Philip M. Dunn

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.

Dequalinium, a selective blocker of the slow afterhyperpolarization in rat sympathetic neurones in culture.

The actions of dequalinium have been investigated in cultured rat sympathetic neurones. It produced a rapid and reversible inhibition of the slow apamin-sensitive component of the afterhyperpolarization (AHP) which follows a single action potential in these cells. The IC50 for this effect was 1.1 microM and in voltage clamp experiments, 1 microM dequalinium produced 45% inhibition of the underlying current IAHP. When the small conductance Ca(2+)-activated K+ channels were blocked by 20 nM apamin the slow component of the AHP was abolished, and dequalinium (10 microM) produced no further change in the residual AHP. Dequalinium (10 microM) had no effect on the voltage-activated Ca2+ current in these cells, suggesting that the inhibition of the AHP was the result of a direct interaction with the K+ channels. The A-current as well as a composite current made up of IK and IC were all unaffected by 10 microM dequalinium. However, at this concentration it did produce 18% inhibition of the M-current. These results show dequalinium to be a potent and selective non-peptide blocker of the apamin-sensitive small conductance Ca(2+)-activated K+ channel in rat sympathetic neurones.

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.

UCL 1684: a potent blocker of Ca2+ -activated K+ channels in rat adrenal chromaffin cells in culture.

The novel K+ channel blocker 6,10-diaza-3(1,3)8,(1,4)-dibenzena-1,5(1,4)-diquinolinacy clodecaphane (UCL 1684) has been tested for its ability to inhibit Ca2+ -activated K+ currents in cultured rat chromaffin cells. Low nanomolar concentrations of UCL 1684 produced a rapid and reversible inhibition of the slow, apamin-sensitive, tail current activated by a depolarizing voltage command. This compound also inhibited the muscarine activated outward current with an IC50 of 6 nM. These results confirm UCL 1684 to be the most potent non-peptidic blocker of the apamin-sensitive Ca2+ -activated K+ channel so far described.

Search for the pharmacophore of the K+ channel blocker, apamin

Abstract The suggestion that the arginine residues, 13Arg and 14Arg, in the octadecapeptide apamin 1 are critically important to its action in blocking Ca2+-dependent K+ channels (and hence part of the ‘pharmacophore’) has been investigated by examining small peptides containing Arg-Arg or Lys-Arg. Bisguanidine derivatives modelled on the Arg-Arg partial pharmacophore have also been synthesised and tested; in particular, N-(2-guanidinoethyl)-3[N1-(2-guanidinoethyl)carbamoyl]-trans-propenamide 11 and its higher homologue 12. None of the compounds showed more than weak activity (Ki > 10−5 M) indicating that although the Arg-Arg fragment may be necessary, it is not a sufficient atom grouping for the pharmacophore.

Synthesis and qsar of dequalinium analogues AS K+ channel blockers. investigations on the role of the 4-amino group

Dequalinium (1) is a potent and selective non-peptidic blocker of the SKCa channel. The contribution of the 4-amino group to activity was investigated by replacing it by other groups R4. The size or lipophilicity of R4 was found to be unimportant and a good correlation was obtained between σR for R4 and the blocking potency of the analogues, suggesting that the role of the NH2 group is electronic.

Bis-quinolinium cyclophanes: A novel class of potent blockers of the apamin-sensitive Ca2+-activated K+ channel

Abstract Based on the structure-activity analysis of two series of blockers of the small conductance Ca2+-activated K+ (SKCa) channel, a novel class of bis-quinolinium cyclophane blockers has been designed and synthesised. These compounds exhibit submicromolar activity; UCL 1530 (4) is a useful agent since it has been shown (elsewhere) to be selective for the neuronal SKCa channel (IC50 = 80 nM) relative to hepatocyte 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‐1H‐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 Ca2+‐activated K+ permeability of rabbit erythrocytes; the contractile response of intestinal smooth muscle to acetylcholine; the Ca2+‐dependent contraction of depolarized intestinal muscle; and the cell volume‐sensitive K+ permeability (Kvol) of liver cells.

Ganglion-blocking activity of dequalinium in frog and rat sympathetic ganglia in vitro

The effect of dequalinium on ganglionic transmission and responses to exogenous acetylcholine receptor agonists were studied in frog and rat sympathetic ganglia. In frog ganglia, dequalinium reduced ganglionic transmission (measured using gross extracellular recording) with an EC50 of 2 microM. At 1 microM, dequalinium produced non-surmountable antagonism of the ganglion depolarization evoked by nicotinic receptor activation. Dequalinium reduced the amplitude of evoked and spontaneous excitatory postsynaptic potentials recorded intracellularly from frog neurones, but had no effect on the action potential elicited by injection of a depolarizing current pulse. Hyperpolarizing and depolarizing responses to muscarine recorded extracellularly from frog ganglia were antagonized by 3 microM dequalinium. In rat ganglia, synaptic transmission and depolarization by an exogenous nicotinic agonist were only slightly inhibited by dequalinium at a concentration of 100 microM. At 30 microM, dequalinium produced a 17% reduction in the depolarization of this tissue by muscarine (1 microM). These results add to the evidence of the diversity of nicotinic and muscarinic acetylcholine receptors, and indicate that dequalinium may be a valuable tool for the study of these receptors. However its usefulness as a probe for calcium-activated potassium channels may be limited by its actions at acetylcholine receptors.