High-affinity Blocker Research Articles

Cytokine Traps: a New Promising Approach to Treatment?

Economides AN, Carpenter LR, Rudge JS et al. Cytokine traps: multi-component, high-affinity blockers of cytokine action. Nature Medicine 2003; 9: 47-52

N-Glycosylation-dependent block is a novel mechanism for drug-induced cardiac arrhythmia.

Voltage-gated potassium channels formed with the cardiac subunit HERG and a polymorphic variant of MinK-related peptide 1 (MiRP1) exhibit increased susceptibility to the antibiotic sulfamethoxazole (SMX) compared with channels formed with wild-type (WT) subunits. Here the molecular bases for SMX high-affinity block are investigated. The polymorphism causes a benign T to A amino acid mutation at position 8 (T8A) that destroys an N-glycosylation site of MiRP1. In vitro disruption of glycosylation by mutagenesis or in vivo by treatment with neuraminidase is associated with increased susceptibility to SMX and to other elementary agents such as divalent cations. Defective glycosylation does not affect the ability of T8A to form stable complexes with HERG, but rather it increases drug susceptibility through structural modifications in the channel complex. We conclude that N-glycosylation may play a key role in the etiology of life-threatening arrhythmia.

Optimal requirements for high affinity and use-dependent block of skeletal muscle sodium channel by N-benzyl analogs of tocainide-like compounds.

Newly synthesized tocainide analogs were tested for their state-dependent affinity and use-dependent behavior on sodium currents (INa) of adult skeletal muscle fibers by means of the Vaseline-gap voltage clamp method. The drugs had the pharmacophore amino group constrained in position alpha [N-(2,6-dimethylphenyl)pyrrolidine-2-carboxamide (To5)] or beta [N-(2,6-dimethylphenyl)pyrrolidine-3-carboxamide (To9)] in a proline-like cycle and/or linked to a lipophilic benzyl moiety as in N-benzyl-tocainide (Benzyl-Toc), 1-benzyl-To5 (Benzyl-To5), and 1-benzyl-To9 (Benzyl-To9). INa were elicited with pulses to -20 mV from different holding potentials (-140, -100, and -70 mV) and stimulation frequencies (2 and 10 Hz). All compounds were voltage-dependent and use-dependent channel blockers. The presence of a proline-like cycle increased the potency; i.e., To5 was 3- and 10-fold more effective than Toc in blocking INa at the holding potential of -140 and -70 mV, respectively. The benzyl group on the amine further enhanced drug effectiveness with the following scale: Benzyl-To9 >/= Benzyl-Toc > Benzyl-To5. At a holding potential of -100 mV and 10-Hz stimulation, Benzyl-To9 blocked INa with a half-maximal concentration of 0.5 microM, being 60 and 400 times more potent than To9 and Toc, respectively. The similar effectiveness of Benzyl-Toc and Benzyl-To9 was paralleled by a similar spatial arrangement by equilibrium geometry modeling. In addition, the latter had a higher pKa value that probably contributed to a slow kinetic during its high use-dependent behavior. Benzyl-To5 had its lowest energy level at a more folded conformation that justifies the less favorable profile among the N-benzylated analogs. The new compounds are the most potent tocainide-like sodium channel blockers so far described and have high therapeutic potentials.

Synthesis and characterization of Pi4, a scorpion toxin from Pandinus imperator that acts on K+ channels.

Pi4 is a 38-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the Chactidae scorpion Pandinus imperator. Together with maurotoxin, Pi1, Pi7 and HsTx1, Pi4 belongs to the alpha KTX6 subfamily of short four-disulfide-bridged scorpion toxins acting on K+ channels. Due to its very low abundance in venom, Pi4 was chemically synthesized in order to better characterize its pharmacology and structural properties. An enzyme-based cleavage of synthetic Pi4 (sPi4) indicated half-cystine pairings between Cys6-Cys27, Cys12-32, Cys16-34 and Cys22-37, which denotes a conventional pattern of scorpion toxin reticulation (Pi1/HsTx1 type). In vivo, sPi4 was lethal after intracerebroventricular injection to mice (LD50 of 0.2 microg per mouse). In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage-gated K+ channel subtypes showed potent inhibition of currents from rat Kv1.2 (IC50 of 8 pm) and Shaker B (IC50 of 3 nm) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels. The sPi4 was also found to compete with 125I-labeled apamin for binding to small-conductance Ca(2+)-activated K+ (SK) channels from rat brain synaptosomes (IC50 value of 0.5 microm). sPi4 is a high affinity blocker of the Kv1.2 channel. The toxin was docked (BIGGER program) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region. The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the interaction and the associated blockage of the Kv1.2 channel.

