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PubMed2004

Domain analysis of the calcium-activated potassium channel SK1 from rat brain. Functional expression and toxin sensitivity.

Two small conductance, calcium-activated potassium channels (SK channels), SK2 and SK3, have been shown to contribute to the afterhyperpolarization (AHP) and to shape the firing behavior in neurons for example in the hippocampal formation, the dorsal vagal nucleus, the subthalamic nucleus, and the cerebellum. In heterologous expression systems, SK2 and SK3 currents are blocked by the bee venom toxin apamin, just as well as the corresponding neuronal AHP currents. However, the functional role and pharmacological profile of SK1 channels from rat brain (rSK1) is still largely unknown, as so far rSK1 homomeric channels could not be functionally expressed. We have performed a domain analysis to elucidate the pharmacological profile and the molecular determinants of rSK1 channel expression by using channel chimeras in combination with immunocytochemistry, immunoblot analysis, and electrophysiology. Our results reveal that the rSK1 subunit is synthesized in cells but does not form functional homomeric channels. Exchanging the carboxyl terminus of rSK1 for that of hSK1 or rSK2 is sufficient to rescue the functional expression of rSK1 channels. Additionally, transplantation of both amino and carboxyl termini of rSK1 onto hSK1 subunits, normally forming functional homomeric channel, hinders their functional expression, while hSK1 channels containing only the rSK1 carboxyl terminus are functional. These results suggest that the lack of functional expression of rSK1 channels is probably due to problems in their assembly and tetramerization but not in their calmodulin-dependent gating. Finally, we show that chimeric channels containing the core domain (S1-S6) of rSK1, unlike hSK1, are apamin-insensitive.

Amino Acid Sequence↗
PubMed1985

An ion's view of the potassium channel. The structure of the permeation pathway as sensed by a variety of blocking ions.

We have studied the block of potassium channels in voltage-clamped squid giant axons by nine organic and alkali cations, in order to learn how the channel selects among entering ions. When added to the internal solution, all of the ions blocked the channels, with inside-positive voltages enhancing the block. Cesium blocked the channels from the outside as well, with inside-negative voltages favoring block. We compared the depths to which different ions entered the channel by estimating the "apparent electrical distance" to the blocking site. Simulations with a three-barrier, double-occupancy model showed that the "apparent electrical distance," expressed as a fraction of the total transmembrane voltage, appears to be less than the actual value if the blocking ion can pass completely through the channel. These calculations strengthen our conclusion that sodium and cesium block at sites further into the channel than those occupied by lithium and the organic blockers. Our results, considered together with earlier work, demonstrate that the depth to which an ion can readily penetrate into the potassium channel depends both on its size and on the specific chemical groups on its molecular surface. The addition of hydroxyl groups to alkyl chains on a quaternary ammonium ion can both decrease the strength of binding and allow deeper penetration into the channel. For alkali cations, the degree of hydration is probably crucial in determining how far an ion penetrates. Lithium, the most strongly hydrated, appeared not to penetrate as far as sodium and cesium. Our data suggest that there are, minimally, four ion binding sites in the permeation pathway of the potassium channel, with simultaneous occupancy of at least two.

Animals↗
PubMed2001

Yeast screen for constitutively active mutant G protein-activated potassium channels.

GIRK2 is a major contributor to G protein-activated inward rectifier potassium channels in the mammalian brain. How GIRK channels open upon contact with Gbetagamma remains unknown. Using a yeast genetic screen to select constitutively active mutants from a randomly mutagenized GIRK2 library, we identified five gating mutations at four residues in the transmembrane domain. Further mutagenesis indicates that GIRK channel opening involves a rotation of the transmembrane segments, bringing one of these residues (V188) to a pore-lining position in the open conformation. Combined with double-mutant studies, these findings suggest that GIRK channels gate by moving from the open conformation inferred from our yeast study of Kir2.1 to a closed conformation perhaps resembling the known KcsA structure.

