Toxins in the characterization of potassium channels.

Several recently characterized toxins (apamin, charybdotoxin, dendrotoxin and noxiustoxin) are proving invaluable for establishing what kinds of potassium channel are expressed in neurones, and what the roles of the channels might be.

How we describe competitive antagonists: three questions of usage.

Competitive antagonists are used by pharmacologists in a definitive manner in the identification of receptor subtypes. However, despite the many decades of their use, there is still some confusion over terms and terminology. Don Jenkinson, a member of the IUPHAR Committee on Receptor Nomenclature, points to terms that are often used imprecisely: pA2 is frequently used interchangeably, but erroneously, for log KB; the term 'competitive' itself has been used ambiguously, and it is even possible that confusion can arise by the expression of the constant from the Schild equation in different ways (affinity/equilibrium/dissociation constants).

Calcium-activated potassium channels in liver cells.

Activation of certain membrane receptors increases the concentration of Ca2+ in the cytosol of hepatocytes. Since in most species these cells possess a PK(Ca) mechanism, the outcome is a rise in PK. This can be blocked by quinine, apamin and certain neuromuscular blocking agents. The binding of labelled apamin to hepatocytes has been studied under physiological conditions, and the relationship between the binding sites and K+ channels is discussed. The physiological role of the PK(Ca) mechanism in hepatocytes is unclear, though it is largely responsible for 'adrenaline hyperkalaemia'.

Synthesis, molecular modeling, and pharmacological testing of bis-quinolinium cyclophanes: potent, non-peptidic blockers of the apamin-sensitive Ca(2+)-activated K(+) channel.

The synthesis and pharmacological testing of two series of novel bis-quinolinium cyclophanes as blockers of the apamin-sensitive Ca(2+)-activated K(+) (SK(Ca)) channel are presented. In these cyclophanes the two 4-aminoquinolinium groups are joined at the ring N atoms (linker L) and at the exocyclic N atoms (linker A). In those cases where A and L contain two or more aromatic rings each, the activity of the compound is not critically dependent upon the nature of the linkers. When A and L each have only one benzene ring, the blocking potency changes dramatically with simple structural variations in the linkers. One of these smaller cyclophanes having A = benzene-1,4-diylbis(methylene) and L = benzene-1, 3-diylbis(methylene) (3j, 6,10-diaza-1,5(1,4)-diquinolina-3(1,3),8(1, 4)-dibenzenacyclodecaphanedium tritrifluoroacetate, UCL 1684) has an IC(50) of 3 nM and is the most potent non-peptidic SK(Ca) channel blocker described to date. Conformational analysis on the smaller cyclophanes using molecular modeling techniques suggests that the differences in the blocking potencies of the compounds may be attributable to their different conformational preferences.

Synthesis and structure-activity relationships of dequalinium analogues as K+ channel blockers. Investigations on the role of the charged heterocycle.

Small conductance Ca(2+)-activated K+ (SKCa) channels occur in many cells but have been relatively little studied. Dequalinium, a bis-quinolinium compound, has recently been shown to be the most potent nonpeptidic blocker of this K+ channel subtype. This paper examines the importance of the quinolinium rings for blocking activity. Analogues of dequalinium were synthesised in which one quinolinium group was removed (1 and 2) or replaced by a triethylammonium group (3). They have been assayed in vitro for their ability to block the after-hyperpolarization (mediated by the opening of SKCa channels) that follows the action potential in rat sympathetic neurones. The compound having one quinolinium and one triethylammonium group (3) showed reduced activity, and it is suggested that the stronger binding to the channel of the quinolinium relative to the triethylammonium group may be related to differences in their electrostatic potential energy maps. Two monoquaternary compounds (1 and 2) were tested, but they exhibited a different pharmacological profile that did not allow definite conclusions to be drawn concerning their potency as blockers of the SKCa channel. Replacement of both quinolinium groups by pyridinium, acridinium, isoquinolinium, or benzimidazolium reduced but did not abolish activity. These results show that compounds having a number of different heterocyclic cations are capable of blocking the SKCa channel. However, among the heterocycles studied, quinoline is optimal. Furthermore, charge delocalization seems to be important: the higher the degree of delocalization the more potent the compound.

