Naunyn-Schmiedeberg's Arch Pharmacol (1996) 353:241 244 © Springer-Verlag 1996

Aleksandar Jovanovic • A l e x e y E. A lekseev • Andre Terz ic

Cardiac ATP-sensitive K + channel: a target for diadenosine 5',5"-p1,W-pentaphosphate

Received: 18 September 1995/Accepted: 16 October 1995

Abstract In numerous studies the intracellular mono- nucleotide-dependent gating of ATP-sensitive K ÷ (KATe) channels has been demonstrated. However, it is not known whether dinucleotide polyphosphates, a family of endogenous compounds structurally-related to ATP, could also modulate this ion conductance. Therefore, in the present study we assessed the direct effect of diadenosine 5',5"-P1,pS-pentaphosphate (ApsA) on cardiac KAxp channel activity using the inside-out configuration of the patch-clamp technique. Addition of ApsA (50 pM) to the internal side of mem- brane patches, excised from guinea-pig ventricular cells, strongly inhibited KATp channel activity. The esti- mated NPo (where N is the number of channels in the patch and Po the open probability of each channel) was 4.16 + 0.50 in the absence and 0.85 + 0.30 in the pres- ence of ApsA (50 ~LM). This effect of ApsA was partially reversible, and the NPo was 2.26 + 0.60 after washout of ApsA. Exposure of I<2AT p channels to increasing concentrations of ApsA revealed that the ApsA-in- duced inhibition is concentration-dependent with the half-maximal effective concentration of 16 I.LM (Hill coefficient: 1.6). On the basis of these results, we con- clude that ApsA is a potent antagonist of the KATp channel activity. This represents a previously unrecognized property of ApsA, as well as the dis- covery of a potentially novel endogenous ligand of myocardial KAw channels.

Key words ATP-sensitive K + channel • Diadenosine pentaphosphate • Diadenosine polyphosphates • Channel gating - Heart - Cardiomyocyte

A. Jovanovic ' A.E. Alekseev - A. Terzic (1~) Division of Cardiovascular Diseases (G-7), Departments of Medicine and Pharmacology, Mayo Clinic, Mayo Foundation, Rochester, MN 55905, USA

Introduction

ATP-sensitive K + (KATp) channels are present in many tissues, and are believed to provide a link between the metabolic status and the electrical excitability of a cell (Noma 1983; Ashcroft and Ashcroft 1990; Davies et al. 1991; Weiss and Venketash 1993; Lazdunski 1994). Although the regulation of KATP channels is rather complex (Edwards and Weston 1993; Terzic et al. 1994c), the dependence of channel gating on intracellu- lar mononucleotides, such as ATP and ADP, is well established (Ashcroft and Ashcroft 1990; Davies et al. 1991; Nichols and Lederer 1991; Findlay 1994). How- ever, less is known as to the regulation of KA~-p channels by other related intracellular nucleotides.

Recently, the family of diadenosine polyphosphates, which are synthesized intracellularly and are struc- turally related to ATP, have been associated with meta- bolic stress (Yakovenko and Formazyuk 1993). In par- ticular, the diadenosine pentaphosphate (ApsA) has received considerable attention in view of its multiple cardiovascular actions (Schluter et al. 1994; Baxi and Vishwanatha 1995). Besides extracellular effects, beli- eved to be mediated through purinoceptors (Pintor et al. 1991), it has been described that intracellular ApsA can inhibit the activity of several nucleotide-binding enzymes, such as adenylate kinase, which are important to maintain the energetic homeostasis of muscle cells (Leinhardt and Secemski 1973; Zeleznikar et al. 1995). The intracellular mode of action of ApsA on nucleo- tide-binding enzymes has been related to the interac- tion of this diadenosine with the intracellular AMP and/or ATP-binding sites on targeted proteins (see Yakovenko and Formazyuk 1993; Baxi and Vish- wanatha 1995).

Taking into consideration the structure of ApsA and its apparent affinity for nucleotide-binding sites (Leinhardt and Secemski 1973; Yakovenko and For- mazyuk 1993; Baxi and Vishwanatha 1995), we

242

hypothesized that this compound may exhibit an affin- ity for nucleotide-gated ion channels, such as the KATp channel, and in turn could modulate channel activity. In view of the ubiquitous intracellular presence of ApsA, evidence for such action of ApsA on KATP channels may be important in the further under- standing of the behavior of this ion conductance. Therefore, we assessed whether ApsA modulate cardiac KATp channel activity.