Drug binding to HERG channels: evidence for a 'non-aromatic' binding site for fluvoxamine.

British Journal of Pharmacology (2003) 139, 883–884. doi:10.1038/sj.bjp.0705336

Molecular determinants of KCNQ1 channel block by a benzodiazepine.

KCNQ1 channels underlie the slow delayed rectifier K+ current, mediate repolarization of cardiac action potentials, and are a potential therapeutic target for treatment of arrhythmia. (E)-(+)-N-[(3R)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl]-3-(2,4-dichlorophenyl)-2-propenamide [L-735821 (L-7)] is a potent blocker of KCNQ1 channels. Here we describe the structural determinants of KCNQ1 that are critical for high-affinity block by L-7 using site-directed mutagenesis to alter specific residues and voltage clamp to record channel currents in Xenopus laevis oocytes. Chimeric channels were constructed by combination of regions from L-7-sensitive KCNQ1 and L-7-insensitive KCNQ2 channel subunits. This approach localized the drug interaction site to the pore and S6 domains of KCNQ1. Substitution of single amino acids identified Thr-312 of the pore domain and Ile-337, Phe-339, Phe-340, and Ala-344 of the S6 domain as the most important molecular determinants of channel block. Some mutations also altered the inactivation properties of KCNQ1, but there was no correlation between extent of inactivation and sensitivity to block by L-7. Modeling was used to simulate the docking of L-7 to the KCNQ1 channel pore. The docking was consistent with our experimental data and predicts that L-7 blocks K+ conductance by physically precluding the occupancy of a K+ ion to a pore helix-coordinated site within the central hydrated cavity, a crucial step in ion permeation.

Coupled movement of permeant and blocking ions in the CFTR chloride channel pore.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel pore is blocked in a voltage-dependent manner by a broad range of anionic substances added to the cytoplasmic side of the membrane. Here we investigate the origin of the voltage dependence of block by intracellular Au(CN)2-, a highly permeant lyotropic anion which also acts as a high-affinity blocker of Cl- permeation. Not only the affinity, but also the voltage dependence of block by intracellular Au(CN)2- ions is strongly dependent on extracellular Cl- concentration; following replacement of most extracellular Cl- by glucose or by impermeant anions, block by Au(CN)2- shows greatly weakened voltage dependence. This suggests that coupled movement of Au(CN)2- and Cl- ions within the pore contributes to the voltage dependence of block. This explanation requires that interactions between different anions take place within the pore, implying simultaneous binding of multiple anions to intrapore sites. Other anions are able to substitute for extracellular Cl- and interact with intracellular Au(CN)2- ions. Analysis of the effects of different extracellular anions on the apparent affinity and voltage dependence of block by intracellular Au(CN)2- ions suggests that extracellular anions do not need to permeate through the channel in order to destabilize Au(CN)2- binding within the pore, implying that this destabilizing effect results from binding to an externally accessible site in the permeation pathway. We propose that multiple anions can bind simultaneously within the CFTR channel pore, and that repulsive interactions between bound anions speeds anion exit from the pore.

Molecular Basis of Isoform-specific μ-Conotoxin Block of Cardiac, Skeletal Muscle, and Brain Na+ Channels