Synthesis and structure-activity relationships of cetiedil analogues as blockers of the Ca(2+)-activated K+ permeability of erythrocytes.

Cetiedil, [2-cyclohexyl-2-(3-thienyl)ethanoic acid 2-(hexahydro-1H-azepin-1-yl)ethyl ester], which blocks the intermediate calcium-activated potassium ion permeability (IK(Ca)) in red blood cells, was used as a lead for investigating structure-activity relationships with the aim of determining the pharmacophore and of synthesizing agents of greater potency. A series of compounds having structures related to cetiedil was made and tested on rabbit erythrocytes. Channel blocking activity within the series was found to correlate well with octanol-water partition coefficients but not with the specific chemical structure of the acid moiety. However, whereas log P for the compounds spans a range of values over 4 orders of magnitude, potency only increases by 2 orders. This suggests that hydrophobic interactions with an active site on the channel are probably not the main determinants of activity. It seems more likely that increased lipophilicity enhances access to the channel, probably from within the cell membrane. In keeping with this interpretation, cetiedil methoiodide was found to be inactive. Triphenylethanoic was found to be a more effective acid grouping than 2-cyclohexyl-2-(3-thienyl)ethanoic, and its 2-(hexahydro-1H-azepin-l-yl)ethyl ester (11) was approximately 3 times more potent than cetiedil. The 9-benzylfluoren-9-yl carboxylic acid ester (21) was found to be approximately 9 times more active than cetiedil, and replacing -CO(2)- in 21 by an ethynyl (-C identical to C-) linkage (compound 26, UCL 1608) increased potency by some 15-fold over that of cetiedil.

Influence of chloride ions on changes in membrane potential during prolonged application of carbachol to frog skeletal muscle.

1. Micro-electrodes were used to follow changes in the membrane potential at the end-plate region of single fibres in narrow strips of frog skeletal muscle exposed to carbachol applied in continuously flowing Ringer solution containing tetrodotoxin (200 nM) and neostigmine (3 muM).2. The depolarizations elicited by carbachol (5-20 muM) usually developed in two phases, the first of which was generally complete within 30 s whereas several min were required for the second.3. Repolarization after carbachol also occurred in two phases, the second of which outlasted the time needed to clear the bath, and varied with the magnitude and duration of the depolarization which carbachol had caused.4. These findings could best be explained in terms of the consequences of net entry of chloride ions into the fibre during the depolarization caused by carbachol. This hypothesis is supported by three lines of evidence:(a) Replacement of the chloride content of the Ringer solution by the less permeant anion isethionate abolished the slow phases of the carbachol response.(b) Reduction of chloride permeability (by lowering pH) caused rapid repolarization during the recovery period after carbachol.(c) When the membrane potential was clamped at the resting level throughout the action of carbachol, so avoiding chloride redistribution, the clamping current records did not show the slow phases attributed to chloride movement.5. Chloride redistribution contributes to the gradual spread of depolarization during prolonged applications of depolarizing agents to skeletal muscle. It also complicates the interpretation of the dose-response relationship, and may make it more difficult to assess the extent to which the receptors become desensitized during the action of agonists applied in the bath.

Interactions between the effects of alpha- and beta-adrenoceptor agonists and adenine nucleotides on the membrane potential of cells in guinea-pig liver slices.

1 The beta-adrenoceptor agonist isoprenaline normally causes only a small and inconsistent increase in the membrane potential of cells in guinea-pig liver slices, in contrast to the large hyperpolarizations seen with alpha-agonists. However, after a selective alpha-adrenoceptor agonist has been applied, the response to isoprenaline becomes greatly enhanced. 2 Simultaneous application of small doses of an alpha- and beta-agonist produce hyperpolarizations larger than the sum of the responses to each agent alone. 3 These interactions occur with a range of sympathomimetic amines, including some which are not substrates for various processes for the uptake and inactivation of catecholamines. 4 Hyperpolarizations caused by externally applied cyclic adenosine-3',5'-monophosphate (cyclic AMP) also become larger after application of an alpha-agonist. 5 The adenine nucleotides adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP) hyperpolarize guinea-pig liver cells in the dose range 0.1-1.0 mM. This response is not increased after an alpha-agonist. However, ADP and ATP are themselves able to enhance the response to beta-agonists. 6 These interactions between alpha-agonists, beta-agonists and adenine nucleotides seem to involve steps subsequent to receptor activation. Changes in the intracellular actions of cyclic AMP may be concerned.