Materials and methods

Cells. Ventricular myocytes were isolated by enzymatic dissociation (Isenberg and Klockner 1982; Kurachi 1985). In guinea-pigs (200-300 g), anesthetized with pentobarbital and artificially ven- tilated, the aorta was cannulated, and following cardiotomy the heart retrogradely perfused at constant temperature (37 ° C) with the following solutions (in raM): first, with NaC1 136.5, KC1 5.4, CaC12 1.8, MgC12 0.53, glucose 5.5, HEPES-NaOH 5.5 (pH 7.4) for 5-10 rain, then With the same solution without Ca 2+, followed by the nominally Ca 2 +-free solution containing collagenase (0.04 g per 100 ml, Sigma type I; Sigma Chemical Co., St. Louis, MO., USA) for 30 min, and finally a high-K+/low-C1 - solution (taurine 10, oxalic acid 10, glutamic acid 70, KC1 25, KH2PO 4 10, glucose 11, EGTA 0.5, and HEPES-KOH 10; pH 7.3 7.4) for 5 min. The heart was then stored in the high-K+/low-C1 - solution at 4 ° C. To isolate single cells a small piece of ventricle was dissected and agitated in the recording chamber. Myocytes were permitted to adhere to the chamber's glass floor, and then superfused at a constant flow rate ( ~ 3-5 ml/min) with (in mM) KC1 140, MgC12 1, EGTA-KOH 5, HEPES-KOH 5 (pH 7.3) in the absence and presence of ApsA (Sigma). Only myocytes with clear striations and a smooth surface were used for experiments.

Sincdle channel recordings. The gigaohm-seal patch clamp technique was employed in the inside-out patch configuration (Hamill et al. 1981). Single channel currents were recorded at 21 23 ° C. The resist- ance of patch electrodes ranged from 5-7 Mf~ and contained KC1 140, CaC12 1, MgC12 1, HEPES-KOH 5 (pH 7.4), and their tips were coated with Sylgard and fire-polished. Channel activity was meas- ured using a patch clamp amplifier (Axopatch-lC, Axon Instru- ments, Foster City, Calif., USA) and monitored on-line on a high- gain digital storage oscilloscope (VC-6025, Hitachi, Tokyo, Japan). The holding potential was - 60 inV. Data were stored on tape using a PCM converter system (VR-10 Instrutech, New York, N.Y. USA), reproduced, low-pass filtered at 1.5 kHz ( - 3 dB) by a Bessel filter (Frequency Devices 902, Haverhill, Mass., USA), sampled at 4 kHz, and analyzed off-line with a software analysis program. The thres- hold for judging the open state was set at half of the single channel amplitude. Using the BioQuest software (developed by A.E.A.), the degree of channel activity was assessed by digitizing segments of current records and forming histograms of baseline and open-level data points. Data were expressed as mean +_ SEM whenever appro- priate. Statistical significance of differences between two means was determined with the Students t test, and a value of P < 0.05 was considered to be statistically significant.

Results

A Inside-oul

c Z

B

3-

0 I • r i i i

25 50 75 100 T ime (s)

Z

4

3

2

0 - -

- Control

• - AP5A 50 ~LM

- Wash-ou t

Fig. 1 Diadenosine 5',5"-PI,PS-pentaphosphate (ApsA)-induced in- hibition of myocardial ATP-sensitive K + channels. A Upper trace: continuous channel record. Lower trace: corresponding NPo values calculated over 2.5-s long intervals. Dotted line: zero current level. Holding potential: - 60 mV. B Average NPo prior to (open column), during (solid column), and after (hatched column) application of 50 ~tM ApsA to the intracellular side of membrane patches

depicted in Fig. 1A, KATp channel activity was sus- tained. Addition of ApsA (50 ~tM), to the intracellular side of the membrane patch, immediately inhibited KAT P channel openings (Fig. 1A). The average value of NPo was 4.16 _+ 0.50 in the absence, and decreased to 0.85 _+ 0.30 in the presence of 50 ~tM ApsA (P < 0.01, n --- 6; Fig. 1B). The effect of ApsA was partially re- versible (Fig. 1A), and the average NPo returned to 2.26 +_ 0.60 following washout of ApsA (n = 6; Fig. 1B). To quantify the action of ApsA on KATp chan- nel activity, excised membrane patches, with sustained KATp channel openings, were exposed to multiple con- centrations of ApsA (Fig. 2A). Such protocol clearly revealed that ApsA inhibited KAT P channels activity in a concentration-dependent manner (Fig. 2). The esti- mated value of the half-maximal effective concentration of ApsA to inhibit NAT P channels was 16 ~tM with a Hill coefficient of 1.6 (Fig. 2B).