Amino Acid Sequence↗
PubMed2001

Evidence for a single nucleotide polymorphism in the KCNQ1 potassium channel that underlies susceptibility to life-threatening arrhythmias.

INTRODUCTION: Congenital long QT syndrome (LQTS) is a genetically heterogeneous arrhythmogenic disorder caused by mutations in at least five different genes encoding cardiac ion channels. It was suggested recently that common polymorphisms of LQTS-associated genes might modify arrhythmia susceptibility in potential gene carriers. METHODS AND RESULTS: We examined the known LQTS genes in 95 patients with definitive or suspected LQTS. Exon-specific polymerase chain reaction single-strand conformation polymorphism and direct sequence analyses identified six patients who carried only a single nucleotide polymorphism in KCNQ1 that is found in approximately 11% of the Japanese population. This 1727G>A substitution that changes the sense of its coding sequence from glycine to serine at position 643 (G643S) was mostly associated with a milder phenotype, often precipitated by hypokalemia and bradyarrhythmias. When heterologously examined by voltage-clamp experiments, the in vitro cellular phenotype caused by the single nucleotide polymorphism revealed that G643S-KCNQ1 forms functional homomultimeric channels, producing a significantly smaller current than that of the wild-type (WT) channels. Coexpression of WT-KCNQ1 and G643S-KCNQ1 with KCNE1 resulted in approximately 30% reduction in the slow delayed rectifier K+ current I(Ks) without much alteration in the kinetic properties except its deactivation process, suggesting that the G643S substitution had a weaker dominant-negative effect on the heteromultimeric channel complexes. CONCLUSION: We demonstrate that a common polymorphism in the KCNQ1 potassium channel could be a molecular basis for mild I(Ks) dysfunction that, in the presence of appropriate precipitating factors, might predispose potential gene carriers to life-threatening arrhythmias in a specific population.

Adult↗
PubMed1992

Electromechanical effects of the putative potassium channel activator celikalim (WAY-120,491) on feline atrial and ventricular muscle.

Celikalim (WAY-120,491) is a putative potassium channel activator that has been shown to lower blood pressure in animal models and humans. In the present study, we have examined the effects of celikalim on contractility and ionic currents in feline cardiac muscle. Celikalim was found to decrease contractility in electrically stimulated (2 Hz frequency) left atrial and right ventricular papillary muscle preparations with IC50 values of 0.95 +/- 0.12 microM (n = 6) and 0.29 +/- 0.07 microM (n = 5), respectively. Glyburide (1 microM) reversed the celikalim-induced negative inotropy (left atrial halves). Celikalim was also shown to activate a glyburide-sensitive current in voltage-clamped isolated ventricular myocytes that reversed close to the calculated value of the potassium equilibrium potential (n = 4 cells). In addition, celikalim was found to inhibit voltage-activated calcium current (L-type) in isolated ventricular myocytes (51 +/- 2% inhibition at 1 microM; n = 4 cells). We conclude that celikalim is a potassium channel activator and hypothesize that both the negative inotropy and the glyburide-sensitive current evoked by this drug are mediated by ATP-regulated potassium channels. Inhibition of voltage-activated calcium channels by celikalim may also contribute to the negative inotropy induced by this drug.

Animals↗
PubMed2004

Abbreviated action potential kinetics in a mouse model of potassium channel overexpression during hippocampal development.

Loss or "gain" of function mutations in voltage-gated ion channels often results in an adverse neurological phenotype. We have examined the electrical characteristics of hippocampal pyramidal cells in a transgenic mouse model to determine how overexpression of a Shaker-type potassium channel subunit during early postnatal development might alter excitability properties of developing neurons. We found that in CA3 neurons potassium channel overexpression led to a transient shortening in duration of single action potentials during the first two postnatal weeks. There was an increase in maximal repolarization rate, without significant effect on the rate of rise. Transgenic CA3 neurons also showed a decrease of firing frequency in response to sustained depolarizing current injection. In contrast, repolarization of action potentials in CA1 neurons was not significantly altered by trangene expression. Western Blot Analysis of membrane-associated transgene protein indicated that transgene protein levels decreased during development, in agreement with functional measures of spike width. Our data indicate that the functional consequences of potassium channel transgene expression are dependent on cellular environment and developmental stage. A transient period of hypoexcitability during a critical period of development for CA3 neurons may contribute to the hyperexcitable phenotype observed in adult animals.