mu-Conotoxins (mu-CTXs) block skeletal muscle Na(+) channels with an affinity 1-2 orders of magnitude higher than cardiac and brain Na(+) channels. Although a number of conserved pore residues are recognized as critical determinants of mu-CTX block, the molecular basis of isoform-specific toxin sensitivity remains unresolved. Sequence comparison of the domain II (DII) S5-S6 loops of rat skeletal muscle (mu1, Na(v)1.4), human heart (hh1, Na(v)1.5), and rat brain (rb1, Na(v)1.1) Na(+) channels reveals substantial divergence in their N-terminal S5-P linkers even though the P-S6 and C-terminal P segments are almost identical. We used Na(v)1.4 as the backbone and systematically converted these DII S5-P isoform variants to the corresponding residues in Na(v)1.1 and Na(v)1.5. The Na(v)1.4-->Na(v)1.5 variant substitutions V724R, C725S, A728S, D730S, and C731S (Na(v)1.4 numbering) reduced block of Na(v)1.4 by 4-, 86-, 12-, 185-, and 55-fold respectively, rendering the skeletal muscle isoform more "cardiac-like." Conversely, an Na(v)1.5--> Na(v)1.4 chimeric construct in which the Na(v)1.4 DII S5-P linker replaces the analogous segment in Na(v)1.5 showed enhanced mu-CTX block. However, these variant determinants are conserved between Na(v)1.1 and Na(v)1.4 and thus cannot explain their different sensitivities to mu-CTX. Comparison of their sequences reveals two variants at Na(v)1.4 positions 729 and 732: Ser and Asn in Na(v)1.4 compared with Thr and Lys in Na(v)1.1, respectively. The double mutation S729T/N732K rendered Na(v)1.4 more "brain-like" (30-fold downward arrow in block), and the converse mutation T925S/K928N in Na(v)1.1 reproduced the high affinity blocking phenotype of Na(v)1.4. We conclude that the DII S5-P linker, although lying outside the conventional ion-conducting pore, plays a prominent role in mu-CTX binding, thus shaping isoform-specific toxin sensitivity.

Cytokine traps: multi-component, high-affinity blockers of cytokine action.

Cytokines can initiate and perpetuate human diseases, and are among the best-validated of therapeutic targets. Cytokines can be blocked by the use of soluble receptors; however, the use of this approach for cytokines such as interleukin (IL)-1, IL-4, IL-6 and IL-13 that use multi-component receptor systems is limited because monomeric soluble receptors generally exhibit low affinity or function as agonists. We describe here a generally applicable method to create very high-affinity blockers called 'cytokine traps' consisting of fusions between the constant region of IgG and the extracellular domains of two distinct cytokine receptor components involved in binding the cytokine. Traps potently block cytokines in vitro and in vivo and represent a substantial advance in creating novel therapeutic candidates for cytokine-driven diseases.

The anti-malarial drug halofantrine and its metabolite N-desbutylhalofantrine block HERG potassium channels.

The antimalarial drug halofantrine has been associated with QT interval prolongation and with fatal and nonfatal arrhythmias in patients without known underlying cardiac abnormalities. A common target for QT interval-prolonging drugs is the human ether-a-go-go gene (HERG) which encodes the pore forming subunit of the rapidly activating delayed rectifier K(+) current (I(Kr)). We studied the effects of halofantrine (0.1-1000 nM) and its major metabolite N-desbutylhalofantrine (3-1000 nM) on wild type HERG K(+) channels stably expressed in HEK 293 cells, using the whole cell patch-clamp recording technique. Halofantrine and N-desbutylhalofantrine blocked HERG K(+) channels in a concentration-dependent manner with a half-maximal inhibitory concentration of 21.6 nM (n=31 cells) and 71.7 nM (n=18 cells), respectively. The development of drug block for both halofantrine and N-desbutylhalofantrine required channel activation indicative of open and/or inactivated state block. Drug washout or cell hyperpolarization resulted in minimal current recovery consistent with virtually irreversible binding. Using a ventricular action potential voltage clamp protocol, halofantrine and N-desbutylhalofantrine block of HERG current was greatest during phases 2 and 3 of the action potential waveform. We conclude that both halofantrine and N-desbutylhalofantrine cause high affinity block of HERG K(+) channels. Although N-desbutylhalofantrine has been suggested to be a safer antimalarial agent compared to halofantrine, our results suggest that the gain in the safety margin for QT interval prolongation-related cardiotoxicity is minimal.

Molecular Determinants of Voltage-dependent Human Ether-a-Go-Go Related Gene (HERG) K+ Channel Block

