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PubMed2001

Delineation of the clotrimazole/TRAM-34 binding site on the intermediate conductance calcium-activated potassium channel, IKCa1.

Selective and potent triarylmethane blockers of the intermediate conductance calcium-activated potassium channel, IKCa1, have therapeutic use in sickle cell disease and secretory diarrhea and as immunosuppressants. Clotrimazole, a membrane-permeant triarylmethane, blocked IKCa1 with equal affinity when applied externally or internally, whereas a membrane-impermeant derivative TRAM-30 blocked the channel only when applied to the cytoplasmic side, indicating an internal drug-binding site. Introduction of the S5-P-S6 region of the triarylmethane-insensitive small conductance calcium-activated potassium channel SKCa3 into IKCa1 rendered the channel resistant to triarylmethanes. Replacement of Thr(250) or Val(275) in IKCa1 with the corresponding SKCa3 residues selectively abolished triarylmethane sensitivity without affecting the affinity of the channel for tetraethylammonium, charybdotoxin, and nifedipine. Introduction of these two residues into SKCa3 rendered the channel sensitive to triarylmethanes. In a molecular model of IKCa1, Thr(250) and Val(275) line a water-filled cavity just below the selectivity filter. Structure-activity studies suggest that the side chain methyl groups of Thr(250) and Val(275) may lock the triarylmethanes in place via hydrophobic interactions with the pi-electron clouds of the phenyl rings. The heterocyclic moiety may project into the selectivity filter and obstruct the ion-conducting pathway from the inside.

Amino Acid Sequence↗
PubMed2000

Cloning, localisation and functional expression of the human orthologue of the TREK-1 potassium channel.

We have cloned human TREK-1, one of the newly emerging mammalian family of 2-P domain potassium channels. The channel has 411 amino acids with a 41-amino-acid extension at the C-terminus when compared with the cloned mouse TREK-1 channel. Expression of hTREK-1 produced a substantial hyperpolarising shift in resting membrane potential accompanied by the induction of large, outwardly rectifying, non-inactivating currents which were potassium selective. Pharmacologically, hTREK-1-mediated currents were only blocked to a limited extent by classic potassium channel blockers or open channel pore blockers known to potently inhibit other channels. The channel was reversibly potentiated by arachidonic acid. CNS distribution of hTREK-1 is widespread with higher levels being observed in caudate, putamen, amygdala, thalamus and spinal cord. Only low levels of expression were seen in the majority of peripheral regions. Thus, hTREK-1, although functionally and pharmacologically similar to mouse TREK-1, appears to have a more CNS-specific distribution.

Amino Acid Sequence↗
PubMed1990

A potassium channel activator modulates both excitatory noncholinergic and cholinergic neurotransmission in guinea pig airways.

The effect of a potassium channel activator, cromakalim (BRL 34915), on excitatory nonadrenergic noncholinergic (e-NANC) and cholinergic neural bronchoconstriction was studied in guinea pigs. We monitored airway opening pressure as an index of airway caliber. After atropine (1 mg/kg iv.) and propranolol (1 mg/kg iv.), bilateral vagal stimulation evoked an e-NANC response. Cromakalim did not alter basal airway caliber, but reduced the e-NANC response to vagal stimulation in a dose-dependent manner, with a maximal inhibition of 71.9 +/- 9.2% (mean +/- S.E.) at 400 micrograms/kg i.v. (P less than .01). Pretreatment with phentolamine (2.5 mg/kg i.v.) had no effect on the inhibitory response produced by cromakalim but glibenclamide (25 mg/kg iv.), an inhibitor of ATP-sensitive potassium channels, blocked its effect. Cromakalim had no inhibitory effect on exogenous substance P (5-25 micrograms/kg i.v.)-induced bronchoconstriction. In animals depleted of tachykinins by capsaicin (50 mg/kg s.c.) pretreatment, cromakalim had an inhibitory effect on both vagalcholinergic and exogenous acetylcholine (0.3-2 micrograms/kg i.v.)-induced bronchoconstriction, although the inhibitory effect was significantly greater on neural stimulation. We conclude that potassium channels modulate both e-NANC and cholinergic neurotransmission, and to a lesser extent acetylcholine-induced bronchoconstriction in guinea pig airways.

