TiPS -March 1992 [Vol. 131

Pharmacology and electrophysiology of ATP-activated ion channels

Extracellular ATE serves as a mess- enger in many tissues*. Specific receptors for ATE mediate a variety of effects, some involv- ing 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 character- istics, including agonist selec- tivity, size, ion selectivity and de- sensitization.

Cell types and functions ATE-activated channels are

present in a subset of neurons in sensory ganglia of the cat, rat and froe3. So far, voltage-clamp recordings have been possible only in cell bodies, where a func- tional role for the channels is unlikely. If the channels are also present in afferent nerve endings, they would elicit firing in re- sponse to ATP, perhaps serving to report tissue damage. ATP- activated channels are also present in cardiac parasympathetic neur- ons4. 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. ATP- activated 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 ATP- activated channels is clearest in a variety of smooth muscle cells. In vas deferens muscle, it is likely that the channels produce a com- ponent of fast, non-adrenergic non-cholinergic excitatory trans- mission by sympathetic neur- ons12-14

. Post-ganglionic sym- pathetic axons contain vesicles in which ATP is co-stored with norepinephrine12. ATE-activated channels may similarly mediate a component of sympathetic trans- mission 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 vaso- dilation, are mediated indirectly by endothelial cell ATE receptors coupled to second messenger sys- tems.) 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 acetyl- choline receptors. Very similar channels of both types are also present in cultured embryonic skeletal muscle2c22.

Kinetics In all these cases, the depolir-

izing, Na+- and K+-permeable channels are directly activated by binding of ATE molecules, as shown by currents that turn on within milliseconds of ATP appli- cation. (By contrast, the G protein- mediated K+ currents in cardiac and skeletal muscle take a few hundred milliseconds to activate.)

87

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 car- diac 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, dose- response 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 super- linea@ (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-ac-

tivated 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 cur- rents are much larger in neurons, PC12 cells and smooth muscle; in quasi-physiological saline sol- ution, the single-channel conduc- tance ranges from 13 pS to 60 pS4,9J3.15~24-27. Opening and clos- ing kinetics can be very complex and have not yet been characterized in any detail.

Ionic selectivity The directly-activated ATP-

gated channels are in all cases equally permeable to small cations such as Na+, K+ and Cs+. Even

@ 1992. Elsevicr Science Publishers Ltd (UK)

n-3

-1 0 1 2

tog W”l

E&T. 1. Hill p&X of wncenttzi~ data &r ourrent activated by external ATP a@ed to a buMtog olnW mot ganglion neuron. The cell was voltage-clamped at -80 mVandk!waniarnent was activated bv aoolioation of ATP (insetI. Maximal current was aotivated by S00 JIM ATP. At low wn&~tions (0.3-1.2 & AT@ the Hill slope is d srggeslin a ~~ of 3 ATP molecules per channet. At high concentrations

very large cations such as tetra- methylammonium, glucosamine, tris and choline are slightly per- meant, at least in sensory neur- ons2, PC12 celb?, hair cells”, skeletal muscleZE and ear artery musclers.

The permeation of Ca’+ is of particular interest because of its importance as a second messenger. Ca” has been shown to carry current in most of the ATP-activated channels studied so far. Estimations of the per- meability of Ca2’ relative to that of Na+ have varied widely; Pca/PN, is estimated as 3 : 1 in ear artery smooth muscle’s and hair celW, 2: 1 in chick skeletal muscle=, 1: 1 in urinary bladder smooth musclei7, and 0.3: 1 in sensory neurons24. The estimations of relative permeability are made using the Gol~an-Hod~in- X&z equation, which depends on assumptions known to be invalid (such as independent movement of individual ions), so that the apparent differences may result partly from different ionic con- ditions. However, even with similar conditions the Ca2’ per- meability of the channels in rabbit ear artery seems clearly higher than in sensory neurons.

