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SPECIAL REPORT

Diadenosine tetraphosphate-induced inhibition of ATP-sensitive K+ channels in patches excised from ventricularmyocytes

Aleksandar Jovanovic & 'Andre Terzic

Division of Cardiovascular Diseases, Departments of Medicine and Pharmacology, Mayo Clinic, Mayo Foundation, Rochester,MN 55905, U.S.A.

Diadenosine 5',5"-P',P"-tetraphosphate (Ap4A) has been termed 'alarmone' due to its role in intracellularsignaling during metabolic stress. It is not known whether Ap4A could modulate ATP-sensitive K+(KATP) channels, a family of channels regulated by the metabolic status of a cell. We applied the single-channel patch-clamp technique to measure the effect of Ap4A on KATP channels. When applied to theintracellular side of patches, excised from guinea-pig ventricular myocytes, Ap4A inhibited KATP channelactivity, in a reversible and concentration-dependent (half-maximal concentration - 17 gM) manner. Weconclude that Ap4A, a naturally occurring diadenosine polyphosphate, is actually an inhibitor of themyocardial KATP channel.

Keywords: ATP-sensitive K+ channel; diadenosine 5',5"-P',P4-tetraphosphate; diadenosine polyphosphate; alarmone; channelgating; guinea-pig; heart; cardiomyocyte

Introduction Diadenosine 5',5"-P',P4 tetraphosphate (Ap4A)has been termed putative 'alarmone' to denote that this dinu-cleotide polyphosphate is synthesized during metabolic chal-lenges and could act homeostatically under stress conditions(Varshavsky, 1983). In several cell types a direct intracellulareffect of Ap4A has been demonstrated on enzymes with nu-cleotide-binding domains which are associated with cellularmetabolism (Yakovenko & Formazyuk, 1993).

ATP-sensitive K+ (KATP) channels are gated by intracellularATP, and provide a link between cellular metabolism andmembrane excitability (Ashcroft & Ashcroft, 1990; Davies etal., 1991; Edwards & Weston, 1993). In the myocardium,modulation of KATP channel activity during metabolic stresshas been related to intracellular mononucleotides, such as ATP(Nichols & Lederer, 1991; Findlay, 1994; Terzic et al., 1994b).It is unknown whether a dinucleotide polyphosphate withputative 'alarmone' properties, such as Ap4A, could also affectKATP channel activity. Therefore, we evaluated the effect ofAp4A on cardiac KATP channels.

Methods Ventricular myocytes were isolated from guinea-pighearts, and the inside-out configuration of the patch-clamptechnique used to record channel activity (Terzic et al., 1994a).Patch pipettes (3-5 MO) were filled with (in mM): KCl 140,CaCl2 1, MgCl2 1, HEPES-KOH 5 (pH 7.4), and the in-tracellular side of excised patches exposed to (in mM):KCl 140, MgCl2 1, EGTA-KOH 5, HEPES-KOH 5 (pH 7.3)in the absence and presence of ATP or AP4A (Sigma). Single-channel recording was conducted at a holding potential of-60 mV (21 - 230C) using a patch-clamp amplifier (AxopatchIC). Data, stored on tape using a PCM converter system(Instrutech), and low-pass filtered at 1 - 1.5 kHz (-3 dB) by aBessel filter (Frequency Devices), were sampled at 4 kHz, andanalyzed with the 'BioQuest' software (developed by Dr A.E.Alekseev). Channel activity was expressed as NP0 (N = numberof channels in the patch; P0= open probability of each chan-

'Author for correspondence.

nel). Data are represented as mean+ s.e.mean. Statistical sig-nificance of differences between two means was determinedwith Student's t test, and P<0.05 considered significant.

Results Upon excision of a patch from a cardiomyocyte,vigorous openings of KATP channels appeared, and could beblocked by 200 giM ATP (Figure la). At equimolar con-centrations ofK + on the external and internal sides of a patch,these channels had a unitary conductance of 90 pS, as de-scribed for myocardial KATP channels (Findlay, 1994; Terzic etal., 1994b). Addition of AP4A (50 gM) to the intracellular sideof a patch, did not affect the magnitude of the unitary currentflowing through a KATP channel (5.7+0.3 vs.5.7+0.3 pA at-60 mV in the absence and presence of AP4A, respectively;P> 0.05, n = 9). Yet AP4A (50 gM) induced immediate inhibi-tion of KATP channels (Figure lb(i)). The NP0, was 4.37 + 1.11in the absence, and 0.60 + 0.22 in the presence of 50 /IM AP4A(P<0.01, n= 9; Figure lb(ii)). The effect ofAP4A was partiallyreversible (Figure b(i), and the NP0 returned to 2.81 + 0.80following washout of AP4A (n = 9; Figure lb(ii)). The in-hibitory effect of AP4A on KATP channels was concentration-dependent. The concentration-response relationship was fittedto a Hill equation with a half-maximal concentration estimatedat 17 gM, and a slope factor of 1.2 (Figure 2).

Discussion This study demonstrates that Ap4A, a naturallyoccurring dinucleotide polyphosphate, inhibits myocardialKATP channels. This represents a previously unrecognizedproperty of Ap4A, that could relate to the proposed in-tracellular potential of this molecule to regulate cellular me-tabolism.