Animals↗
PubMed1994

Interactive regulation of the ATP-sensitive potassium channel of cardiac muscle.

A variety of ligands, including intracellular nucleotides, pharmaceutical products, and neurohormones, influence the adenosine triphosphate (ATP)-sensitive potassium channel of cardiac muscle. In this review it is emphasized that these ligands act not only on the channel-gating mechanism but also influence each others' effects or effectiveness in a quite complex series of interactions. Thus, ATP, which directly closes the channel-gating mechanism, also enhances the efficacy of sulfonylurea drugs and is necessary for the action of certain potassium-channel-opening drugs and neurohormones. Intracellular nucleoside diphosphates that, depending on the conditions, either act on the channel-gating mechanism or influence the effectiveness of ATP, also facilitate and reduce the effectiveness of potassium-channel opening and sulfonylurea drugs, respectively. Our knowledge of the mechanisms that underlie the interactions between these different ligands is limited. The cloning of the channel and the characterization of the different ligand-binding sites will be a great step forward.

Adenosine↗
PubMed2001

Role of NO pathway, calcium and potassium channels in the peripheral pulmonary vascular tone in dogs.

Because hypoxic pulmonary vasoconstriction occurs mainly in the small pulmonary arteries, the authors investigated the effects of drugs acting on the nitric oxide (NO) pathway and the calcium and potassium channels in the peripheral pulmonary circulation, without interference with the overall pulmonary or systemic circulation. Mixed venous blood was infused in wedged areas to study the pressure/flow relationship and to compute peripheral pulmonary vascular resistance (PPVR). The authors studied the effects of Nomega-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, sodium nitroprusside (SNP, an NO donor), the calcium channel blockers verapamil, nifedipine and nicardipine, and the potassium channel opener levcromakalim, during normoxia and acute mild normocapnic hypoxia. In the peripheral pulmonary circulation, L-NAME caused an increase in PPVR during normoxia (+95%; p<0.001) and hypoxia (+60%; p<0.01). Following the increase by L-NAME, SNP decreased PPVR during normoxia (-24%; p<0.05) and hypoxia (-23%; p<0.05). Verapamil, nifedipine and nicardipine did not modify PPVR during normoxia but during hypoxia they decreased PPVR (-28%, nonsignificant; -27%, p<0.01 and -33%, p<0.05, respectively). Levcromakalim did not modify PPVR during normoxia or hypoxia. In conclusion, the nitric oxide pathway and voltage-dependent calcium channels, and not adenosine triphosphate sensitive potassium channels, play an important role in the control of peripheral pulmonary circulation in dogs.

Animals↗
PubMed2000

Clinical, genetic, and expression studies of mutations in the potassium channel gene KCNA1 reveal new phenotypic variability.

Episodic ataxia type 1 (EA1) is an autosomal dominant central nervous system potassium channelopathy characterized by brief attacks of cerebellar ataxia and continuous interictal myokymia. Point mutations in the voltage-gated potassium channel gene KCNA1 on chromosome 12p associate with EA1. We have studied 4 families and identified three new and one previously reported heterozygous point mutations in this gene. Affected members in Family A (KCNA1 G724C) exhibit partial epilepsy and myokymia but no ataxic episodes, supporting the suggestion that there is an association between mutations of KCNA1 and epilepsy. Affected members in Family B (KCNA1 C731A) exhibit myokymia alone, suggesting a new phenotype of isolated myokymia. Family C harbors the first truncation to be reported in KCNA1 (C1249T) and exhibits remarkably drug-resistant EA1. Affected members in Family D (KCNA1 G1210A) exhibit attacks typical of EA1. This mutation has recently been reported in an apparently unrelated family, although no functional studies were attempted. Heterologous expression of the proteins encoded by the mutant KCNA1 genes suggest that the four point mutations impair delayed-rectifier type potassium currents by different mechanisms. Increased neuronal excitability is likely to be the common pathophysiological basis for the disease in these families. The degree and nature of the potassium channel dysfunction may be relevant to the new phenotypic observations reported in this study.

