Class Of Potassium Channels Research Articles

Membrane potential drives the exit from pluripotency and cell fate commitment via calcium and mTOR

Transitioning from pluripotency to differentiated cell fates is fundamental to both embryonic development and adult tissue homeostasis. Improving our understanding of this transition would facilitate our ability to manipulate pluripotent cells into tissues for therapeutic use. Here, we show that membrane voltage (Vm) regulates the exit from pluripotency and the onset of germ layer differentiation in the embryo, a process that affects both gastrulation and left-right patterning. By examining candidate genes of congenital heart disease and heterotaxy, we identify KCNH6, a member of the ether-a-go-go class of potassium channels that hyperpolarizes the Vm and thus limits the activation of voltage gated calcium channels, lowering intracellular calcium. In pluripotent embryonic cells, depletion of kcnh6 leads to membrane depolarization, elevation of intracellular calcium levels, and the maintenance of a pluripotent state at the expense of differentiation into ectodermal and myogenic lineages. Using high-resolution temporal transcriptome analysis, we identify the gene regulatory networks downstream of membrane depolarization and calcium signaling and discover that inhibition of the mTOR pathway transitions the pluripotent cell to a differentiated fate. By manipulating Vm using a suite of tools, we establish a bioelectric pathway that regulates pluripotency in vertebrates, including human embryonic stem cells.

Shank promotes action potential repolarization by recruiting BK channels to calcium microdomains.

Mutations altering the scaffolding protein Shank are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. Among its many binding partners, Shank directly binds CaV1 voltage activated calcium channels. Here, we show that the Caenorhabditis elegans SHN-1/Shank promotes CaV1 coupling to calcium activated potassium channels. Mutations inactivating SHN-1, and those preventing SHN-1 binding to EGL-19/CaV1 all increase action potential durations in body muscles. Action potential repolarization is mediated by two classes of potassium channels: SHK-1/KCNA and SLO-1 and SLO-2 BK channels. BK channels are calcium-dependent, and their activation requires tight coupling to EGL-19/CaV1 channels. SHN-1's effects on AP duration are mediated by changes in BK channels. In shn-1 mutants, SLO-2 currents and channel clustering are significantly decreased in both body muscles and neurons. Finally, increased and decreased shn-1 gene copy number produce similar changes in AP width and SLO-2 current. Collectively, these results suggest that an important function of Shank is to promote microdomain coupling of BK with CaV1.

A sensitive regulator of cellular potassium

Ion Channels A class of potassium channels called K2P channels modulates resting membrane potential in most cells. The channels are regulated by multiple ligands, including the antidepressant drug Prozac, as well as factors such as mechanical stretch and voltage. Dong et al. determined the structure

Targeting two-pore domain potassium channels — A promising strategy for treating T cell mediated autoimmunity

Two-pore domain potassium channels, such as TASK, are essential players in the regulation of the resting membrane potential. A recent publication in Experimental Neurology revealed that the pharmacological blockade of the two-pore domain potassium channel TASK1 improved clinical disease severity, the number of cellular infiltrates, as well as the degree of demyelination in experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. This effect was driven by a reduction in the generation of proinflammatory cytokines and proliferation of T lymphocytes. Thus, functional activity of T lymphocytes was identified to be TASK1 dependent suggesting that this class of potassium channels may represent an interesting target for treating T cell-driven autoimmunity directed against the central nervous system.

External Ba2+ Block of the Two-pore Domain Potassium Channel TREK-1 Defines Conformational Transition in Its Selectivity Filter

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba(2+) blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba(2+) at the assumed K(+) binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba(2+) was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K(+)-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K(+) and proton.

Electrophysiological and molecular characterization of the two‐pore‐domain Potassium channels in murine fibroblasts of renal cortex

BackgroundPotassium channels have been proposed to play a key role in the progression of organ fibrosis. Recently, it has been shown that potassium channels control crucial fibroblast functions like proliferation and migration (Grgic., 2009).Our aims in this study are to characterize the role of new classes of potassium channels in murine renal fibroblasts (TFBs) and determine the regulation of the activity of these newly characterized potassium channels by different growth factors that are important in the pathophysiology of renal fibrosis.MethodChannel characterization were determined by whole cell patch‐clamp experiments of TFBs. In addition to channel function we tested the expression with real‐time‐PCR and the localization with immunofluorescence experiments.ResultsIn murine renal fibroblasts we detected the expression of six members of the two‐pore‐domain potassium channels (K2P) family. In the quantitative RT‐PCR experiments, TREK‐1, TREK‐2 and THIK‐1 were the most dominant arachidonic‐acid (AA) sensitive K2Ps.In whole cell experiments, we tested activators or inhibitors like changes in pH and temperature, mechanical stress, volatile anaesthetics, unsaturated fatty acids, metallic substances, antiarrhythmic and antidepressive agents. Our data suggest that THIK‐1 has a significant contribute to AA‐sensitive current.Mitogenic stimulation of fibroblast with profibrotic agents like b‐FGF and TGF‐β led to a downregulation of the channel activity in more than 70 %.ConclusionAside from previously described Ca2+‐activated potassium channels renal fibroblasts also possess a distinct array of K2P‐potassium channels. These channels are also regulated in the presence of profibrotic stimuli. In the clinical context K2P‐channels should be tested as pharmacological target in renal fibrosis.