The structural determinants for the voltage-dependent block of ion channels are poorly understood. Here we investigate the voltage-dependent block of wild-type and mutant human ether-a-go-go related gene (HERG) K(+) channels by the antimalarial compound chloroquine. The block of wild-type HERG channels expressed in Xenopus oocytes was enhanced as the membrane potential was progressively depolarized. The IC(50) was 8.4 +/- 0.9 microm when assessed during 4-s voltage clamp pulses to 0 mV. Chloroquine also slowed the apparent rate of HERG deactivation, reflecting the inability of drug-bound channels to close. Mutation to alanine of aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of HERG greatly reduced the potency of channel block by chloroquine (IC(50) > 1 mm at 0 mV). However, mutation of Tyr-652 also altered the voltage dependence of the block. In contrast to wild-type HERG, block of Y652A HERG channels was diminished by progressive membrane depolarization, and complete relief from block was observed at +40 mV. HERG channel block was voltage-independent when the hydroxyl group of Tyr-652 was removed by mutating the residue to Phe. Together these findings indicate a critical role for Tyr-652 in voltage-dependent block of HERG channels. Molecular modeling was used to define energy-minimized dockings of chloroquine to the central cavity of HERG. Our experimental findings and modeling suggest that chloroquine preferentially blocks open HERG channels by cation-pi and pi-stacking interactions with Tyr-652 and Phe-656 of multiple subunits.

The Antipsychotic Drugs Sertindole and Pimozide Block erg3, a Human Brain K+ Channel

The antipsychotic drugs sertindole and pimozide are known to prolong the QT interval on the electrocardiogram via a high affinity block of the cardiac K+ channel known as HERG (human ether-a-go-go-related gene; erg1). We wished to test whether these drugs also displayed high affinity for the related neuronal K+ channel erg3. The cDNA encoding erg3 channel was cloned from a human brain library. Northern analysis confirmed that the channel was localized to brain relative to other tissues including heart, liver and lung. Within the brain, erg3 was expressed in higher amounts in the frontal lobe and cerebellum relative to the temporal, parietal and occipital lobes. Transient expression of erg3 in Chinese hamster ovary cells produced outwardly directed K+ currents that activated at approximately −50 mV and produced a large transient component at positive membrane potentials. Inward tail currents measured at −100 mV were blocked in a dose-dependent fashion by sertindole resulting in an IC50 value of 43 nM. Significant inhibition was observed at concentrations as low as 3 nM. Block of erg3 by sertindole also displayed a positive voltage-dependence. Pimozide blocked erg3 channel currents with an IC50 of 103 nM and significant inhibition was noted at concentrations of 10 nM and higher. We conclude that erg3 can be blocked by certain antipsychotic drugs like sertindole and pimozide. Inhibition of erg3 or related K+ channels in the brain may contribute to the efficacy/side effect profiles of some antipsychotic drugs.

Dual incorporation of photoaffinity ligands on dopamine transporters implicates proximity of labeled domains.

We have recently developed novel high-affinity blockers for the dopamine transporter (DAT) by carrying out structure-activity studies of GBR 12909 molecule piperidine analogs. To investigate the molecular basis of binding of these compounds in comparison to known sites of action of GBR 12909, cocaine, and benztropine analogs, we developed a piperidine-based photoaffinity label [(125)I]4-[2-(diphenylmethoxy)ethyl]-1-[(4-azido- 3-iodophenyl)methyl]-piperidine [(125)I]AD-96-129), and used proteolysis and epitope-specific immunoprecipitation to identify the protein domains that interact with the ligand. [(125)I]AD-96-129 became incorporated into two different regions of the DAT primary sequence, an N-terminal site containing transmembrane domains (TMs) 1 to 2, and a second site containing TMs 4 to 6. Both of these regions have been identified previously as sites involved in the binding of other DAT photoaffinity labels. However, in contrast to the previously characterized ligands that showed nearly complete specificity in their binding site incorporation, [(125)I]AD-96-129 became incorporated into both sites at comparable levels. These results suggest that the two domains may be in close three-dimensional proximity and contribute to binding of multiple uptake blockers. We also found that DATs labeled with [(125)I]AD-96-129 or other photoaffinity labels displayed distinctive sensitivities to proteolysis of a site in the second extracellular loop, with protease resistance related to the extent of ligand incorporation in the TM4 to 6 region. These differences in protease sensitivity may indicate the relative proximity of the ligands to the protease site or reflect antagonist-induced conformational changes in the loop related to transport inhibition.