Action Potentials↗
PubMed1994

Effect of diabetes mellitus on response of the basilar artery to activation of ATP-sensitive potassium channels.

Our goal was to determine whether responses of the basilar artery to activation of ATP-sensitive potassium channels are altered during diabetes mellitus. We measured changes in diameter of the basilar artery in vivo in non-diabetic and diabetic rats (streptozotocin; 50-60 mg/kg i.p.) in response to activation of ATP-sensitive potassium channels using aprikalim (RP 52891) and levcromakalim (BRL 38227). Aprikalim (1.0 microM) dilated the basilar artery in non-diabetic rats by 27 +/- 6%, but by only 11 +/- 3% in diabetic rats (means +/- S.E.; P < 0.05). Levcromakalim (1.0 microM) dilated the basilar artery in non-diabetic rats by 45 +/- 11%, but by only 20 +/- 5% in diabetic rats (P < 0.05). Nitroglycerin (1.0 microM) dilated the basilar artery by 20 +/- 5% in non-diabetic rats and 17 +/- 2% in diabetic rats (P > 0.05). Thus, impaired dilatation of pial arterioles in diabetic rats in response to aprikalim and levcromakalim is not related to a non-specific effect of diabetes mellitus on vasodilatation. The findings of the present study suggest that ATP-sensitive potassium channels are functional in the rat basilar artery in vivo and are altered during diabetes mellitus.

Adenosine Triphosphate↗
PubMed1990

Characterization and functional expression of a rat genomic DNA clone encoding a lymphocyte potassium channel.

Low stringency hybridization screening of a rat genomic DNA library with a previously described cDNA clone encoding a rat voltage-gated potassium channel has resulted in the characterization of a member of the potassium channel family, RGK5. An uninterrupted nucleotide sequence encodes a protein 525 amino acids in length, revealing that the entire coding region resides on a single exon. RGK5 transcripts are present in both mouse thymus and rat brain, as determined by Northern blot analysis. RNA transcribed in vitro from RGK5 genomic DNA directs the expression of functional potassium currents after injection into Xenopus oocytes. The currents are activated by depolarization, being half-activated at -14 mV, and inactivate almost completely during depolarizations of 1 to 2 s. The properties of the currents strongly resemble those of the type n potassium channel present on both immature thymocytes and Th lymphocytes.

Animals↗
PubMed2001

Hippocampal heterotopia lack functional Kv4.2 potassium channels in the methylazoxymethanol model of cortical malformations and epilepsy.

Human cortical malformations often result in severe forms of epilepsy. Although the morphological properties of cells within these malformations are well characterized, very little is known about the function of these cells. In rats, prenatal methylazoxymethanol (MAM) exposure produces distinct nodules of disorganized pyramidal-like neurons (e.g., nodular heterotopia) and loss of lamination in cortical and hippocampal structures. Hippocampal nodular heterotopias are prone to hyperexcitability and may contribute to the increased seizure susceptibility observed in these animals. Here we demonstrate that heterotopic pyramidal neurons in the hippocampus fail to express a potassium channel subunit corresponding to the fast, transient A-type current. In situ hybridization and immunohistochemical analysis revealed markedly reduced expression of Kv4.2 (A-type) channel subunits in heterotopic cell regions of the hippocampus of MAM-exposed rats. Patch-clamp recordings from visualized heterotopic neurons indicated a lack of fast, transient (I(A))-type potassium current and hyperexcitable firing. A-type currents were observed on normotopic pyramidal neurons in MAM-exposed rats and on interneurons, CA1 pyramidal neurons, and cortical layer V-VI pyramidal neurons in saline-treated control rats. Changes in A-current were not associated with an alteration in the function or expression of delayed, rectifier (Kv2.1) potassium channels on heterotopic cells. We conclude that heterotopic neurons lack functional A-type Kv4.2 potassium channels and that this abnormality could contribute to the increased excitability and decreased seizure thresholds associated with brain malformations in MAM-exposed rats.