Following excision of a membrane patch from a ven- tricular myocyte, multiple openings of KATP channels (single channel conductance ~ 90 pS) appeared (Fig. 1A; see also Terzic et al. 1994a). In the patch

Discussion

The present study demonstrates that ApsA, an ubiqui- tous intracellular dinucleotide polyphosphate, inhibits

243

A Inside-out AP5A 0.5 HM, 5 p M 50pM

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0 50 100 150 200

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80

60

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n = 1 . 6

, . . . . ,,:1

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10.0 100.0

[ApsA] (,[.LM)

Fig. 2 Concentration-dependent effect of diadenosine 5',5"-P1,P 5- pentaphosphate (ApsA)-induced inhibition of myocardial ATP-sen- sitive K + channels. A Upper trace: continuous channel record. Lower trace: corresponding NPo values calculated over 2.5-s long intervals. Dotted line: zero current level. Holding potential: - 6 0 inV. B At different concentrations of ApsA, relative channel activity expressed as NPo was obtained with reference to values recorded in the absence of ApsA. Data are from 3-6 membrane patches for each data point. Solid line was drawn according to the equation, y = 1/{1 + ([ApsA~/Ki)n}, in which y = relative NP0 at each ApsA concentration, [ApsA] = concentration ofApsA, Ki = the concentration of Ap5A at half-maximal inhibition of the channel, and n = Hill coefficient

m y o c a r d i a l KAT P channel activity. This represents a previously undescribed property of Ap~A, and indi- cates that KAT P channel activity can be regulated, not only by mononucleotides, but also by a dinucleotide polyphosphate.

The apparent potency of ApsA to inhibit myocar- dial KAT P channels is around 2-fold higher than is that of ATP. Intracellular ATP usually inhibits cardiac KATe channels with a half-maximal effective concentra- tion in the range of 30 pM (Nichols and Lederer 1991; Terzic et al. 1994a), although much higher concentra- tions of ATP are needed to inhibit KATe channels in certain patches (Findlay and Faivre 1991). It also ap- pears that the apparent potency of ApsA to inhibit KATP channels is several-fold higher than the potency of other conventional mononucleotides, such as ADP or

GTP (Nichols and Lederer 1991; Findlay 1994). This could indicate that the apparent sensitivity of the myo- cardial KAT P channel towards ApsA-induced channel inhibition is somewhat higher than that of previously recognized endogenous mononucleotide ligands.

The inhibitory effect of ApsA on KATe channel activity was observed following application of the dinucleotide to the intracellular side of membrane patches. Since ApsA is a molecule known to be poorly membrane-permeable (see Yakovenko and Formazyuk 1993; Baxi and Vishwanatha 1995), it is conceivable that the site of action of ApsA is intracellular. ApsA could have acted either on the KATe channel itself or on associated proteins. The inside-out patch clamp tech- nique does not allow to distinguish between these two possibilities. It should be, however, pointed out that the effect of ApsA occurred in the absence of GTP, thus eliminating the possible involvement of a GTP-binding protein in transducing the inhibitory effect of ApsA on KAT P channels. A GTP-independent KAT P channel gat- ing has been previously reported in the case of intracel- lular mononucleotides (Ashcroft and Ashcroft 1990; Nichols and Lederer 1991), and potassium channel openers (Quast et al. 1988; Edwards and Weston 1993), in contrast to the adenosine-dependent KAT P channel regulation which is mediated through a GTP-binding protein (Terzic et al. 1994b). The saturable nature and the concentration-dependence of the action of ApsA on KATP channels suggest that a specific binding site with an apparent micromolar affinity for ApsA is essential in transducing this action. The structure of the KAT e chan- nel is still controversial, yet based on functional studies it appears that the KATP channel protein may possess several nucleotide-binding sites (Tung and Kurachi 1991; Terzic et al. 1994a) to which, in principle, ApsA could directly bind to. However, the present study does not exclude the existence of a, previously unrecognized, site selective for diadenosine polyphosphates and also associated with a KAT P channel. Regardless of the mechanism of action of ApsA, the present study dem- onstrates that ApsA can antagonize the activity of cardiac KATP channels. This raises the possibility of an endogenous dinucleotide polyphosphate-dependent gating of KATP channels.

In mammalian tissues, including human ceils, basal levels of ApsA have been estimated within the range of 10 to 100 nM (see Yakovenko and Formazyuk 1993; Baxi and Vishwanatha 1995). Thus, it is unlikely that, under resting conditions, ApsA could play a direct role in modulating KATe channel activity. It should be pointed out that several pathophysiological conditions, including metabolic stress, have been associated with a rise in the intracellular concentration of diadenosine polyphosphates (see Yakovenko and Formazyuk 1993; Baxi and Vishwanatha 1995). Thus, it is tempting to speculate that ApsA could play a modulatory role in the regulation of the behavior of myocardial KAT e channels under certain conditions.

244

Acknowledgements This work was supported by grants from the American Heart Association, and the National Heart Foundation, a program of the American Health Assistance Foundation. A.T. is a recipient of the Ruth Salta Young Investigator Award from the National Heart Foundation and of the Faculty Developmental Award from the Pharmaceutical Research and Manufacturers of America Foundation.

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