sulfhydryl redox modulates atp-sensitive k+channels in rabbit ventricular myocytes

JOBNAME: BBRC 219#3 PAGE: 1 SESS: 7 OUTPUT: Tue Apr 2 10:39:00 1996/xypage/worksmart/tsp000/68953f/39

Sulfhydryl Redox Modulates ATP-Sensitive K+ Channels in RabbitVentricular Myocytes

Jin Han,* Euiyong Kim,* Won-Kyung Ho,† and Yung E. Earm†,1

*Department of Physiology and Biophysics, College of Medicine, Inje University, Korea; and †Department ofPhysiology and Biophysics, College of Medicine, Seoul National University, Seoul, Korea

Received January 22, 1996

The properties of sulfhydryl redox modulation of the ATP-sensitive K+ (KATP) channel have been examinedin rabbit ventricular myocytes, using the patch-clamp technique. The sulfhydryl oxidizing agent 5,59-dithio-bis-(2-nitro-benzoic acid) (DTNB) induced an inhibition of the channel activity without change in the single channelconductance. DTNB had no effect on the inhibitory action by ATP. Analysis of the open and closed timedistributions showed that DTNB decreased the life time of bursts and increased the interburst interval withoutchanges in open and closed time distributions shorter than 5 ms. N-ethylmaleimide (NEM), a substance thatreacts with sulfhydryl groups of cysteine residues in proteins, induced an irreversible closure of the channel. Theresults suggested that changes in the sulfhydryl redox also modulate KATP channel activity of the KATP channelin rabbit ventricular myocytes. © 1996 Academic Press, Inc.

When activated pharmacologically ATP-sensitive K+ channels (KATP channels) drastically re-duce action potential duration (1), and it has been suggested that they may be responsible for actionpotential shortening in metabolically compromised ischemic muscle (2). The regulatory link be-tween channel activity and cellular metabolism remains controversial. Many studies demonstratedthat the channels in the heart modulated by intracellular metabolites generated during ischemiaother than ATP (3,4).The sulfhydryl groups (SH groups) are essential for the function of many biologically active

proteins. It has been also suggested that assessable free sulfhydryl groups are present on KATP

channel in pancreaticb-cells (5) and skeletal muscle cells (6). It remains, however, unclear whetherchanges of sulfhydryl redox state modulate KATP channels in cardiac muscle. In the present study,we have investigated the effect of sulfhydryl modification on the KATP channels in rabbit ven-tricular myocytes using the patch clamp technique. Our data suggest that changes in the sulfhydrylredox are involved in the modulation of KATP channel activity in rabbit ventricular myocytes.

MATERIALS AND METHODSCell preparation.Rabbit ventricular myocytes were isolated by using a Langendorff column at 37°C for coronary

perfusion and collagenase (5 mg/50cc, Yakurt, Japan) for enzymatic dispersion as described previously (3). Isolated cellswere stored in a high K+, low Cl −storage medium (7) (composition in mM: taurine 10, glutamic acid 70, KCl 25, KH2PO410, glucose 22, EGTA 0.5, pH 7.4 with KOH).Single channel recordings.Single-channel currents were measured at room temperature (24 ± 2°C) in inside-out con-

figurations of the patch-clamp technique (8) with a patch-clamp amplifier (EPC-7, LIST, Darmstadt, Germany; Axopatch-1D, Axon Instruments, Foster City, CA, USA). The solution facing the outside of the patch membrane contained (in mM):140 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, pH 7.4 with KOH. The solution facing the inside of the patch membranecontained (in mM): 127 KCl, 13 KOH, 1 MgCl2, 5 EGTA, 10 glucose, 10 HEPES, pH 7.4 with KOH. Data acquisition andanalysis were performed as previous described (3). All drugs were obtained from Sigma (St. Louis, MO, USA).

RESULTS AND DISCUSSION

The effects of DTNB on the KATP channels were examined by the inside-out patch configurationthat was performed with symmetrical transmembrane K+ concentration of 140 mM (Fig. 1). On

1 Correspondence to Professor Yung E. Earm, Department of Physiology and Biophysics, College of Medicine, SeoulNational University, Yonkeun-Dong, Chongno-Ku, Seoul 110-799, Korea. Fax: +82-2-763-9667.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS219,900–903 (1996)ARTICLE NO. 0330

9000006-291X/96 $18.00Copyright © 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.