Action Potentials↗
PubMed1991

Effects of some potassium channel blockers on the ionic currents in myelinated nerve.

The effects of some potassium channel blockers on the ionic currents and on the so-called K(+)-depolarization in intact myelinated nerve fibres were studied. 4-AP, and in particular, Flaxedil, proved to be selective K(+)-current blockers. However, TEA, a crown ether (DCH18C6), a longchained triethylammonium compound (C10-TriEA), capsaicin, and the extract from the medicinal herb Ruta graveolens proved not to be selective K(+)-current blockers; they all block Na(+)-currents as well, although to a lesser extent. The sodium inactivation curve did not change under TEA and Flaxedil but was shifted on the potential axis in negative direction by DCH18C6, 4-AP, capsaicin and the Ruta extract whereas C10-TriEA caused a shift of both sodium inactivation and activation parameters in positive direction. Regarding to the kinetics of the persisting K(+)-current fraction, two different kinds of blockade were found: 1. Unchanged K(+)-kinetic which is typical for the effects of TEA, 4-AP, Flaxedil, and C10-TriEA. 2. Clearly changed K(+)-kinetic, characterized by K(+)-transients; which is typical for the effects of capsaicin and in particular, for those of DCH18C6 and of the Ruta extract. The possibly different modes of action of both groups of blockers are discussed in terms of current models for the action of potassium channel blockers.

4-Aminopyridine↗
PubMed1995

Effect of four potassium channel agonists on bupivacaine-induced toxicity in mice.

The influence of four potassium channel agonists i.e. diazoxide (D), levcromakalim (L), nicorandil (N) and pinacidil (P) on bupivacaine-induced acute toxicity was evaluated by measuring the convulsant activity, the time of latency to convulse and the mortality rate. Four different dosages (i.e. 0.1, 1, 10 and 100 mg/kg/i.p. for D, N and P and 0.01, 0.1, 1 and 5 mg/kg/i.p. for L) were injected to a total of 200 male NMRI adult mice: 16 groups of 10 mice each were previously treated by a single i.p. dose of each potassium channel agonist while controls (n = 40) received saline injection. Thus, 15 minutes later, all groups were injected with a 50 mg/kg/i.p. single dose of bupivacaine. The convulsant activity of bupivacaine was significantly modified by only high doses of L in a dose-dependent manner. Compared to the controls, the period of latency was significantly increased for most of the doses of P, N, D and L in a dose dependent manner for L and P. The anesthetic-induced mortality (47.5% for controls) was not significantly modified by D, but decreased by N and increased by high doses of L and P which is probably related to a delayed mortality.

Animals↗
PubMed2003

Localization of KCNC1 (Kv3.1) potassium channel subunits in the avian auditory nucleus magnocellularis and nucleus laminaris during development.

The KCNC1 (previously Kv3.1) potassium channel, a delayed rectifier with a high threshold of activation, is highly expressed in the time coding nuclei of the adult chicken and barn owl auditory brainstem. The proposed role of KCNC1 currents in auditory neurons is to reduce the width of the action potential and enable neurons to transmit high frequency temporal information with little jitter. Because developmental changes in potassium currents are critical for the maturation of the shape of the action potential, we used immunohistochemical methods to examine the developmental expression of KCNC1 subunits in the avian auditory brainstem. The KCNC1 gene gives rise to two splice variants, a longer KCNC1b and a shorter KCNC1a that differ at the carboxy termini. Two antibodies were used: an antibody to the N-terminus that does not distinguish between KCNC1a and b isoforms, denoted as panKCNC1, and another antibody that specifically recognizes the C terminus of KCNC1b. A comparison of the staining patterns observed with the panKCNC1 and the KCNC1b specific antibodies suggests that KCNC1a and KCNC1b splice variants are differentially regulated during development. Although panKCNC1 immunoreactivity is observed from the earliest time examined in the chicken (E10), a subcellular redistribution of the immunoproduct was apparent over the course of development. KCNC1b specific staining has a late onset with immunostaining first appearing in the regions that map high frequencies in nucleus magnocellularis (NM) and nucleus laminaris (NL). The expression of KCNC1b protein begins around E14 in the chicken and after E21 in the barn owl, relatively late during ontogeny and at the time that synaptic connections mature morphologically and functionally.