4-Aminopyridine↗
PubMed1992

Differential effects of endothelin-1 on the vasorelaxant properties of benzopyran and non-benzopyran potassium channel openers.

1. The effects of endothelin-1 (ET-1) on the vasorelaxant properties of structurally different potassium channel openers (PCOs), BRL-38227, Ro 31-6930, SDZ PCO 400, EMD-52692, RP-49356 and pinacidil, were studied. 2. All PCOs evoked concentration-related relaxations of ET-1 (10 nM) or KCl (20 mM) contracted rat isolated aortic rings denuded of endothelium. BRL-38227, EMD 52692, SDZ PCO 400 and Ro 31-6930 were 11-42 times less potent in relaxing contractions to ET-1 than KCl. In contrast, this differential potency was not observed with RP-49356 or pinacidil. 3. BRL-38227 (0.06-3.0 microM), RP-49356 (0.3-3.0 microM) and pinacidil (0.3-3.0 microM) displaced KCl concentration-response curves to the right of controls, without modifying the maximum response. A subcontractile concentration of ET-1 (0.1 nM) prevented the inhibitory effects of low concentrations of BRL-38227 (0.06-0.1 microM) on KCl responses, but failed to modify those to RP-49356, pinacidil or high concentrations of BRL-38227 (0.3-3.0 microM). The inhibitory effects of BRL-38227 (0.1 microM) were also not changed by ET-3 (1.0 nM) or angiotensin II (0.1 nM). 4. In anaesthetized spontaneously hypertensive rats (SHR), cumulative bolus intravenous administrations of BRL-38227 (1-1000 micrograms kg-1, i.v.), Ro 31-6930 (1-1000 micrograms kg-1, i.v.), RP-49356 (10-1000 micrograms kg-1, i.v.) or nitrendipine (0.1-30 micrograms kg-1, i.v.) produced dose-dependent falls in diastolic blood pressure (DBP).ET-1 (i.v.) evoked a transient fall in DBP (1 pg kg- = 58 + 1 mmHg) which returnedto pre-administration levels within 4 min.5. Pretreatment of anaesthetized SHR with ET-l (1 pg kg-', i.v.) significantly increased the ED,5 (dose to evoke a 15% fall in DBP) values for BRL-38227 and Ro 31-6930. However, ET-l failed to modify the ED,5 values for RP-49356 or nitrendipine. The ED50 values for all of the vasodilators studied were not modified by ET-1.6. Infusion of BRL-38227 (2 pgkg-'min-', i.v.) or RP-49356 (4 pgkg-'min', i.v.) to anaesthetized SHR evoked dose-related falls in DBP, with a corresponding increase in descending aortic blood flow (DABF) and a decrease in total lower body vascular resistance (TLBVR). Pretreatment with ET-1 (1 ptg kg-', i.v.) significantly attenuated the decreases in DBP and TLBVR observed with low doses of BRL-38227, but not RP-49356 or high doses of BRL-38227. In contrast, ET-3 (3 pig kg-, i.v.) failed to modify the effects of BRL-38227 on DBP or TLBVR.7. In conscious SHR, the fall in DBP to BRL-38227 (30 pgkg-', p.o.) was significantly reduced following ET-1 (1 pig kg-', i.a.) treatment. ET-1 (1 pg kg-', i.a.) pretreatment, however, failed to modify the decrease in DBP induced by an equieffective oral dose of RP-49356 (1001pgkg-1).8. In conclusion, ET-1 selectively attenuated the vasorelaxant effects of the potassium channel opener,BRL-38227 and other substituted benzopyrans. The results are compatible with the hypothesis that benzopyran PCOs and ET-1 have affinity for a site that does not recognise RP-49356 or pinacidil. Thus,ET-l can differentiate between structurally unrelated potassium channel openers. The cardiovascular effects of some, but not all, PCOs might be radically modified in the clinical setting by elevated endogenous levels of ET-1 associated with certain diseased states.

Animals↗
PubMed2005

1,4-Diazabicyclo[2.2.2]octane derivatives: a novel class of voltage-gated potassium channel blockers.