A global synergy of ions and voltage?

Membrane voltage and intracellular ion concentrations change under various cell states. Changes of both membrane potential and ion concentration can be sensed by the same elements in cell membranes. The calcium-activated potassium channels are a typical example. In hippocampal neurons, action potential waveforms are adapted to repetitive electrical inputs based on calcium-dependent repolarization. This may occur quickly or slowly. In the fast process, the duration of a single action potential becomes shorter. In the slow process, spike frequency and patterns are changed. The fast process is mainly achieved by activation of a class of Kv channel, a BK-type calcium-activated potassium channel. Its activities are regulated by both increase of intracellular calcium and depolarization. In fact, the BK channel has a voltage sensor domain and a cytoplasmic calcium-sensing domain. BK channels are often co-localized with voltage-gated calcium channels and can sense local and rapid calcium increase following opening of a single voltage-gated calcium channel during an action potential. The slower process of modification of neuronal firing depends on another class of potassium channel, the small conductance calcium-activated potassium channel (the SK channel), which binds calmodulin on the intracellular side. In contrast with the BK channel, the SK channel lacks intrinsic voltage dependence. Synergy between membrane voltage change and ions is not restricted to neurons and myocytes. In phagocytes such as the neutrophil or macrophage, reactive oxygen species are produced by the actions of NADPH oxidase. This activity induces remarkable depolarization since superoxide anions are transferred to the external space leaving protons inside. Thus both depolarization and intracellular acidification occur by the oxidase's activity. The voltage-gated proton (Hv) channel serves as an ideal player for cancelling both outcomes of the oxidase activity. The Hv channel is activated by both depolarization and intracellular acidification. Voltage dependence of steady-state open probability measured as the conductance–voltage (G–V) curve is shifted dependent on both intracellular and extracellular pH values. Recently, the molecular correlate of the Hv channel was identified. Knockout mice for the Hv channel exhibit reduced production of superoxide anions in neutrophils, consistent with the view that the Hv channel plays a role in promoting NADPH oxidase activity. More roles of the Hv channel in other cell types are also emerging. A study of knockout mice for the Hv channel led to the identification of unexpected roles in the signals of antibody production in B-lymphocytes. Another surprise was its presence and potential role in human sperm. Many ion channel species are expressed in sperm, but how ion channels contribute to sperm physiology, such as motility, has remained unknown. Patch clamping of sperm has now brought about a revolution in this field. This series of reviews covers three ion channel topics that explore the sites of synergy between voltage and ions. Jianmin Cui reviews recent findings of the structure–function relationship of the BK-type calcium-activated potassium channel (Cui, 2010). The interaction between S4, a key transmembrane segment of the voltage sensor domain, and the cytoplasmic metal ion-binding sites is one focus and the recently resolved X-ray structure of the cytoplasmic domain for calcium sensing is also discussed. Demaurex & Chemaly (2010) review the historical background of the Hv channel in leukocytes and recent molecular studies. Finally, Lishko & Kirichok (2010) review the recent discovery of the Hv channel in human sperm and its functional coupling with the CatSper channel, another class of voltage sensor containing channel that operates as an alkalization-activated calcium channel.

Tuning of the Outer Hair Cell Motor by Membrane Cholesterol

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.

In vitro synthesis, tetramerization and single channel characterization of virus-encoded potassium channel Kcv

Chlorella virus-encoded membrane protein Kcv represents a new class of potassium channel. This 94-amino acids miniature K + channel consists of two trans-membrane α-helix domains intermediated by a pore domain that contains a highly conserved K + selectivity filter. Therefore, as an archetypal K + channel, the study of Kcv may yield valuable insights into the structure–function relationships underlying this important class of ion channel. Here, we report a series of new properties of Kcv. We first verified Kcv can be synthesized in vitro. By co-synthesis and assembly of wild-type and the tagged version of Kcv, we were able to demonstrate a tetrameric stoichiometry, a molecular structure adopted by all known K + channels. Most notably, the tetrameric Kcv complex retains its functional integrity in SDS (strong detergent)-containing solutions, a useful feature that allows for direct purification of protein from polyacrylamide gel. Once purified, the tetramer can form single potassium-selective ion channels in a lipid bilayer with functions consistent to the heterologously expressed Kcv. These finding suggest that the synthetic Kcv can serve as a model of virus-encoded K + channels; and its newly identified properties can be applied to the future study on structure-determined mechanisms such as K + channel functional stoichiometry.