Interactions between receptors that increase cytosolic calcium and cyclic AMP in guinea-pig liver cells.

The action of agonists which increase the K+ permeability of liver cells was studied by using a K+-sensitive electrode to record the net movement of K+ between guinea-pig isolated hepatocytes and their suspension medium. Two types of agonist were examined. Type 1 comprised angiotensin II, ATP, noradrenaline and amidephrine, all of which are thought to raise cytosolic Ca2+ in hepatocytes. The Type 2 agonists were isoprenaline and glucagon, which activate adenylate cyclase. Each type of agonist initiated K+ loss from the hepatocytes though the response to Type 2 agonists was more variable than that to Type 1, and sometimes absent. Simultaneous application of a small concentration of an agonist from each class caused a loss of K+ which was much larger than the sum of that seen with each agonist alone, i.e. potentiation occurred. The alpha-adrenoceptor antagonist, WB 4101, abolished potentiation if applied after an alpha-agonist, and before a Type 2 agonist, showing that both receptors have to be active for potentiation to occur. Simultaneous application of a maximal concentration of each type of agonist caused a larger loss of K+ (approximately 17% of the cell total within 45 s) than did a maximal concentration of a Type 1 agonist alone (approximately 10%). Since the K+ loss caused by these agonists is thought to be a consequence of a rise in cytosolic Ca2+, the influence of both types of agonist on 45Ca and 42K efflux from guinea-pig liver slices was studied. The effect of isoprenaline on 45Ca and 42K efflux became much greater following a previous application of the alpha-adrenoceptor agonist, amidephrine. In the presence of apamin, the potentiated effect of isoprenaline on 42K efflux was greatly reduced whereas that on 45Ca efflux was little affected. The effects of Type 1 and Type 2 agonists separately and together on the cyclic AMP content of isolated hepatocytes were examined. Type 2 agonists increased cyclic AMP in the expected way. The increase became slightly smaller, if anything, when a Type 1 agonist was applied at the same time. Hence potentiation could not be ascribed to changes in cyclic AMP formation. Possible mechanisms for potentiation are discussed. Our evidence suggests, albeit indirectly, that it is a consequence of an interaction between the effects of the two types of agonist on cytosolic Ca2+.

Compounds that block both intermediate-conductance (IK(Ca)) and small-conductance (SK(Ca)) calcium-activated potassium channels.

1. Nine bis-quinolinyl and bis-quinolinium compounds related to dequalinium, and previously shown to block apamin-sensitive small conductance Ca(2+)-activated K(+) channels (SK(Ca)), have been tested for their inhibitory effects on actions mediated by intermediate conductance Ca(2+)-activated K(+) channels (IK(Ca)) in rabbit blood cells. 2. In most experiments, a K(+)-sensitive electrode was employed to monitor the IK(Ca)-mediated net loss of cell K(+) that followed the addition of the Ca(2+) ionophore A23187 (2 microM) to red cells suspended at an haematocrit of 1% in a low K(+) (0.12 - 0.17 mM) solution. The remainder used an optical method based on measuring the reduction in light transmission that occurred on applying A23187 (0.4 or 2 microM) to a very dilute suspension of red cells (haematocrit 0.02%). 3. Of the compounds tested, the most potent IK(Ca) blocker was 1,12 bis[(2-methylquinolin-4-yl)amino]dodecane (UCL 1407) which had an IC(50) of 0.85+/-0.06 microM (mean+/-s.d. mean). 4. The inhibitory action of UCL 1407 and its three most active congeners was characterized by (i) a Hill slope greater than unity, (ii) sensitivity to an increase in external [K(+)], and (iii) a time course of onset that suggested use-dependence. Also, the potency of the nonquaternary compounds tested increased with their predicted lipophilicity. These findings suggested that the IK(Ca) blocking action resembles that of cetiedil rather than of clotrimazole. 5. Some quaternized members of the series were also active. The most potent was the monoquaternary UCL 1440 ((1-[N-[1-(3, 5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino]-10-[N'-(2-me thylqu inolinium-4yl)amino] decane (trifluoroacetate) which had an IC(50) of 1.8+/-0.1 microM. The corresponding bisquaternary UCL 1438 (1, 10-bis[N-[1-(3,5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino] decane bis(trifluoroacetate) was almost as active (IC(50) 2.7+/-0.3 microM). 6. A bis-aminoquinolium cyclophane (UCL 1684) had little IK(Ca) blocking action despite its great potency at SK(Ca) channels (IC(50) 4.1+/-0.2 nM). 7. The main outcome is the identification of new intermediate-conductance Ca(2+)-activated K(+) channel blockers with a wide range of IK(Ca)/SK(Ca) selectivities.