Voltage-gated potassium (Kv) channels are targets for therapeutic drugs in the treatment of pathologic conditions including cardiac arrhythmia and epilepsy. In this study, we synthesized three classes of novel polyammonium compounds incorporating the bicyclic unit 1,4-diazabicyclo[2.2.2]octane (DABCO) and tested their action on three representative mammalian Kv channels (Kv2.1, Kv3.4, and Kv4.2) expressed in Xenopus laevis oocytes. Nonsubstituted DABCO did not block the Kv channels tested. Simple DABCO monostrings and diDABCO strings inhibited Kv2.1 and Kv3.4 channels, with potency increasing with string length for both these DABCO classes. Both Kv2.1 and Kv3.4 were most sensitive to C16 monostrings, with IC50 values of 1.9 and 0.6 microM, respectively. For compounds comprising two DABCO groups separated by an aromatic ring, inhibition depended upon relative positioning of the two DABCO groups, and only the para form (JC638.2alpha) was active, blocking Kv2.1 with an IC50 of 186 microM. Kv4.2 channels were relatively insensitive to all compounds tested. Unlike the tetraethylammonium ion (TEA), neither JC638.2alpha nor C16 monostring TA279 produced block when applied intracellularly via the recording electrode to Kv2.1 channels expressed in Chinese hamster ovary cells, suggesting against an internal site of action. However, JC638.2alpha protected an introduced cysteine (K356C) in the Kv2.1 outer pore from permanent modification by methanethiosulfonate ethyltrimethylammonium (MTSET). These data suggest that JC638.2alpha occupies an external binding site similar to that of TEA in the Kv2.1 outer pore, but with much higher affinity than TEA. These DABCO salts represent a new class of Kv channel blockers, some with higher potencies than any previously described quaternary ammonium ions. The potential for synthesis of an array of modular derivatives suggests that DABCO compounds hold promise as probes of Kv channel structure and identity and as potential therapeutic agents.

Animals↗
PubMed1995

[Potassium channel openers antagonize 4-aminopyridine induced histamine release from rat peritoneal mast cells].

4-Aminopyridine (4-AP) was shown to promote histamine release from isolated rat peritoneal mast cells (PMC) in a dose-dependent manner. Potassium channel openers, minoxidil (Min) 200 mg.kg-1 ig, diazoxide (Dia) 500 mumol.L-1 in vitro and calcium antagonist nifedipine (Nif) 125 mg.kg-1 ig were found to inhibit 4-AP induced histamine release from rat PMC. Electron microscopy revealed that 4-AP caused partial degranulation of PMC and extrusion of granules and Min 200 mg.kg-1 ig retarded this phenomenon. These results provide evidence that potassium channels are present in rat mast cell membrane and indicate that the mechanism of histamine release by 4-AP may be related to its potassium channel blocking effect. As a result of this effect, the calcium channels open and Ca2+ influx to the mast cells increases, thus eliciting histamine release. Potassium channel openers seemed to be a new group of inhibitors of histamine release from mast cells.

4-Aminopyridine↗
PubMed2003

[The role of delayed rectifier potassium channels in the regulation of bronchial smooth muscle tension in asthmatic rats].