A functional assay for paralytic shellfish toxins that uses recombinant sodium channels

Saxitoxin (STX) and its derivatives are highly toxic natural compounds produced by dinoflagellates commonly present in marine phytoplankton. During algal blooms (“red tides”), shellfish accumulate saxitoxins leading to paralytic shellfish poisoning (PSP) in human consumers. PSP is a consequence of the high-affinity block of voltage-dependent Na channels in neuronal and muscle cells. PSP poses a significant public health threat and an enormous economic challenge to the shellfish industry worldwide. The standard screening method for marine toxins is the mouse mortality bioassay that is ethically problematic, costly and time-consuming. We report here an alternative, functional assay based on electrical recordings in cultured cells stably expressing a PSP target molecule, the STX-sensitive skeletal muscle Na channel. STX-equivalent concentration in the extracts was calibrated by comparison with purified STX, yielding a highly significant correlation (R=0.95; N=30) between electrophysiological determinations and the values obtained by conventional methods. This simple, economical, and reproducible assay obviates the need to sacrifice millions of animals in mandatory paralytic shellfish toxin screening programs.

Receptor blockers — general aspects with respect to their use in domestic animal reproduction

Receptor blockers compete with the respective agonist for binding to a given receptor without inducing complete signal transduction. In recent years, major interest has focused on sex-steroid hormone receptor blockers (antagonists). Indications have been obtained that inadequate changes in receptor conformation and subsequent failure of transcriptional activation are major events preventing hormonal activity. However, various subtypes and variants of receptors and receptor mutations have also been identified. Expression of antihormonal effects may vary depending on the type of receptor the blocker is bound to. Hence, receptor blockers may also have an inherent agonistic activity. Aglepristone® is the first antiprogestin registered for veterinary use with the indication “interruption or prevention of pregnancy”; similarly, these types of compounds were successfully used for induction of parturition in the dog and cat and for conservative treatment of pyometra in the dog. Moreover, application of antiprogestins has clearly demonstrated the role of progesterone as a major factor controlling overt pseudopregnancy in dogs. With respect to farm animals, parturition was induced in cows without an increased incidence of retained fetal membranes. Other than antiprogestins, antioestrogens and antiandrogens are still in a more experimental phase. In particular for use in humans, high-affinity blockers binding to the oxytocin/vasopressin receptor are in development; they exert distinct tocolytic activities. Also, the release of GnRH can be inhibited by respective antagonists; however, their use in reproduction is still hampered by the high dose requirement and the side effects observed.

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

Dihydropyridine enantiomers block recombinant L-type Ca2+ channels by two different mechanisms.

1. The molecular basis of the state-dependent block of L-type Ca2+ channels by dihydropyridines is still poorly understood. Therefore, we studied the enantioselectivity of Ca2+ channel block by isradipine enantiomers at three holding potentials (-80, -60 and -40 mV) in Chinese hamster ovary (CHO) cells stably expressing the rabbit lung alpha1C-b-subunit. 2. The extent of enantioselectivity did not markedly change with the holding potential (IC50 ratios of 104-138), whereas the potency of both isradipine enantiomers increased with depolarisation of the holding potential. 3. In addition to its block of the peak Ca2+ channel current, Ipeak, (-)-isradipine inhibited the relative current at the end of the test pulse, the so-called Ilate, normalised to Ipeak (Ilate/Ipeak). This effect was unaffected by the holding potential and revealed distinct kinetics compared to the development of conventional block of Ipeak. 4. When these effects were studied using an alpha1C-b-mutant lacking the high-affinity dihydropyridine binding site, expressed in human embryonic kidney (HEK 293) cells, both enantiomers blocked Ilate/Ipeak to a similar degree. 5. Our data are discussed within the framework of the 'guarded receptor' and the 'modulated receptor' hypotheses. The very different properties of the block of Ilate/Ipeak compared to those of the conventional high-affinity block of Ipeak suggest the existence of an additional mechanism possibly mediated via a second, distinct binding site.

Amino acid substitutions in the pore of rat glutamate receptors at sites influencing block by polyamines.

