(r-I(del 10-25)) or 10-39
(r-I(del 10-39)) in the extracellular domain were
subsequently tested for their sensitivity to Hg. Both
mutants displayed similar general activation properties as wild-type
r-I. However, while Hg still positively
regulated r-I(del 10-25) (Fig. 4, A and C), superfusion with Hg resulted in
an inhibition of I(del 10-39) of -38.7 ± 4.5% (Fig. 3, B and C). This result points to an
interaction of Hg with the extracellular I protein domain from amino acids 26-39. Hg is known to form chelates with dibasic amino acids, and 3 out of
4 extracellular dibasic amino acids are located between amino acids 26
and 39, while no dibasic amino acid is present between amino acids 10
and 25. It is therefore possible that Hg indeed forms
chelates of I protein subunits by interacting with dibasic
amino acids and that such chelation causes a stabilization of activated
channels. The formation of such Hg-I protein chelates was dependent on prior activation of
I, suggesting that during I activation a
conformational change of the extracellular region occurs. However, it
appears that only the mobility of the extracellular domain is important
for I channel function but not the extracellular
Hg binding region itself, because the deletion of
this domain does not render the channel nonfunctional. Similarly, it
appears that also the mobility of the intracellular domain must be
warranted for I channels to be functional. There,
oxidation of a conserved Cys residue and the presumed formation of
disulfide bonds may result in an impaired mobility of the intracellular
tail, prompting the inhibition of I. Because this Cys
residue is not necessary for normal I channel function, it
may represent an ``off-switch'' for the I channel under certain pathophysiological conditions.
on
r-I mutants with deletions in the extracellular domain. A, Hg produces a qualitatively similar
effect on the mutant r-I(del 10-25) as on wild-type (WT) I. B, Hg had only
inhibitory effects on a mutant with an extended deletion in the
extracellular domain of the I protein containing amino
acids 26-39 (r-I(del 10-39)). This inhibitory
effect was qualitatively similar to the use-independent effect of
Hg on wild-type r-I. C,
relative effects (± S.E.) of Hg on potassium
outward currents induced by wild-type r-I and several
mutants. The double mutant r-I(del 10-39)/A94- is
insensitive to both positive regulation through
Hg-mediated extracellular chelation and negative
regulation caused by HO-mediated intracellular
oxidation.
(A) type potassium channels (13) , but
the physiological relevance of such regulation is not clear yet.
However, in isolated heart models(14) , peroxides were shown to
account for reperfusion induced arrhythmias, and peroxides caused a
strong inhibition of I combined with an action potential
duration prolongation in isolated guinea pig cardiocytes(6) .
The results of this study suggest that oxidation of the I protein participates in such events. The possible involvement of
I proteins in the genesis of arrhythmias may shed a new
light on their putative role as targets for novel
antiarrhythmics(5) . protein activates an
endogenous oocyte potassium channel(15) . However, there is
accumulating evidence that the I protein is at least an
integral part of a different type of potassium channel itself. It was
demonstrated that the I protein itself represents the
molecular basis for La
blockade(16) ,
regulation through protein kinase C (17) , and ion selectivity
of I(18) . Here, we provide evidence that the
I protein itself is the target for peroxide and
Hg-mediated I regulation (Fig. 5). Recently, several studies have suggested that I protein density and/or mobility play a role for activation and
deactivation of this unique K channel(11, 19, 20) . The data
presented in this study suggest indeed that the mobility of the
external and internal I protein domains is important for
regular channel function.
)
We are indebted to Drs. A. Müller,
P. Hausen, and J. P. Ruppersberg for many fruitful discussions. We
acknowledge the expert preparation and handling of Xenopus oocytes by B. Noll and R. Vesenmeier.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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