The dinucleotide polyphosphate, Ap4A, was effective whenapplied in micromolar concentrations to the intracellular sideof excised patches. The effect of Ap4A was concentration-de-pendent suggesting the involvement of a saturable binding site.The potency and efficacy of Ap4A in blocking KATP channelswas comparable to that described for the ATP-evoked KATPchannel inhibition (Nichols & Lederer, 1991; Findlay, 1994;Terzic et al., 1994b). Since Ap4A inhibited KATP channels in theabsence of intracellular GTP, it implies that a GTP-binding

British Journal of Pharmacology (1996) 117, 233-235

234 ~~~~~~~~~~~A.Jovanovic & A. Terzic Special Report

ATP 200 gJma

I II L.I. I

0-z

II I II II'I II 1 =

0 20 40 60 80 100 120 140Time (s)

AP4A 50 gIm

II

3-

a- 2-z

0-III I I

100 150 200Time (s)

b (ii)6 1

5-

4-

z

2-

1 -

0-

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Figure 1 ATP-(a) and AP4A- (b) induced inhibition of KATP channels. Upper traces in (a) and (b(i)): channel records. Lower tracesin (a) and (b(ii)): corresponding NP0 values calculated over 2.5-s long intervals. Dotted lines: zero current level. (b(ii)) Average NP0prior to (open column), during (solid column), and after (hatched column) application of 50 mM Ap4A to the intracellular side ofpatches.

protein is not required to transduce this effect. These findingsprobably exclude the possibility that the effect of ApNA onKATP channels was due to an extracellular action on pur-inoceptors (Baxi & Vishwanatha, 1995). Rather, Ap4A couldhave acted directly on intracellular binding sites either on theKATP channel itself or associated proteins. Previously, inter-actions of ApNA with intracellular nucleotide-binding enzymeshave been associated with the binding of Ap4A to nucleotide-binding sites (Baxi & Vishwanatha, 1995). Thus, the site ofaction of Ap4A could include the putative ATP-binding in-hibitory or other nucleotide-binding site(s) of KATP channels(Edwards & Weston, 1993; Findlay, 1994; Terzic et al., 1994a).Regardless of the site of action of Ap4A, the present study

suggests that cardiac KATP channels could be gated not only byATP and related mononucleotides, but also by the dinucleo-tide polyphosphate, ApNA.

The physiological importance of the action of ApNA onKATP channels is, at present, unknown. Ap4A is synthesizedintracellularly, yet the concentrations of Ap4A in cells at restare in the pico- to nanomolar range (Yakovenko & For-mazyuk, 1993). However, endogenous Ap4A has a con-siderably longer intracellular half-life than ATP (Baxi &Vishwanatha, 1995), and under stress conditions, Ap4A canrise to concentrations > > 1 mM (Varshavsky, 1983). Providedthat the affinity of KATP channels for ApNA in intact myocytesis similar to that measured in excised patches, it is under stress

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234

b (i)0......................................................................

It 11 ti I .. IL, ki Id.]

A. Jovanovic & A. Terzic Special Report 235

a 0.4

0.2

0.01 0.1 1 10 100 1000[AP4P], 1M

Figure 2 Concentration-dependent inhibition of KATP channels byAp4A. At different concentrations of Ap4A, relative channel activitywas obtained with reference to values recorded in the absence ofAp4A. Data are from 6-9 patches for each point. Solid line wasdrawn according to the equation: y = I /{ 1 + ([Ap4A]/K,)fI}; y = re-lative NP0 at each Ap4A concentration ([Ap4A]), Ki = [Ap4A] at half-maximal inhibition of channels = 17 gM; nH = Hill coefficient = 1.2.

conditions that sufficient levels of intracellular Ap4A could besynthesized to affect channel activity. Hence, the putative roleof Ap4A in the intracellular regulation of KATP channels awaitsdefinition.

The authors acknowledge the expert contribution of Dr A.E.Alekseev with computer programming. A.T. is a recipient of theRuth Salta Young Investigator Award from the American HealthAssistance Foundation and of the Faculty Developmental Awardfrom the Pharmaceutical Research and Manufacturers of AmericaFoundation.

References

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BAXI, M.D. & VISHWANATHA, J.K. (1995). Diadenosine polypho-sphates: their biological and pharmacological significance. J.Pharmacol Methods, 33, 121 - 128.

DAVIES, N.W., STANDEN, N.B. & STANFIELD, P.R. (1991). ATP-dependent potassium channels of muscle cells: Their properties,regulation, and possible function. J. Bionerg. Biomembr., 23,509- 535.

EDWARDS, G. & WESTON, A.H. (1993). The pharmacology of ATP-sensitive potassium channels. Annu. Rev. Pharmacol. Toxicol., 33,597 - 637.

FINDLAY, I. (1994). Interactive regulation of the ATP-sensitivepotassium channel of cardiac muscle. J. Cardiovasc. Pharmacol.,24, S6 - S 11.

NICHOLS, C.G. & LEDERER, W.J. (1991). Adenosine triphosphate-sensitive potassium channels in the cardiovascular system. Am. J.Physiol., 261, H 1675 -H 1686.

TERZIC, A., FINDLAY, I., HOSOYA, Y. & KURACHI, Y. (1994a).Dualistic behaviour of ATP-dependent K + channel towardsintracellular nucleoside diphosphates. Neuron, 12, 1049- 1058.

TERZIC, A., TUNG, R.T. & KURACHI, Y. (1994b). Nucleotideregulation of ATP-sensitive K+ channels. Cardiovasc. Res., 28,746 - 753.

VARSHAVSKY, A. (1983). Diadenosine 5',5"'-Pl, P4-tetraphosphate:a pleotropically acting alarmone? Cell, 34, 711 -712.

YAKOVENKO, I.N. & FORMAZYUK, V.E. (1993). Diadenosineoligophosphates: metabolic pathways and role in regulation ofthe functional activity of cells. Biokhimiya, 58, 3-24.

(Received September 20, 1995Accepted October 4, 1995)