Under physiologic~ conditions, ATP gating of channels probably leads to an increase in internal Ca2+ by two mechanisms: direct entry of Ca2’ through the chan- nels and activation of voltage- dependent Ca’+ channels by depolarization of the cell. Direct entry of Ca2’ has been shown to be quite large in ear artery smooth muscler6, urinary bladder smooth musclel’ and cochlear hair cellslo.

Although Ca*+ is permeant, addition of Ca2* to the external solution actually reduces the cur- rent carried by Naf ions9,‘53.6,27*2g. This seemingly paradoxical obser- vation can be understood if the pore of the channel possesses a binding site for Ca**, so that Ca2+ ions pause (and block permeation of Na+ ions) as they pass through the channel. Consistent with this idea, the channel in skeletal muscle can be blocked by Cd2+, Zn2’, Mn2+ and La2+ ions, com- petitors for many Ca’+-binding site.@.

Because most of the current is clearly carried by cations, it has been believed in most cases that the ATP-activated channels are purely cation selective. In the case of the skeletal muscle channel, however, Thomas and Hume22

TiPS - March 1992 [Vol. 131

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 evi- dence against some anion per- meability 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 deriva- tives4,1’,15,1s,20,30. Adenosine and AMP are completely ineffective in most cells, while ADF is a weak agonist. UTP and GTP are ineffec- tive; CTP apparently has some agonist activity in neurons31 but not skeletal muscle30.

ATPyS (adenosine 5’-O-(3-thio- ~phosphate) is roughly equi- potent with ATP in cardiac muscle*a, skeletal muscle3’ and PC12 cells”, as is 2-methylthio- ATP tested in skeletal muscle3’ and neurons4. Although the recep- tor seems broadly similar in the different cell types, clear differ- ences are made evident by a[,@- methylene-ATP, which is a potent agonist in ear artery smooth muscler5, a somewhat weaker agonist in vas deferens smooth muscle14 and neurons4, but has only antagonistic activity in car- diac muscle18.

The receptor is of the P2 type, based on its selectivity for ATP over adenosine. However, its agonist selectivity does not fall neatly into any of the subclassifi- cations of P2 receptors that have been proposedi. Unlike the agonist selectivity for the PzX receptor, ar,B-methylene-ATP methylene-ATP are no%ore $s tent than ATP for activating any of the channels studied. Unlike the agonist selectivity for the Pzy receptor, 2-methylthio-ATP is not more potent than ATP.

Antagonists The most potent antagonist

found so far for ATP-activated channels is the dye reactive blue 2, proposed as a P2v receptor antag- onist in other systems. Reactive blue 2 inhibits ATP-activated cur- rents with an ICsc of l-10 M in neurons4 and PC12 3” cells3 ; the inhibition in PC12 cells is com- petitive. It will be interesting to see if reactive blue 2 also serves as

TiPS - March 1992 [Vol. 131 89

an antagonist at the smooth muscle, cardiac muscle and skeletal muscle channels. Sura- min is another competi- tive 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,Y- dichloromethylene)-tri- phosphonate is the most potent of these (GO -21 PM) at neur- onal channels31.

ar,P-Methylene ATP inhibits the effect of ATP in cardiac atria1 myocytes18 and para- sympathetic neurvns4, but not in skeletal muscle20,30, sensory

'L .et’s see what mischief these guys can do outside the cell.’

neuron+ or vas deferens muscle14. Because it is also effective as an agonist at some channels, the in- hibitory effect of cw,P-methylene- ATP might result from induction of long-term desensitization of the channels.

The distilbene DIDS and 2’,3’- dialdehyde-ATP are potent irre- versible inhibitors of the skeletal muscle channe130. DIDS, which probably forms covalent bonds, also irreversibly blocks ATP- induced 45Ca2+ entry in rat par- otid acinar cells33.

Channel blockers There has been little effort to

identify channel-blocking mol- ecules. Since the ion selectivity of the channel resembles that of well-studied channels such as the nicotinic acetylcholine receptor, glutamate-activated channels, 5-I-ITS-activated channels, and cation channels of the rod outer segment, it will be interesting to test blockers of these channels such as local anesthetics (QX314, (1X222), dizocilpine, phencyclidine and (-)-diltiazem.