OBJECTIVE: To investigate the role of delayed rectifier potassium channels (K(V)) in the regulation of bronchial smooth muscle tension in asthmatic rats and their effects on bronchial reactivity in vitro. METHODS: By using a specific potassium channel blocker, the bronchial contraction induced by spasmogens was observed in normal and asthmatic rats with the isometric tension recording technique. RESULTS: (1) K(V) blocker 4-aminopyridine (4-AP) caused concentration-dependent bronchial contraction in vitro. The concentration-response curve to 4-AP in the asthmatic group showed a left displacement with bigger pD(2) (the negative logarithm of the drug concentration causing 50% of maximal effect, 2.58 +/- 0.07, n = 10, P < 0.001) and no change in E(max) [maximal effect, (32 +/- 5) mg/mg, P > 0.05], compared with pD(2) (2.12 +/- 0.04, n = 10) and E(max) [(31 +/- 6) mg/mg] obtained in the control group; (2) In the control group, pre-treatment with 0.1 mmol/L 4-AP displaced the concentration-response curves to ET-1 and histamine to the left. pD(2) (for ET-1 and histamine) before and after 4-AP treatment was 6.27 +/- 0.38, 5.59 +/- 0.27 and 6.80 +/- 0.47, 6.42 +/- 0.14 respectively (P < 0.01). E(max) before and after 4-AP treatment was (36 +/- 8) mg/mg, (36 +/- 8) mg/mg and (40 +/- 8) mg/mg, (39 +/- 9) mg/mg respectively (P > 0.05); (3) In the asthmatic group, pre-treatment with 0.1 mmol/L 4-AP had no effect on the concentration-response curve to ET-1 and histamine. There was no significant difference (P > 0.05) in pD(2) before (6.51 +/- 0.07, 5.86 +/- 0.14 respectively) and after (6.48 +/- 0.16, 5.96 +/- 0.08 respectively) 4-AP treatment, and in E(max) before [(61 +/- 8) mg/mg, (54 +/- 11) mg/mg respectively] and after [(65 +/- 10) mg/mg, (55 +/- 9) mg/mg respectively] 4-AP treatment. CONCLUSION: The activity of K(V) decreases in bronchial smooth muscle isolated from asthmatic rats compared with that obtained from normal ones. This change may be involved in the bronchial hyperreactivity to some spasmogens in vitro in asthmatic rats.

Animals↗
PubMed2001

Distribution and expression of TREK-1, a two-pore-domain potassium channel, in the adult rat CNS.

TREK-1 is a member of the two-pore-domain potassium channel family which is expressed predominantly in the CNS. Using an anti-peptide polyclonal antiserum, we have determined the distribution of TREK-1 in the brain and spinal cord of adult rats. Specificity of the antiserum was tested using a TREK-1-transfected cell line and confirmed with c-myc-tagged TREK-1. In thin tissue sections, immunoreactivity was widespread throughout the rat brain and spinal cord. TREK-1-like signals were observed in the cerebral cortex, basal ganglia, hippocampus, and various other subcortical nuclei in the hypothalamus, thalamus, mesencephalon and rhombencephalon. TREK-1 labelling appeared to be over the entire cell membrane, including the cell body and processes. Cells that morphologically resembled projection neurones and interneurones but not glial cells were labelled. As interneurones and known GABAergic projection neurones were the predominant population labelled, we investigated the possibility that TREK-1 is expressed in GABA-containing neurones using a specific anti-GABA antiserum. Expression of TREK-1 in GABA-containing neurones was observed in a number of areas, including the isocortex, hippocampus and thalamus. Thus, TREK-1 expression defines a unique and specific subset of interneurones and principal cells. These studies indicate a widespread distribution of TREK-1 potassium channels throughout the rat brain and spinal cord, with expression in a number of areas being demonstrated to be present on GABA-containing neurones.

Animals↗
PubMed1996

An essential binding surface for ShK toxin interaction with rat brain potassium channels.

An "Ala scan" analysis of ShK toxin, a 35-residue basic peptide possessing three disulfide bonds, identifies seven side chains which influence binding to brain delayed rectifier potassium channels. Additional analogs were synthesized and tested to further decipher the roles of these residues, particularly Tyr23. The inhibitory effects of these analogs on 125I-labeled dendrotoxin binding to rat brain membranes showed that replacement of Tyr23 with Ala drastically lowered the affinity of the toxin for the Kv1.2 channels. Ala substitution of Phe27 reduced potency more than 15-fold. Monosubstituted Ala analogs for Ile7, Ser20, or Lys30 each displayed 5-fold reductions in potency. Thus, aromaticity at position 23 is important for effective delayed rectifier brain K channel binding. In contrast, the aromatic residue at position 27 was not critical, since cyclohexylalanine substitution increased affinity. The solution structure of ShK toxin clusters Ile7, Arg11, Ser20, Lys22, Tyr23, and Phe27 in close proximity, forming the potassium channel binding surface of the toxin. We propose an essential binding surface on the toxin in which Lys22 and Tyr23 are major contributors, through ionic and aromatic (hydrophobic) interactions, with the potassium channel.

