Pharmacology and electrophysiology of ATP-activated ion channels

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Pharmacology and electrophysiology of ATP-activated ion channels Extracellular ATE serves as a messenger in many tissues*. Specific receptors for ATE mediate a variety of effects, some involving second messenger pathways. In excitable cells, where ATP acts as a true neurotransmitter, one mechanism is the direct activation of ion channels. Since the initial discovery in sensory neurons by Krishtal, Marchenko and Pidoplichko’ in 1983, channels activated by external ATE have been found in a variety of cells. In the past few years, a picture has emerged of a family of channels, located in cell membranes, that are activated by binding of ATP on the extracellular side. In all cases, the channels are permeable to Na+, K+ and Ca*+, and their opening has a depolarizing, excitatory effect on the cell. Channels in different cells differ in detailed characteristics, including agonist selectivity, size, ion selectivity and desensitization. Cell types and functions ATE-activated channels are present in a subset of neurons in sensory ganglia of the cat, rat and So far, voltage-clamp froe3. recordings have been possible only in cell bodies, where a functional role for the channels is unlikely. If the channels are also present in afferent nerve endings, they would elicit firing in response to ATP, perhaps serving to report tissue damage. ATPactivated channels are also present in cardiac parasympathetic neurons4. In the CNS, they have been described in dorsal horn neurons’ as well as in a small subset of cultured hippocampal neuror&. In all cases, ATP excites the neurons, but the physiological significance is unknown. ATPactivated channels similar to those in neurons are found in the rat phaeochromocytoma-derived PC12 cell line74, where they can be easily studied. AT&activated channels are present in hair cells of the outer

cochlealo~ll, where they mediate

substantial direct Ca2’ entry as well as depolarizing the cell. The functional role of ATPactivated channels is clearest in a variety of smooth muscle cells. In vas deferens muscle, it is likely that the channels produce a component of fast, non-adrenergic non-cholinergic excitatory transmission by sympathetic neurons12-14 . Post-ganglionic sympathetic axons contain vesicles in which ATP is co-stored with norepinephrine12. ATE-activated channels may similarly mediate a component of sympathetic transmission to some vascular smooth muscle cells; in rabbit ear artery smooth muscle cells, ATE-activated channels produce contraction both by exciting action potentials and by directly admitting Ca” ions15J6. (Other effects of ATE on blood vessels, including vasodilation, are mediated indirectly by endothelial cell ATE receptors coupled to second messenger systems.) Urinary bladder smooth muscle also contains ATE-activated channels much like those in vas deferens muscle*‘. Two types of ATE-activated channel are known in cardiac atria1 muscle cells’8*19. One is directly activated by ATP, is permeable to both Na+ and K+, and depolarizes the cell. The other is activated less directly via a G protein, is highly K+-selective, and hyperpolarizes the cell; these channels are the same as those opened by muscarinic acetylcholine receptors. Very similar channels of both types are also present in cultured embryonic skeletal muscle2c22. Kinetics In all these cases, the depolirizing, Na+- and K+-permeable channels are directly activated by binding of ATE molecules, as shown by currents that turn on within milliseconds of ATP application. (By contrast, the G proteinmediated K+ currents in cardiac and skeletal muscle take a few hundred milliseconds to activate.)

In most cells, the current declines or desensitizes with maintained application of ATE, typically with a half-time of a few seconds. Desensitization is slow in frog sensory neurons3 and may be lacking in guinea-pig hair ceW”J1. The rate of desensitization is weakly voltage-dependent in cardiac muscle18; otherwise there are few clues to its mechanism. Stoichiometry Like all known ligand-gated ion channels, ATE-activated channels seem to require binding of more than one agonist molecule in order to open. Sometimes, doseresponse curves averaged from many cells can be fitted tolerably well by a 1: 1 binding mode12JJl, but in most cases where the data have been examined closely, the response of a single cell to low concentraticns of ATE is superlinea@ (see Fig. 1). In sensory neurons, the dose-response data fit the assumption that ATE must bind to each of three identical, non-interacting binding sites in order to open the channe123. Data from vas deferens muscle and cardiac muscle are fitted by a model having two binding sites with positive cooperativity’4*18. These results suggest that the channels may be composed of multiple subunits, as are other ligand-gated channels whose structures are known. Single-channel behavior Differences between ATE-activated channels are most evident at the single-channel level. In cardiac muscle and skeletal muscle, the single-channel conductance (~1 pS) is too small to be detected in patch-clamp recordings and can only be estimated by noise analysis’8,22. Single-channel currents are much larger in neurons, PC12 cells and smooth muscle; in quasi-physiological saline solution, the single-channel conductance ranges from 13 pS to 60 pS4,9J3.15~24-27. Opening and closing kinetics can be very complex and have not yet been characterized in any detail. Ionic selectivity The directly-activated ATPgated channels are in all cases equally permeable to small cations such as Na+, K+ and Cs+. Even