formation of the inside-out patches in ATP-free internal solution at −50 mV, the channel activityrevealed an open state with overlaps of up to three unitary currents of 3.12 pA. The currents wereinhibited by ATP and glibenclamide (not shown), indicating that it was the KATP channel. Appli-cation of the sulfhydryl oxidizing agent DTNB caused an inhibition of the KATP channel. Theinhibition was reversed by the disulfide reducing agent DTT (Fig. 1A). The inhibitory action ofDTNB on the KATP channel activity was dose-dependent, with a DTNB concentration for halfinhibition of 36.3mM (Fig 1B). There was no statistical difference in the single channel conduc-tance before (78.2 ± 2.6, n4 9) and after (80.9 ± 1.8, n4 9) the application of DTNB (P > 0.05,Fig. 1C). DTNB had no effect on the inhibitory action by ATP (Fig 1D). In the absence of DTNB,the ATP concentration at the half-maximal inhibition (Ki) and Hill coefficient (n) were 76.3 ± 17.0mM and 1.2 ± 0.1 (n4 7). In the presence of DTNB, Ki andn were 83.1 ± 13.7mM and 1.2 ±0.2 (n4 6).In two experiments single channel recordings were obtained and the open- and closed-time of

KATP channel were successfully analyzed in the absence and the presence of DTNB. Fig. 2 showsrepresentative results of the experiment at a membrane potential of −50 mV. The fast open andclosed kinetics within the bursts was analyzed from the records sampled at 10 kHz. In the absenceof DTNB, the open time distribution was described by a single exponential with a time constant (to)of 2.59 ms. The histogram of closed time within bursts was also fitted by a single exponentialfunction. This analysis was performed with closed times longer than 5 ms to be discarded. Theclosed time constant (tc) within bursts was 0.25 ms. The values ofto (2.15) andtc (0.27) were notchanged markedly by DTNB. The duration of each bursting opening was measured from therecords sampled at 0.1 kHz. The histogram of burst duration consisted of a single exponential

FIG. 1. Effects of DTNB on the KATP channel activity. A. Single-channel recordings from inside-out patch configura-tion. The solution-exchange protocol for DTNB (0.5 mM), DTT (1 mM) and ATP (1 mM) is shown above current traces.c, closed level. Patch membrane potential was held at −50 mV. Low-pass filter, 5 kHz. B. The dose-response relationshipfor the inhibitory action of DTNB. C. Current-voltage relationship of the KATP channel. D. Dose-response relationship forchannel inhibition by ATP in the absence (circle) and the presence (triangle) of DTNB.

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distribution. Its time constant (tb) was markedly decreased from 38 to 15 ms by DTNB. Theinterburst time histograms were fitted with two exponential functions. The time constant (tc1) ofthe fast exponential component was unaffected by DTNB (from 8 to 11 ms). The time constant (tc2)of the slow exponential component was increased from 65 to 332 ms by DTNB. These findingssuggest that DTNB decreases the channel activity by a decrease in burst durations and an increasein interburst intervals.Fig. 3 shows the inhibitory effect of a substance that reacts with sulfhydryl groups of cysteine

residues in proteins, N-ethylmaleimide (NEM) on the KATP channel. On addition of NEM (2 mM),there was a pronounced reduction in channel activity.Our results indicate that inhibition of the channel activity was caused by sulfhydryl redox

modulation because a common property of DTNB and NEM is the modification of sulfhydrylgroups in proteins. In addition, the effect of DTNB was reversed by addition of excess of thedisulfide reducing agent DTT, indicating that inhibition of channel activity was caused by sulf-hydryl oxidation and not due to a nonspecific effect on the channel. The present study suggests thata sulfhydryl group on KATP channel is critical for activity in rabbit ventricular myocytes as in thecases of mouse skeletal muscle (6) and pancreaticb-cell (5). Further experiments need to be carriedout to investigate the molecular basis of the underlying mechanism of KATP channel modulation bysulfhydryl modification.Although the role of sulfhydryl groups in regulating the KATP channel activity in the heart is

unclear, it is possible that the sulfhydryl groups are essential for the cardioprotection in ischemichearts. Recently, it has been shown that the sulfhydryl-containing compounds protect the ischemicmyocardium (9, 10) and that the cardioprotective activity was abolished by KATP channel blockers(10), indicating that cardioprotection with the sulfhydryl-containing compounds is via modulationof KATP channel.

FIG. 2. Effects of DTNB on the kinetic property of the KATP channel. The histograms of the open and closed timeswithin bursts were analyzed from the current records filtered at 10 kHz. The histograms of the burst and interburst durationswere analyzed from the current records filtered at 0.1 kHz.

FIG. 3. Effect of NEM on the KATP channel activity. NEM (2 mM) was added to the bath solution for the periodindicated by the bar. c, closed level. Currents were recorded at −50 mV. Low-pass filter, 5 kHz.


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ACKNOWLEDGMENTS

This work was supported by Basic Medical Research Fund (1994) from the Ministry of Education and Grant 02–94–225from the Seoul National University Hospital Fund.

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sulfhydryl redox modulates atp-sensitive k+channels in rabbit ventricular myocytes

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