Neuromuscular blocking agents inhibit receptor-mediated increases in the potassium permeability of intestinal smooth muscle.

The neuromuscular blocking agents tubocurarine, atracurium and pancuronium have been tested for their ability to inhibit receptor-mediated increases in the K+ permeability of intestinal smooth muscle. All three agents, as well as the bee venom peptide apamin, reduced both the resting efflux of 86Rb and the increase in efflux caused by the application of either bradykinin (1 microM) or an alpha 1-adrenoceptor agonist, amidephrine (20 microM), to depolarized strips of guinea-pig taenia caeci. This suggested that like apamin, the neuromuscular blocking agents inhibit the Ca2+-dependent K+ permeability (PK(Ca] mechanism which in this tissue is activated by a variety of membrane receptors. The concentrations (IC50S) of atracurium, pancuronium and (+)-tubocurarine which reduced the effect of amidephrine on 86Rb efflux by 50% were 12, 37 and 67 microM respectively. Also in keeping with an ability to block PK(Ca), the neuromuscular blockers and apamin reduced the inhibition by amidephrine and bradykinin of physalaemin-mediated contractions of the taenia caeci. The IC50 values were 15, 31 and 120 microM for atracurium, tubocurarine and pancuronium respectively, and 2.3 nM for apamin. Each of the neuromuscular blockers, and apamin, increased the spontaneous contractions of the rabbit duodenum and blocked the inhibitory effect of amidephrine thereon. It is concluded that the PK(Ca) mechanism in the longitudinal smooth muscle of the intestine It is concluded that the PK(Ca) mechanism in the longitudinal smooth muscle of the intestine resembles that of hepatocytes and sympathetic ganglion cells in its susceptibility to inhibition by neuromuscular blocking agents, as well as by apamin.

Discrimination between subtypes of apamin-sensitive Ca(2+)-activated K+ channels by gallamine and a novel bis-quaternary quinolinium cyclophane, UCL 1530.