@ 1992.ElsevicrSciencePublishers Ltd (UK)

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have found that the channel is also permeable to small anions, with NOa- and I+ calculated to be equally permeant with Na+. In retrospect, the experimental evidence against some anion permeability in other cells is weak, and this point should probably be re-examined.

Agonist selectivity and

receptor type The receptor on the channels is highly selective for ATP over most other adenosine derivatives4,1’,15,1s,20,30. Adenosine and AMP are completely ineffective in most cells, while ADF is a weak agonist. UTP and GTP are ineffective; CTP apparently has some agonist activity in neurons31 but not skeletal muscle30. 2 1 0 -1 ATPyS (adenosine 5’-O-(3-thiotog W”l ~phosphate) is roughly equipotent with ATP in cardiac E&T. 1.Hillp&X of wncenttzi~ data &r ourrent activated by external ATP a@ed to a buMtog olnW mot ganglionneuron. The cell was voltage-clamped at muscle*a, skeletal muscle3’ and -80 mVandk!waniarnent was activatedbv aoolioationof ATP (insetI. Maximal current PC12 cells”, as is 2-methylthiowas aotivatedby S00JIMATP. At low wn&~tions (0.3-1.2 & AT@ the Hill slope is ATP tested in skeletal muscle3’ d srggeslin a ~~ of 3 ATP moleculesper channet.At high concentrations and neurons4. Although the receptor seems broadly similar in the different cell types, clear differences are made evident by a[,@Under physiologic~ conditions, very large cations such as tetramethylene-ATP, which is a potent ATP gating of channels probably methylammonium, glucosamine, agonist in ear artery smooth leads to an increase in internal tris and choline are slightly perweaker muscler5, a somewhat Ca2+ by two mechanisms: direct meant, at least in sensory neuragonist in vas deferens smooth ons2, PC12 celb?, hair cells”, entry of Ca2’ through the chanmuscle14 and neurons4, but has skeletal muscleZE and ear artery nels and activation of voltageonly antagonistic activity in carmusclers. dependent Ca’+ channels by diac muscle18. depolarization of the cell. Direct The permeation of Ca’+ is The receptor is of the P2 type, of particular interest because of entry of Ca2’ has been shown to based on its selectivity for ATP its importance as a second be quite large in ear artery smooth over adenosine. However, its messenger. Ca” has been shown muscler6, urinary bladder smooth agonist selectivity does not fall to carry current in most of the musclel’ and cochlear hair cellslo. neatly into any of the subclassifiATP-activated channels studied Although Ca*+ is permeant, cations of P2 receptors that have so far. Estimations of the peraddition of Ca2* to the external been proposedi. Unlike the agonist meability of Ca2’ relative to that solution actually reduces the curselectivity for the PzX receptor, of Na+ have varied widely; rent carried by Naf ions9,‘53.6,27*2g. ar,B-methylene-ATP Pca/PN, is estimated as 3 : 1 in ear This seemingly paradoxical obsermethylene-ATP are no%ore $s artery smooth muscle’s and hair vation can be understood if the tent than ATP for activating any celW, 2: 1 in chick skeletal pore of the channel possesses a of the channels studied. Unlike muscle=, 1: 1 in urinary bladder binding site for Ca**, so that Ca2+ the agonist selectivity for the Pzy smooth musclei7, and 0.3: 1 in ions pause (and block permeation receptor, 2-methylthio-ATP is not sensory neurons24. The estimations of Na+ ions) as they pass through more potent than ATP. of relative permeability are made the channel. Consistent with this using the Gol~an-Hod~inidea, the channel in skeletal Antagonists X&z equation, which depends on muscle can be blocked by Cd2+, The most potent antagonist assumptions known to be invalid Zn2’, Mn2+ and La2+ ions, comfound so far for ATP-activated (such as independent movement petitors for many Ca’+-binding channels is the dye reactive blue 2, of individual ions), so that the site.@. proposed as a P2v receptor antagapparent differences may result Because most of the current is onist in other systems. Reactive partly from different ionic conclearly carried by cations, it has blue 2 inhibits ATP-activated curditions. However, even with been believed in most cases that rents with an ICsc of l-10 M in similar conditions the Ca2’ perthe ATP-activated channels are ; the neurons4 and PC12 cells33” meability of the channels in rabbit purely cation selective. In the case inhibition in PC12 cells is comear artery seems clearly higher of the skeletal muscle channel, petitive. It will be interesting to than in sensory neurons. however, Thomas and Hume22 see if reactive blue 2 also serves as n-3

