©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Basis of I Protein Regulation by Oxidation or Chelation (*)

(Received for publication, September 6, 1994; and in revised form, December 1, 1994)

Andreas E. Busch (1)(§) Siegfried Waldegger (1) Tobias Herzer (1) Gertraud Raber (1) Erich Gulbins (1) Toru Takumi (2) Koki Moriyoshi (3) Shigetada Nakanishi (3) Florian Lang (1)

From the  (1)Physiological Institute I, University of Tübingen, D-72076 Tübingen, Federal Republic of Germany, the (2)Department of Pharmacology, Faculty of Medicine, Osaka University, Suita, Osaka 565, Japan, and the (3)Institute of Immunology, Kyoto University, Faculty of Medicine, Kyoto 606, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Slowly activating I 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.


INTRODUCTION

Expression of the I 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.


EXPERIMENTAL PROCEDURES

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 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(2), 1 MgCl(2), 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)(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.


RESULTS AND DISCUSSION

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 H(2)O(2) (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 H(2)O(2) was enhanced by prolonged superfusion periods (>30 min). Addition of the reducing agent Dithioerythritol (DTE; 5 mM) completely reversed the H(2)O(2)-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 H(2)O(2) (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).


Figure 1: Inhibition of I by DTNP and H(2)O(2). A and B, DTNP (100 µM) and H(2)O(2) (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, H(2)O(2) 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 H(2)O(2) 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.).



The membrane-permeable, thiol group oxidizing reagent DTNP inhibited I similar to H(2)O(2) (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.

In the intracellular domain of the I 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 H(2)O(2). Under H(2)O(2) (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 H(2)O(2) (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.


Figure 5: Determinants for I 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 on I

In contrast to H(2)O(2), 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).


Figure 2: Effects of Hg 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 H(2)O-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.



The described positive regulatory effects of Hg 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.


Figure 3: Use-dependent and use-independent effects of Hg 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.



Because the predicted chelation with Hg 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.


Figure 4: Effects of Hg 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 H(2)O(2)-mediated intracellular oxidation.



Redox reactions at a Cys have also been shown to regulate the inactivation of neuronal I(K)(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) .

The results presented here cannot exclude the possibility that the I 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.


FOOTNOTES

*
This work was supported in part by Deutsche Forschungsgemeinschaft Grants Bu 704/3-1 (to A. E. B.) and La 315/4-1 (to F. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Helmholtz fellowship. To whom correspondence should be addressed: Physiological Institute I, University of Tübingen, D-72076 Tübingen, Federal Republic of Germany. Tel.: 49-7071-293071; Fax.: 49-7071-293073.

(^1)
The abbreviations and trivial names used are: NE 10064, 1-[[[5(4-chlorophenyl)-2-furanyl]methylene]amino]-3-[4-(4-methyl-1-piperazinyl)butyl]-2,4-imidazolidinedione dihydrochloride; DTE, dithioerythritol; DTNP, 2,2`-dithiobis(5-nitropyridine); DIDS, 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.