1. Gallamine, dequalinium and a novel bis-quaternary cyclophane, UCL 1530 (8,19-diaza-3(1,4),5(1,4)-dibenzena-1 (1,4),7(1,4)-diquinolina-cyclononadecanephanedium) were tested for their ability to block actions mediated by the small conductance, apamin-sensitive Ca(2+)-activated K+ (SKCa) channels in rat cultured sympathetic neurones and guinea-pig isolated hepatocytes. 2. SKCa channel block was assessed in sympathetic neurones by the reduction in the slow afterhyperpolarization (AHP) that follows an action potential, and in hepatocytes by the inhibition of the SKCa mediated net loss of K+ that results from the application of angiotensin II. 3. The order of potency for inhibition of the AHP in sympathetic neurones was UCL 1530 > dequalinium > gallamine, with IC50 values of 0.08 +/- 0.02, 0.60 +/- 0.05 and 68.0 +/- 8.4 microM respectively, giving an equi-effective molar ratio between gallamine and UCL 1530 of 850. 4. The same three compounds inhibited angiotensin II-evoked K+ loss from guinea-pig hepatocytes in the order dequalinium > UCL 1530 > gallamine, with an equi-effective molar ratio for gallamine to UCL 1530 of 5.8, 150 fold less than in sympathetic neurones. 5. Dequalinium and UCL 1530 were as effective on guinea-pig as on rat sympathetic neurones. 6. UCL 1530 at 1 microM had no effect on the voltage-activated Ca2+ current in rat sympathetic neurones, but inhibited the hyperpolarization produced by direct elevation of cytosolic Ca2+. 7. Direct activation of SKCa channels by raising cytosolic Ca2+ in hepatocytes evoked an outward current which was reduced by the three blockers, with dequalinium being the most potent. 8. These results provide evidence that the SKCa channels present in guinea-pig hepatocytes and rat cultured sympathetic neurones are different, and that this is not attributable to species variation. UCL 1530 and gallamine should be useful tools for the investigation of subtypes of apamin-sensitive K+ channels.

Differences in the actions of some blockers of the calcium-activated potassium permeability in mammalian red cells.

1. The actions of some inhibitors of the Ca2+-activated K+ permeability in mammalian red cells have been compared. 2. Block of the permeability was assessed from the reduction in the net loss of K+ that followed the application of the Ca2+ ionophore A23187 (2 microM) to rabbit red cells suspended at a haematocrit of 1% in a low potassium solution ([K]0 0.12-0.17 mM) at 37 degrees C. Net movement of K+ was measured using a K+-sensitive electrode placed in the suspension. 3. The concentrations (microM +/- s.d.) of the compounds tested causing 50% inhibition of K+ loss were: quinine, 37 +/- 3; cetiedil, 26 +/- 1; the cetiedil congeners UCL 1269, UCL 1274 and UCL 1495, approximately 150, 8.2 +/- 0.1, 0.92 +/- 0.03 respectively; clotrimazole, 1.2 +/- 0.1; nitrendipine, 3.6 +/- 0.5 and charybdotoxin, 0.015 +/- 0.002. 4. The characteristics of the block suggested that compounds could be placed in two groups. For one set (quinine, cetiedil, and the UCL congeners), the concentration-inhibition curves were steeper (Hill coefficient, nH, > or = 2.7) than for the other (clotrimazole, nitrendipine, charybdotoxin) for which nH approximately 1. 5. Compounds in the first set alone became less active on raising the concentration of K+ in the external solution to 5.4 mM. 6. The rate of K+ loss induced by A23187 slowed in the presence of high concentrations of cetiedil and its analogues, suggesting a use-dependent component to the inhibitory action. This was not seen with clotrimazole. 7. The blocking action of the cetiedil analogue UCL 1274 could not be overcome by an increase in external Ca2+ and its potency was unaltered when K+ loss was induced by the application of Pb2+ (10 microM) rather than by A23187. 8. These results, taken with the findings of others, suggest that agents that block the red cell Ca2+-activated K+ permeability can be placed in two groups with different mechanisms of action. The differences can be explained by supposing that clotrimazole and charybdotoxin act at the outer face of the channel whereas cetiedil and its congeners may block within it, either at or near the K+ binding site that determines the flow of K+.

Blockade by WB 4101 of alpha-adrenoceptors in the rat vas deferens and guinea-pig taenia caeci.

The effectiveness of WB 4101, a recently described alpha-adrenoceptor antagonist, in blocking an excitatory and two inhibitory responses to alpha-receptor activation was studied. One of the inhibitory responses was the reduction by the selective alpha-agonist amidephrine of carbachol contractures of isolated guinea-pig taenia caeci. WB 4101 antagonised this inhibition with a Schild plot slope of 0.99 and a pA2 of 8.9. The same pA2 value was obtained for blockade of the contractile effect of amidephrine and noradrenaline on the rat vas deferens. WB 4101 was, however, several hundred times less active in antagonising the inhibitory effect of clonidine on the twitch response of the vas deferens to field stimulation. Incidental observations were that the twitch was increased by low concentrations of amidephrine, and by relatively high concentrations of WB 4101. Because of its potency and postsynaptic selectivity, WB 4101 should be useful for adrenoceptor classification.