TiPS - March 1992 [Vol. 131 an antagonist at the smooth muscle, cardiac muscle and skeletal muscle channels. Suramin is another competitive antagonist, with an IC& of 30 p in PC12 cells7*32. Many adenosine derivatives without agonist activity act as competitive inhibitors, mostly rather weak. Adenosine 5’-(P,Ydichloromethylene)-triphosphonate is the most potent of these (GO -21 PM) at neuronal channels31. ar,P-Methylene ATP inhibits the effect of ATP in cardiac atria1 myocytes18 and parasympathetic neurvns4, but not in skeletal 'L.et’ssee whatmischief these guys can do outside the cell.’ muscle20,30, sensory neuron+ or vas deferens muscle14. focussed on effects of ATP at nicoBecause it is also effective as an tinic synapses, and ATP has been agonist at some channels, the infound to potentiate the effects of hibitory effect of cw,P-methyleneacetylcholine on both skeletal ATP might result from induction and sympathetic neurof long-term desensitization of the ZZ?e3’ channels. There are conflicting data about The distilbene DIDS and 2’,3’whether ATP by itself can activate dialdehyde-ATP are potent irreacetylcholine receptor channels. In versible inhibitors of the skeletal cell-attached patch recording from muscle channe130. DIDS, which skeletal muscle, ATP induces probably forms covalent bonds, activity of what are, apparently, also irreversibly blocks ATPnicotinic acetylcholine receptor induced 45Ca2+ entry in rat parchannels as identified by size, otid acinar cells33. reversal potential and cw-bungarotoain sensitivi@G38. However, Channel blockers wh?n outside-out patches are forn:ed from skeletal muscle, appliThere has been little effort to cation of ATP does not activate identify channel-blocking molchannels, even in patches that ecules. Since the ion selectivity possess high channel activity in of the channel resembles that of response to acetylcholine20*38.Also, well-studied channels such as the acetylcholine receptor channels nicotinic acetylcholine receptor, would not appear generally to be glutamate-activated channels, activated by ATP, because many 5-I-ITS-activated channels, and sympathetic and sensory neurons cation channels of the rod outer give large currents in response to segment, it will be interesting to acetylcholine but have no response test blockers of these channels at all to ATP. The discrepancy may such as local anesthetics (QX314, be partly one of resolution. Even (1X222), dizocilpine, phencyclidine with high ATP concentrations, the and (-)-diltiazem. channel activity in cell-attached patches is very low, orders of ATF and nicotinic receptor magnitude lower than the channel channels activity induced by even low There are interesting unresolved acetylcholine concentrations37,38. puzzles concerning interactions of In fact, the effect of ATP could ATP with niotinic acetylcholine be regarded as enhancing rare receptors in skeletal muscle and openings of channels that occur neurons. Because ATP is co-stored even in the complete absence of with acetylcholine in some synany transmitter. ATP enhances the aptic vesicles, attention has