Animals↗
PubMed2003

Characterization of potassium channel modulators with QPatch automated patch-clamp technology: system characteristics and performance.

Planar silicon chips with 1-2-microm etched holes (average resistance: 2.04 +/- 0.02 MOmega in physiological buffer, n = 274) have been developed for patch-clamp recordings of whole-cell currents from cells in suspension. An automated 16-channel parallel screening system, QPatch 16, has been developed using this technology. A single-channel prototype of the QPatch system was used for validation of the patch-clamp chip technology. We present here data on the quality of patch-clamp recordings and from actual drug screening studies of human potassium channels expressed in cultured cell lines. Using Chinese hamster ovary (CHO) and human embryonic kidney cells (HEK), gigaseals of 4.1 +/- 0.4 GOmega (n = 146) and high-quality whole-cell current recordings were obtained from hERG and KCNQ4 potassium channels. Success rates for gigaseal recordings varied from 40 to 95%, and 67% of the whole-cell configurations lasted for >20 min. Cells were maintained in suspension up to 4 h in a cell storage facility that is integrated in the QPatch 16. No decline in patchability was observed during this time course. A series of screens was conducted with known inhibitors of the hERG and KCNQ4 potassium channels. Dose-response relationship characterizations of verapamil and rBeKm-1 blockage of hERG currents provided IC(50) values similar to values reported in the literature.

Animals↗
PubMed1996

The weaver mutation changes the ion selectivity of the affected inwardly rectifying potassium channel GIRK2.

The weaver mutation in mice has recently been identified as a single base-pair mutation in the Girk2 gene, which encodes a G-protein-activated inwardly rectifying potassium channel, GIRK2. The mutation results in a Gly to Ser substitution at residue 156, in the putative pore-forming region of the potassium channel. In the present study, we used Xenopus oocytes to express mutant GIRK2, and to characterize the effects of the mutation on the channel. The mutation results in a loss of the normal high selectivity for K+ over Na+, with little effect on other channel properties such as activation by the mu opioid receptor. The resulting increase in basal Na+ permeability causes a marked depolarization of oocytes expressing the mutant GIRK2 protein. This result was observed even when the mutant GIRK2 was coexpressed with GIRK1, a situation more analogous to that seen in vivo. Thus, the increased Na+ permeability and resulting depolarization may contribute to the pathology of cerebellar granule cells and substantia nigra dopaminergic neurons observed in the weaver mice.

Analgesics↗
PubMed2001

Regulated expression of adenosine triphosphate-sensitive potassium channel subunits in pancreatic beta-cells.