Antagonism of an indirectly acting agonist: block by propranolol and sotalol of the action of tyramine on rat heart.

Some agonists act indirectly in the sense that they cause the release of a second substance that brings about the response finally observed. An antagonist which competitively inhibits the action of the intermediate substance will also reduce the response to the indirectly acting agonist, provided that the receptors are freely accessible. A simple mass-law model for indirect antagonism of this kind is presented, and its predictions are compared with the results obtained in an experimental study of the influence of propranolol and sotalol on the inotropic response of isolated rat atria to tyramine. While there is reasonable qualitative agreement, the fit is not exact and reasons for this are discussed.

Dequalinium: a potent inhibitor of apamin-sensitive K+ channels in hepatocytes and of nicotinic responses in skeletal muscle.

The bisquaternary compound dequalinium has been tested for its ability to inhibit the loss of K+ which angiotensin II causes in guinea-pig hepatocytes and which occurs through apamin-sensitive Ca(2+)-activated K+ (SKCa) channels. Dequalinium blocked angiotensin II-evoked K+ loss with an IC50 of 1.5 +/- 0.1 microM and also inhibited 125I-monoiodoapamin binding with a KI of 1.1 +/- 0.1 microM. It is the most active non-peptide SKCa blocker so far described. The neuromuscular blocking agent vecuronium was also tested, and proved to be considerably less effective (IC50, 4.5 +/- 0.3 microM; KI, 3.6 +/- 0.5 microM). Dequalinium was also examined for its actions at nicotinic receptors in skeletal muscle and was found to be a potent, non-competitive antagonist of carbachol contractions of the frog rectus abdominis. In the frog cutaneous pectoris muscle, end-plate depolarizations induced by carbachol became smaller and more transient in the presence of dequalinium at 10 nM. However, contractions of the frog sartorius and rat diaphragm in response to nerve stimulation were inhibited only by concentrations > 1 microM. These apparently discrepant effects of dequalinium on nicotinic responses could be explained either by open channel block of slow onset or by 'stabilization' of the desensitized state of the receptor. The potency of dequalinium will make it a useful agent for the study of nicotinic receptors as well as of SKCa channels.

The synthesis and some pharmacological actions of the enantiomers of the K(+)-channel blocker cetiedil.

Cetiedil ((+/-)-2-cyclohexyl-2-(3-thienyl)ethanoic acid 2-(hexahydro-1 H-azepin-1-yl) ethyl ester) possesses anti-sickling and analgesic, antispasmodic, local anaesthetic and vasodilator activities. A total synthesis and circular dichroism spectra of the enantiomers of cetiedil is described, together with a comparison of their effectiveness as blockers of the Ca(2+)-activated K+ permeability of rabbit erythrocytes; the contractile response of intestinal smooth muscle to acetylcholine; the Ca(2+)-dependent contraction of depolarized intestinal muscle; and the cell volume-sensitive K+ permeability (Kvol) of liver cells. The enantiomers did not differ substantially in their ability to block the Ca(2+)-activated K+ permeability of rabbit red cells or in their effectiveness as blockers of the contractile response of depolarized smooth muscle to externally applied Ca2+. There was a clear difference in the muscarinic blocking activity of the enantiomers, as assessed by inhibition of the contractile response of intestinal smooth muscle to acetylcholine; (+)-cetiedil was 7.7 +/- 0.2 (s.d.) times more active than the (-) from. The enantiomers also differed in their potency as blockers of the increase in membrane conductance which occurs when liver cells swell. The concentration of (+)-cetiedil needed to reduce the conductance increase by 50% was 2.04 +/- 0.54 (s.d.) microM; (-)-cetiedil was 2.6 +/- 0.8 (s.d.) times less active (IC50 of 5.2 +/- 1.2 microM). Differences in the biological actions of the enantiomers of cetiedil indicate that a more extensive study could be rewarding in relation to the use of the enantiomers both in therapeutics and in the study of K+ channels.