89

spontaneous activity about tenfold, while acetylcholine induces activity at least 10 OOO-fold greater%. If ATF’ has an efficacy 1000 times lower than acetylcholine, its action might not be evident in many recording conditions. More important than any agonist activity of its own, may be ATP’s reported ability to enhance synergistically the effect of low acetylcholine concentrations. For example, micromolar concentrations of ATP were reported to produce a two- to threefold enhancement of channel activity in response to 0.1 nM acetylcholine in Xenopus skeletal muscle37. It would be interesting to explore the effects of ATP on acetylcholine using potency systematically whole-cell or outside-out patch recording. It is not known whether direct binding of ATP to the acetylcholine receptor is involved; there is some evidence for a second messenger system=. In PC12 cells, currents reversing near 0 mV are activated by both ATP and acetylcholine. Although the currents are clearly activated by distinct receptors (effects of ATP being antagonized by reactive blue 2 and suramin and those of acetylcholine by o-tubocurarine), Nakazawa and colleagues made the surprising discovery that the ATP- and acetylcholine-activated currents are not additive’. Similar currents were induced by either

TiPS -March

90 transmitter alone or both applied together, suggesting that the same channels can be activated by either transmitter. An intriguing possibility is that ‘heteromeric’ channels exist, formed by association of different subunits with binding sites for acetylcholine and ATP, with the channel liable to activation by either. q

q

cl

ATP-activated channels constitute a class of ligand-gated channels present primarily in muscle cells and neurons. The channels are controlIed by P-&ype receptors, with some differences in agonist selectivity between different cell types. Reactive blue 2 is the most potent competitive antagonist, and little is known about potential channel blockers. Nothing is known about the structure of the channels; one might guess that they may have a subunit structure similar to nicotinic acetylcholine receptor channels, especially if heteromeric acetylcholine/ATP channels can be formed. BRUCEP.BEAN &mhnent of Neurobiology, Hnmnrd Medical School, 220 Longwood Avenue, Boston, MA 02115, USA.

References 1 Burnstock, G. (1990) Ann. NY Acad. Sci. 603,1-17 2 Krishtai, 0. A., Marcbenko, S. M. and Pidoplicbko, V. I. (1983) Neurosci. Left. 35,41-45 3 Bean, 8. P. (1990) J. Neurosci. 10, l-10 4 Fieber, L. A. and Adams, D. J. (1991) J. Physiol. ILond.) 434, 239-256 5 Jahr, C. E. and Jessell, T. M. (1983) Nature 304.730-733 6 Inoue, K., Nakazawa, K., Fujimori, K., Watano, T. and Takanaka. A. Ncurosci. Letf. (in press) 7 Nakazawa, K., Fujimori, K., Takanaka, A. and Inoue, K. (1990) BT.J. Pharv~acol. 101,224-226 8 Nakazawa, K., Fujimori, K., Takanaka, A. and Inoue, K. (1991) J. Physiol: (Loud.) 434.647660 9 Net&us, R.; f&b&, B. F. X. and Reuter, H. (1991) I. Neurosci. 11.3984-3990 10 Ashmo~,~ J. F. and Ohmori, H. (1990) j. Physiof. (Land.) 428, 109-131 11 Nakagawa, T., Akaike, N., Kimitsuki, T., Komune, S. and Arima, T. (1990) J. Neurophysiol. 63,1068-1074 l2 von Kugegen, I. and Starke, K. (1991) Trends Pharmacoi. Sci. 12,319-324 13 Nakazawa, K. and Matsuki, N. (1987) Pflug. Arch. 409,644646 14 Friel, D. D. (1988) 1. Physiol. (Land.) 401, 361-380 15 Benham, C. D. and Tsien, R. W. (1987) Nature 328,275-278