The regulation of glucose-dependent insulin secretion in pancreatic beta-cells is linked to the expression and function of the ATP-sensitive potassium channel (K(ATP)), which is composed of a sulfonylurea receptor (SUR1) and an inwardly rectifying potassium channel (Kir6.2). Previous animal and human genetic studies have demonstrated that disruption or defective expression of K(ATP) subunit genes has a profound impact on the regulation of insulin secretion. Little is known about how SUR1 and Kir6.2 gene expression is regulated. Here we show that high glucose concentrations lead to a marked decrease (approximately 70%) in Kir6.2 messenger RNA (mRNA) levels in isolated rat pancreatic islets as well as in the INS-1 beta-cell line. This effect is reversible, because exposure to low glucose reinduces Kir6.2 transcript levels. The cognate K(ATP) channel subunit SUR1 showed similar down-regulation at high glucose concentration. The K(ATP) channel activity of INS-1 cells cultivated at high glucose was reduced by 33-51%. In contrast, glucagon-like peptide-1 (GLP-1) induced Kir6.2 mRNA steady state levels and was able to prevent glucose-dependent inhibition of Kir6.2 mRNA and K(ATP) channel activity. To provide further insight into the mechanisms by which glucose and GLP-1 regulate beta-cell K(ATP) channel genes, we have cloned and initiated the characterization of the Kir6.2 gene transcriptional regulatory regions contained within the entire 4.5 kb flanked by the SUR1 and Kir6.2 genes. Transient transfection experiments with five deletion constructs in a pancreatic beta-cell line (INS-1) showed that the proximal 988 bp of the Kir6.2 promoter sequence contributes only 25-30% to the total basal promoter activity. The minimal promoter region -67/+140, also encompassing parts of the 5'-untranslated region, confers sensitivity to GLP-1, which stimulates transcriptional activity of the Kir6.2 minigene by about 2-fold. We propose that glucose- and GLP-1-dependent regulation of K(ATP) subunit genes may be important in the adaptation of beta-cells to changes in secretory demands in physiological and diseased states.

ATP-Binding Cassette Transporters↗
PubMed2001

Multiple sites of interaction between the intracellular domains of an inwardly rectifying potassium channel, Kir6.2.

The amino-terminal and carboxy-terminal domains of inwardly rectifying potassium channel (Kir) subunits are both intracellular. A direct physical interaction between these two domains is involved in the response of Kir channels to regulatory factors such as G-proteins, nucleotides and intracellular pH. We have previously mapped the region within the N-terminal domain of Kir6.2 that interacts with the C-terminus. In this study we use a similar in vitro protein-protein interaction assay to map the regions within the C-terminus which interact with the N-terminus. We find that multiple interaction domains exist within the C-terminus: CID1 (amino acids (aa) 279-323), CID2 (aa 214-222) and CID3 (aa 170-204). These domains correlate with regions previously identified as making important contributions to Kir channel assembly and function. The highly conserved nature of the C-terminus suggests that a similar association with the N-terminus may be a feature common to all members of the Kir family of potassium channels, and that it may be involved in gating of Kir channels by intracellular ligands.

Amino Acid Sequence↗
PubMed2002

The voltage-gated potassium channel KCNQ2 in Taiwanese children with febrile convulsions.

Mutations in the voltage-gated potassium channel genes KCNQ2 and KCNQ3 have been found to cause benign familial neonatal convulsions. Recent studies provided evidence that KCNQ2 and KCNQ3 contribute to the M-current, which regulates the subthreshold electrical excitability in the CNS. Febrile convulsions represent the majority of childhood seizures, and show a strong family history, suggesting a genetic predisposition. By performing an association study, we investigated whether KCNQ2 gene polymorphisms can be used as markers of susceptibility to febrile convulsions. These data suggest that the KCNQ2 gene might not be a useful marker for prediction of the susceptibility of febrile convulsions.

Child↗
PubMed2002

Three-dimensional structure of the S4-S5 segment of the Shaker potassium channel.

The propagation of action potentials during neuronal signal transduction in phospholipid membranes is mediated by ion channels, a diverse group of membrane proteins. The S4-S5 linker peptide (S4-S5), that connects the S4 and S5 transmembrane segments of voltage-gated potassium channels is an important region of the Shaker ion-channel protein. Despite its importance, very little is known about its structure. Here we provide evidence for an amphipathic alpha-helical conformation of a synthetic S4-S5 peptide of the voltage-gated Drosophila melanogaster Shaker potassium channel in water/trifluoroethanol and in aqueous phospholipid micelles. The three-dimensional solution structures of the S4-S5 peptide were obtained by high-resolution nuclear magnetic resonance spectroscopy and distance-geometry/simulated-annealing calculations. The detailed structural features are discussed with respect to model studies and available mutagenesis data on the mechanism and selectivity of the potassium channel.