ATF and nicotinic receptor channels

There are interesting unresolved puzzles concerning interactions of ATP with niotinic acetylcholine receptors in skeletal muscle and neurons. Because ATP is co-stored with acetylcholine in some syn- aptic vesicles, attention has

focussed on effects of ATP at nico- tinic synapses, and ATP has been found to potentiate the effects of acetylcholine on both skeletal

ZZ?e3’ and sympathetic neur-

There are conflicting data about whether ATP by itself can activate acetylcholine receptor channels. In cell-attached patch recording from skeletal muscle, ATP induces activity of what are, apparently, nicotinic acetylcholine receptor channels as identified by size, reversal potential and cw-bungaro- toain sensitivi@G38. However, wh?n outside-out patches are forn:ed from skeletal muscle, appli- cation of ATP does not activate channels, even in patches that possess high channel activity in response to acetylcholine20*38. Also, acetylcholine receptor channels would not appear generally to be activated by ATP, because many sympathetic and sensory neurons give large currents in response to acetylcholine but have no response at all to ATP. The discrepancy may be partly one of resolution. Even with high ATP concentrations, the channel activity in cell-attached patches is very low, orders of magnitude lower than the channel activity induced by even low acetylcholine concentrations37,38.

In fact, the effect of ATP could be regarded as enhancing rare openings of channels that occur even in the complete absence of any transmitter. ATP enhances the

spontaneous activity about ten- fold, while acetylcholine induces activity at least 10 OOO-fold greater%. If ATF’ has an efficacy 1000 times lower than acetyl- choline, its action might not be evident in many recording conditions.

More important than any agon- ist activity of its own, may be ATP’s reported ability to enhance synergistically the effect of low acetylcholine concentrations. For example, micromolar concen- trations of ATP were reported to produce a two- to threefold en- hancement 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 potency systematically using whole-cell or outside-out patch recording. It is not known whether direct binding of ATP to the acetyl- choline 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

90 TiPS -March 2992 [Vol. 131

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 associ- ation of different subunits with binding sites for acetylcholine and ATP, with the channel liable to activation by either.

q q cl

ATP-activated channels consti- tute a class of ligand-gated chan- nels present primarily in muscle cells and neurons. The channels are controlIed by P-&ype recep- tors, with some differences in agonist selectivity between dif- ferent cell types. Reactive blue 2 is the most potent competitive antagonist, and little is known about potential channel blockers. Nothing is known about the struc- ture of the channels; one might guess that they may have a sub- unit structure similar to nicotinic acetylcholine receptor channels, especially if heteromeric acetyl- choline/ATP channels can be formed.

BRUCEP.BEAN

&mhnent of Neurobiology, Hnmnrd Medical School, 220 Longwood Avenue, Boston, MA 02115, USA.

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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 de- velopment of severe ventricular arrhythmias, sustained ischaemic myocardial damage and sub- sequent cellular necrosis. In 1983, Barber attempted to demonstrate that serial occlusions of the left anterior descending coronary artery in dogs produced consist- ent electrocardiographic changesl. However, it was observed that if occlusions were separated by relatively short periods of time (three minutes), then the electro- cardiographic 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 ultra- structural changes resulting from prolonged ischaemia* (Fig. 1).

It was therefore suggested that the initial ischaemic stimulus in this type of protocol ‘precon- ditions’ the heart against a sub- sequent, more severe ischaemic

@ 1992, Elaevier Science Publishers Ltd (UK)

insult. A very recent suggestion to explain these effects is that Ai- adenosine receptors are activated in response to the precondition- ing 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 summar- ize the current level of under- standing of the mechanisms involved.

It was initially believed that the preconditioning phenomenon was a result of opening up of coronary collateral vessels. How- ever, 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