16 Benham, C. D. (1989) /. Physiol. (Loud.) 419,68%701 17 Schneider, P., Hopp, H. H. and Isenberg, G. (1991) 1. PhysioJ. (Loud.) 440,479-496 18 Friel, D. D. and Bean, B. P. (1988) J. Gen. Physiol. 91, l-27 19 Friel, D. D. and Bean, B. P. (1990) Pflug. Arch. 415,651-657 20 Hume, R. I. and Honig. M. G. (1986) J. Neurosci. 6,681-690 21 Hume, R. 1. and Thomas, S. A. (1988) J. Physiof. (Land.) 406, 503-524 22 Thomas, S. A. and Hume, R. I. (1990) 1. Gen. Phusiol. 95. 569-590 23 ‘Kleuss, C.-et al. (1991) Nature 353,43-48 24 Bean, B. P., Williams, C. A. and Ceelen, P. W. (1990) I. Neurosci. 10; 11-19 25 Bean, B. P. and Friel, D. D. (1990) in Ion Channels (Vol. 2) (Narabashi. T.. ed.). .pp. 16%203, Plenum Press 26 Krishtal, 0. A., Marchenko, S. M. and Obukbov, A. G. (1988) Neuroscience 27, 995-1000 27 Nakazawa, K., Inoue, K., Fujimori, K. and Takanaka, A. (1990) Neurosci. Letf. 119,5-8 28 Hagiwara, S. and Byerly, L. (1981) Annu. Rev. Neurosci. 4, 6%125 29 Nakazawa, K., Fujimori, K., Takanaka,

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A. and Inoue, K. (1990) J. Physiol. (Loud.) 428.257-272 Thomas, S:A, Zawisa, M. J., Lin, X. and Hume. R. I. (1991) Br. 1. Pharmncof. 103, 1963-1969 ’ . Krishtal, 0. A., Marcbenko, S. M., Obukbov. A. G. and Volkova. T. M. (1988) Br..J. Pharmacol. 95,1057~1062 Nakazawa, K., Inoue, K., Fujimori, K. and Takanaka, A. (1991) Pflug. Arch. 418,214219 Soltoff, S. P., McMiBian, M. K., Takuno, B. R. and Cantley, L. C. (1990) Ann. NY Acad. Sci. 603, 446-447 Ewald, D. A. (1976) 1. Membr. Biol. 29, 47-65 Akasu, T. and Koketsu, K. (1985) Br. 1. Pharmacol. 84,525-531 (Abstr.) Kolb, H-A. and Wakelam, M. J. 0. (1983) Nature 306,621-623 Igusa, Y. (1988) J. Physiol. (Land.) 405, 169-185 Lu, Z. and Smith, D. 0. (1991) 1. Physiol. (Land.) 436,45-56

DIDS: 4,4’-diisocyanatostilbene-2,2’disulphonic acid QX222: N’-(trimethylaminomethyl)~2’,6’xylidide QX314: N,N,N-trimethy12’,6’-xylidide

Myocardial preconditioning as the heart’s self-protecting response against the consequences of ischaemia Prolonged occlusion of a coronary artery is associated with the development of severe ventricular arrhythmias, sustained ischaemic myocardial damage and subsequent cellular necrosis. In 1983, Barber attempted to demonstrate that serial occlusions of the left anterior descending coronary artery in dogs produced consistent electrocardiographic changesl. However, it was observed that if occlusions were separated by relatively short periods of time (three minutes), then the electrocardiographic changes seen during the second occlusion were markedly diminished compared to the first occlusion. This effect was subsequently observed on myocardial damage in dogs, where a similar series of coronary occlusions decreased the ultrastructural changes resulting from prolonged ischaemia* (Fig. 1). It was therefore suggested that the initial ischaemic stimulus in this type of protocol ‘preconditions’ the heart against a subsequent, more severe ischaemic @ 1992, Elaevier Science Publishers Ltd (UK)

insult. A very recent suggestion to explain these effects is that Aiadenosine receptors are activated in response to the preconditioning stimulus and this in some way increases the tolerance of the heart to further injury. The aim of this article is to describe the process of preconditioning and to summarize the current level of understanding of the mechanisms involved. It was initially believed that the preconditioning phenomenon was a result of opening up of coronary collateral vessels. However, the demonstration of similar protection in low collateral flow models such as rabbits3, rats4 and pigs5 has disproved this theory. Preconditioning is also inducible by means other than regional myocardial ischaemia, such as rapid ventricular pacing to induce global ischaemiae. A similar preconditioning phenomenon may exist in humans, as there are documented reports of repeated coronary vasospasm where some incidents showed