Amino Acid Sequence↗
PubMed2003

Inhibition of hEAG1 and hERG1 potassium channels by clofilium and its tertiary analogue LY97241.

1 We investigated the inhibition of hEAG1 potassium channels, expressed in mammalian cells and Xenopus oocytes, by several blockers that have previously been reported to be blockers of hERG1 channels. 2 In the whole-cell mode of mammalian cells, LY97241 was shown to be a potent inhibitor of both hEAG1 and hERG1 channels (IC(50) of 4.9 and 2.2 nM, respectively). Clofilium, E4031, and haloperidol apparently inhibited hEAG1 channels with lower potency than hERG1 channels, but they cannot be considered hERG1-specific. 3 The block of hEAG1 channels by LY97241 and clofilium was time-, use-, and voltage-dependent, best explained by an open-channel block mechanism. 4 Both drugs apparently bind from the intracellular side of the membrane at (a) specific site(s) within the central cavity of the channel pore. They can be trapped by closure of the activation gate. 5 In inside-out patches from Xenopus oocytes, hEAG1 block by clofilium was stronger than by LY97241 (IC(50) of 0.8 and 1.9 nM, respectively). In addition, hEAG1 block by clofilium was much faster than by LY97241 although there was no difference in the voltage dependence of the on-rate of block. 6 Physico-chemical differences of clofilium and the weak base LY97241 determine the access of the drugs to the binding site and thereby the influence of the recording mode on the apparent block potencies. This phenomenon must be considered when assessing the inhibitory action of drugs on ion channels.

Animals↗
PubMed2007

The role of S4 charges in voltage-dependent and voltage-independent KCNQ1 potassium channel complexes.

Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 alpha subunits to generate the voltage-dependent I(Ks) channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium "leak" currents in gastrointestinal epithelia. Other Kv alpha subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments revealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitutive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels. The results demonstrate that the unique S4 charge paucity of KCNQ1 facilitates its unique conversion to a leak channel by ancillary subunits such as MiRP2.

Alanine↗
PubMed2003

Expression of the ether-a-go-go (ERG) potassium channel in smooth muscle of the equine gastrointestinal tract and influence on activity of jejunal smooth muscle.

OBJECTIVE: To determine whether ether-a-go-go (ERG) potassium channels are expressed in equine gastrointestinal smooth muscle, whether ERG channel antagonists affect jejunal muscle contraction in vitro, and whether plasma cisapride concentrations in horses administered treatment for postoperative ileus (POI) are consistent with ERG channels as drug targets. SAMPLE POPULATION: Samples of intestinal smooth muscle obtained from 8 horses free of gastrointestinal tract disease and plasma samples obtained from 3 horses administered cisapride for treatment of POI. PROCEDURE: Membranes were prepared from the seromuscular layer of the duodenum, jejunum, ileum, cecum, large colon, and small colon. Immunoblotting was used to identify the ERG channel protein. Isolated jejunal muscle strips were used for isometric stress response to ERG channel blockers that included E-4031, MK-499, clofilium, and cisapride. Plasma concentrations of cisapride were determined in 3 horses administered cisapride for treatment of POI after small intestinal surgery. RESULTS: Immunoblotting identified ERG protein in all analyzed segments of the intestinal tract in all horses. The selective ERG antagonist E-4031 caused a concentration-dependent increase in jejunal contraction. Clofilium, MK-499, and cisapride also increased jejunal contraction at concentrations consistent with ERG channel block; effects of E-4031 and cisapride were not additive. Peak plasma cisapride concentrations in treated horses were consistent with ERG block as a mechanism of drug action. CONCLUSIONS AND CLINICAL RELEVANCE: The ERG potassium channels modulate motility of intestinal muscles in horses and may be a target for drugs. This finding may influence development of new prokinetic agents and impact treatment of horses with POI.

Animals↗