Amino Acid Sequence↗
PubMed1996

Identification of ATP-sensitive potassium channel in frog ventricular myocytes.

ATP-sensitive potassium channels were found in frog ventricular myocytes using the inside-out patch-clamp technique. The channel was selectively permeable to K+. Single-channel conductance was 32.6 pS at 3.0 mM of [K+]o and 132 mM [K+]i and 77.3 pS at 114 mM [K+]o and 132 mM [K+]i. ATP did not affect single-channel conductance. The open probability of the channel was decreased by intracellular application of ATP in both the presence and absence of 2 mM MgCl2. The coexistence of Mg2+ with ATP shifts the dose-response curve for the open probability of ATP-sensitive K+ channel against ATP rightward. The shift of the curve indicates that Mg-ATP is less effective than free ATP in inhibiting the channel. An open-time histogram was fitted by a single exponential function with a time constant of 1.63 +/- 0.17 msec (n = 5) in an ATP-free medium. Mean open time (1.57 +/- 0.10 msec; n = 5) was not altered but the inter-burst time (closed time between bursts) lengthened in 10 microM ATP.

Adenosine Triphosphate↗
PubMed2001

Insulin release and suppression by tacrolimus, rapamycin and cyclosporin A are through regulation of the ATP-sensitive potassium channel.

AIM: By focusing on the pancreatic beta cell response to tacrolimus, cyclosporin A (CsA) and rapamycin we hoped to identify immunophilin, calcineurin and/or novel mechanism involvement and advance the understanding of immunosuppressant regulated insulin control. METHODS: A glucose responsive beta cell model was established in which the glucose response was blocked by immunosuppressant treatment and this model was used to further characterise this effect. Quantification of insulin release to immunosuppressants and specific inhibitors was used to identify the mechanism involved. RESULTS: It was found that upon the addition of tacrolimus, rapamycin, or CsA, rapid and significant exocytosis of cellular insulin was seen. A dose response study of this effect revealed optimal concentration windows of 50- 80 nm for tacrolimus, 100-300 nm for rapamycin, and 7-12 mm for CsA in RIN-5F cells. Optimal insulin release for HIT-T15 cells was similar. Additional experiments demonstrate that immunosuppressant pretreatment blocked the subsequent immunosuppressant induced insulin release but not that of a thapsigargin control, suggesting that suppression and release are non-toxic, specific and in the same pathway. Further experiments showed that this insulin release was a calcium dependent process, which was blocked by inhibitors of l-type calcium channels. Continued studies showed that the specific ATP-sensitive potassium channel agonist diazoxide (150 mm) also blocked immunosuppressant-induced insulin release. CONCLUSIONS: A model that fits this data is a novel calcineurin-independent immunophilin mediated partial closing of the ATP-sensitive potassium channel, which would lead to an initial insulin release but would reduce subsequent responses through this pathway.

Animals↗
PubMed1997

Reversible antisense inhibition of Shaker-like Kv1.1 potassium channel expression impairs associative memory in mouse and rat.

Long-term memory is thought to be subserved by functional remodeling of neuronal circuits. Changes in the weights of existing synapses in networks might depend on voltage-gated potassium currents. We therefore studied the physiological role of potassium channels in memory, concentrating on the Shaker-like Kv1.1, a late rectifying potassium channel that is highly localized within dendrites of hippocampal CA3 pyramidal and dentate gyrus granular cells. Repeated intracerebroventricular injection of antisense oligodeoxyribonucleotide to Kv1.1 reduces expression of its particular intracellular mRNA target, decreases late rectifying K+ current(s) in dentate granule cells, and impairs memory but not other motor or sensory behaviors, in two different learning paradigms, mouse passive avoidance and rat spatial memory. The latter, hippocampal-dependent memory loss occurred in the absence of long-term potentiation changes recorded both from the dentate gyrus or CA1. The specificity of the reversible antisense targeting of mRNA in adult animal brains may avoid irreversible developmental and genetic background effects that accompany transgenic "knockouts".

Animals↗