1. The effect on polyamine block of mutations at the Q/R site and the conserved negative charge +4 site in AMPA and kainate receptors was studied using the rat glutamate receptor GluR6 expressed in Xenopus oocytes and human embryonic kidney (HEK) cells. 2. Introduction of negative charge at the Q/R site increased the equilibrium dissociation constant at 0 mV (Kd(0)) for spermine from 1.3 to 4.0 microM (Q590E); the smaller side chains Q590D and Q590N had Kd(0) values of 47 and 20 microM. Reductions in spermine affinity were also obtained for the small hydrophobic residues Q590V and Q590A, with Kd(0) values of 3.6 and 8.8 microM. Positively charged side chains produced outward rectifying responses similar to those recorded for GluR6(Q) with polyamine-free conditions, suggesting a complete absence of voltage-dependent block by spermine. 3. Substitution of tryptophan at the Q/R site produced high-affinity block with a Kd(0) of 190 pM. In Xenopus oocytes no outward current was observed at potentials up to +200 mV. A much smaller increase in affinity was observed for Q590F and Q590Y, which had Kd(0) values of 0.28 and 0.83 microM respectively. 4. The Q590H mutant gave weakly birectifying responses strikingly different from those for other mutants. When ionization of the His group was increased by raising the external hydrogen ion concentration, responses became outward rectifying. The ratios of the conductance at 100 mV over that at -100 mV for Q590H were 0.52 at pH 8.3 and 2.5 at pH 5.3. 5. Neutralization of charge or aromatic residues at the +4 site produced a large reduction of spermine affinity, with Kd(0) values for E594N, E594Q and E594W of 109, 1020 and 2150 microM, respectively. In the absence of polyamines, E594K and E594R produced strongly inward rectifying responses while E594Q, E594A and E594W were birectifying. 6. A model for permeant block allowed quantitative comparisons between mutants. Despite large changes in well depth and barrier heights, there was little change in the voltage dependence of block for both Q/R and +4 site mutants. We propose a model with a distributed binding site for polyamines in which the +4 site is located near the entrance to the channel.

Sulfonylurea and K(+)-channel opener sensitivity of K(ATP) channels. Functional coupling of Kir6.2 and SUR1 subunits.

The sensitivity of K(ATP) channels to high-affinity block by sulfonylureas and to stimulation by K(+) channel openers and MgADP (PCOs) is conferred by the regulatory sulfonylurea receptor (SUR) subunit, whereas ATP inhibits the channel through interaction with the inward rectifier (Kir6.2) subunit. Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) profoundly antagonized ATP inhibition of K(ATP) channels expressed from cloned Kir6.2+SUR1 subunits, but also abolished high affinity tolbutamide sensitivity. By stabilizing the open state of the channel, PIP(2) drives the channel away from closed state(s) that are preferentially affected by high affinity tolbutamide binding, thereby producing an apparent loss of high affinity tolbutamide inhibition. Mutant K(ATP) channels (Kir6. 2[DeltaN30] or Kir6.2[L164A], coexpressed with SUR1) also displayed an "uncoupled" phenotype with no high affinity tolbutamide block and with intrinsically higher open state stability. Conversely, Kir6. 2[R176A]+SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block. In addition to antagonizing high-affinity block by tolbutamide, PIP(2) also altered the stimulatory action of the PCOs, diazoxide and MgADP. With time after PIP(2) application, PCO stimulation first increased, and then subsequently decreased, probably reflecting a common pathway for activation of the channel by stimulatory PCOs and PIP(2). The net effect of increasing open state stability, either by PIP(2) or mutagenesis, is an apparent "uncoupling" of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with poly-L-lysine.

Differential sensitivity of beta-cell and extrapancreatic K(ATP) channels to gliclazide.

To investigate the tissue specificity of gliclazide for cloned beta-cell, cardiac and smooth muscle ATP-sensitive K-channels (K(ATP) channels). These channels share a common pore-forming subunit, Kir6.2, which associates with different sulphonylurea receptor isoforms (SUR1 in beta-cells, SUR2A in heart, SUR2B in smooth muscle). Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes, and channel activity was measured by recording macroscopic currents in giant inside-out membrane patches. Gliclazide was added to the intracellular membrane surface. We reported previously that Kir6.2-SUR1 currents are blocked at two sites by tolbutamide: a high-affinity site on SUR1 and a low-affinity site on Kir6.2. We now show that gliclazide also inhibits beta-cell K(ATP) channels at two sites: a high-affinity site, which is half-maximally blocked (Ki) at 50 +/- 7 nmol/l (n = 8) and a low-affinity site with a Ki of 3.0 +/- 0.6 mmol/l (n = 4). The high-affinity site on SUR1 was thus about 40-fold more sensitive to gliclazide than to tolbutamide (Ki approximately 2 micromol/l). Cloned cardiac and smooth muscle K(ATP) channels did not show high-affinity block by gliclazide. Kir6.2-SUR2A currents exhibited a single low-affinity site with a Ki of 0.8 +/- 0.1 mmol/l (n = 5), which is likely to reside on the Kir6.2 subunit. Our results show that gliclazide is a sulphonylurea with high affinity and strong selectivity for the beta-cell type of K(ATP) channel.