Donald H. Jenkinson

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.

SK3 is an important component of K+ channels mediating the afterhyperpolarization in cultured rat SCG neurones

1 Our aim was to identify the small‐conductance Ca2+‐activated K+ channel(s) (SK) underlying the apamin‐sensitive afterhyperpolarization (AHP) in rat superior cervical ganglion (SCG) neurones. 2 Degenerate oligonucleotide primers designed to the putative calmodulin‐binding domain conserved in all mammalian SK channel sequences were employed to detect SK DNA in a cDNA library from rat SCG. Only a single band, corresponding to a fragment of the rSK3 gene, was amplified. 3 Northern blot analysis employing a PCR‐generated rSK3 fragment showed the presence of mRNA coding for SK3 in SCG as well in other rat peripheral tissues including adrenal gland and liver. 4 The same rSK3 fragment enabled the isolation of a full‐length rSK3 cDNA from the library. Its sequence was closely similar to, but not identical with, that of the previously reported rSK3 gene. 5 Expression of the rSK3 gene in mammalian cell lines (CHO, HEK cells) caused the appearance of a K+ conductance with SK channel properties. 6 The application of selective SK blocking agents (including apamin, scyllatoxin and newer non‐peptidic compounds) showed these homomeric SK3 channels to have essentially the same pharmacological characteristics as the SCG afterhyperpolarization, but to differ from those of homomeric SK1 and SK2 channels. 7 Immunohistochemistry using a rSK3 antipeptide antibody revealed the presence of SK3 protein in the cell bodies and processes of cultured SCG neurones. 8 Taken together, these results identify SK3 as a major component of the SK channels responsible for the afterhyperpolarization of cultured rat SCG neurones.

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

The effectiveness of WB 4101, a recently described α-adrenoceptor antagonist, in blocking an excitatory and two inhibitory responses to α-receptor activation was studied. One of the inhibitory responses was the reduction by the selective α-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.

Discrimination between subtypes of apamin‐sensitive Ca2+‐ 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 Ca2+‐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 XXXX 8.4 μm 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 μm 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.

Potassium channels – multiplicity and challenges

The development of our knowledge of the function, structure and pharmacology of K+ channels is briefly outlined. This is the most diverse of all the ion channel families with at least 75 coding genes in mammals. Alternative splicing as well as variations in the channel subunits and accessory proteins that co‐assemble to form the functional channel add to the multiplicity. Whereas diversity of this order suggests that it may be possible to develop new classes of drug, for example, for immunomodulation and some diseases of the central nervous system, the ubiquity of K+ channels imposes stringent requirements for selectivity. Animal toxins from the snake, bee and scorpion provide useful leads, though only in a few instances (e.g. with apamin) it has been possible to produce non‐peptidic analogues of high potency. The scale of the resources needed to identify, and characterize fully, specific K+ channel as targets and then develop modulators with the required selectivity presents a challenge to both academic and applied pharmacologists.

Antagonism of an indirectly acting agonist: Block by propranolol and sotalol of the action of tyramien 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.

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

1 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. 2 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 μM) or an α1‐adrenoceptor agonist, amidephrine (20 μM), 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. 3 The concentrations (IC50s) of atracurium, pancuronium and (+)‐tubocurarine which reduced the effect of amidephrine on 86Rb efflux by 50% were 12, 37 and 67 μM respectively. 4 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 μM for atracurium, tubocurarine and pancuronium respectively, and 2.3 nM for apamin. 5 Each of the neuromuscular blockers, and apamin, increased the spontaneous contractions of the rabbit duodenum and blocked the inhibitory effect of amidephrine thereon. 6 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.

Peripheral actions of apamin

Abstract Apamin abolishes certain actions of α-adrenoceptor agonists and ATP on smooth muscle and liver cell membranes. This appears to be because it can block not the receptors but rather the increases in potassium permeability that follow receptor activation. Apamins selectivity and potency should make it useful for the study of receptor-controlled changes in membrane permeability.

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 μm). 2 The depolarizations elicited by carbachol (5–20 μm) 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:

Compounds that block both intermediate-conductance (IKCa) and small-conductance (SKCa) calcium-activated potassium channels

Nine bis‐quinolinyl and bis‐quinolinium compounds related to dequalinium, and previously shown to block apamin‐sensitive small conductance Ca2+‐activated K+ channels (SKCa), have been tested for their inhibitory effects on actions mediated by intermediate conductance Ca2+‐activated K+ channels (IKCa) in rabbit blood cells. In most experiments, a K+‐sensitive electrode was employed to monitor the IKCa‐mediated net loss of cell K+ that followed the addition of the Ca2+ ionophore A23187 (2 μM) 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 μM) to a very dilute suspension of red cells (haematocrit 0.02%). Of the compounds tested, the most potent IKCa blocker was 1,12 bis[(2‐methylquinolin‐4‐yl)amino]dodecane (UCL 1407) which had an IC50 of 0.85±0.06 μM (mean±s.d.mean). 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 IKCa blocking action resembles that of cetiedil rather than of clotrimazole. 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‐methylquinolinium‐4yl)amino] decane (trifluoroacetate) which had an IC50 of 1.8±0.1 μM. 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 (IC50 2.7±0.3 μM). A bis‐aminoquinolium cyclophane (UCL 1684) had little IKCa blocking action despite its great potency at SKCa channels (IC50 4.1±0.2 nM). The main outcome is the identification of new intermediate‐conductance Ca2+‐activated K+ channel blockers with a wide range of IKCa/SKCa selectivities.