Focus Issue: Tuning in to the Ion Channel

In this second Focus Issue in an ongoing series of articles on the regulation and function of ion channels, Science 's STKE presents four articles that highlight various aspects of ion channel function. A Review by Faundez and Hartzell concerns the functions of intracellular chloride channels in endosomes and related organelles, concluding with an intriguing scenario whereby chloride channels in the membranes of synaptic vesicles could help determine their neurotransmitter content. A Perspective by DeCoursey on the functions of ion channels in phagocytes critically evaluates the possible contributions of proton channels and large-conductance calcium- and voltage-activated potassium channels to phagosomal destruction of microbes. Trauner and Kramer discuss various examples of metabolic regulation of potassium channels, speculating that other classes of potassium channels may be similarly subject to regulation by various small metabolites. Wolf discusses recent research implicating the TRPM7 "chanzyme" in magnesium homeostasis, research that has turned our previous concepts of the regulation of magneisum homeostasis upside down.

Potassium channels and membrane potential in the modulation of intracellular calcium in vascular endothelial cells.

The endothelium plays a vital role in the control of vascular functions, including modulation of tone; permeability and barrier properties; platelet adhesion and aggregation; and secretion of paracrine factors. Critical signaling events in many of these functions involve an increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)). This rise in [Ca(2+)](i) occurs via an interplay between several mechanisms, including release from intracellular stores, entry from the extracellular space through store depletion and second messenger-mediated processes, and the establishment of a favorable electrochemical gradient. The focus of this review centers on the role of potassium channels and membrane potential in the creation of a favorable electrochemical gradient for Ca(2+) entry. In addition, evidence is examined for the existence of various classes of potassium channels and the possible influence of regional variation in expression and experimental conditions.

Ion channels: structural bioinformatics and modelling.

Ion channels are membrane proteins of key physiological and pharmacological importance. As is the case for many integral membrane proteins, X-ray structures are known for a few bacterial channels, yet structures of human homologues are required for analysis of channel-associated diseases and for drug design. Homology modelling can be used to help remedy this deficit. In combination with molecular dynamics simulations and associated calculations, modelling provides a powerful approach to understanding structure/function relationships in human ion channels. Modelling techniques have been applied to two classes of potassium channels: voltage-gated (Kv) and inward rectifier (Kir) channels. Kir channel models, based on the structure of the bacterial channel KcsA, have been used as a starting point for detailed simulation studies that have increased our understanding of ion permeation and selectivity mechanisms. The transmembrane domain of GluR0, a bacterial homologue of mammalian glutamate receptors, also may be modelled using the KcsA structure as a template. Models of the nicotinic acetylcholine receptor may be constructed in a modular fashion. The snail acetylcholine-binding protein provides a template for the extracellular ligand-binding domain. The transmembrane pore region can be modelled on the basis of NMR structures of the pore-lining M2 helix.

Conditional repression of a calcium-activated potassium channel reveals its role in the hypercapnic ventilatory response

An increase in tidal volume is an important feature of the ventilatory response to hypercapnia. The neural substrate for tidal volume augmentation is the frequency of action potentials per inspiratory burst. Small-conductance, calcium-activated potassium channels, which are gated by the increases in intracellular calcium during activation, contribute to the interval between action potentials by generating an afterhyperpolarization current. The generation of mice in which one member of this class of potassium channels (SK3) can be regulated by dietary doxycycline (dox) [1] has enabled its role in the ventilatory response to carbon dioxide to be examined. Experiments were carried out in awake mice at 2 weeks of age. A control period in 100% oxygen was followed by 5 min in 5% CO2, 95% O2. Animals targeted for repression of SK3 (SK3 T/T) on dox showed a greater increase in tidal volume than either SK3 T/T not exposed to dox or wild type. There was no difference in the increase in respiratory rate between the three groups of animals. These results show that SK3 is an important regulator of action potential frequency during CO2 stimulation. They also suggest that medullary neurons which contribute to respiratory pattern may be characterized by SK3 channels while those responsible for rhythm may not.

Exciting times for PIP2.

One class of potassium channels, the KATP channels, are key players in a variety of functions-insulin secretion, blood vessel tone, and the response of the heart to lack of oxygen. Ashcroft describes in her Perspective how new results in this issue explain a long-standing puzzle about the true sensitivity of these channels to ATP. Baukrowitz et al. and Shyng et al. show that the phospholipid PIPregulates the ATP sensitivity of these channels.

Structure, chromosome localization, and tissue distribution of the mouse twik K + channel gene

We have recently discovered a new class of potassium channels with two pore-forming domains and four membrane-spanning domains. When heterologously expressed, these channels produce time- and voltage-independent currents that classify them as background or leak channels. TWIK (for t andem of P domains in a w eak i nwardly rectifying K + channel) was the first member of this family to be cloned. Here, we describe the genomic organization of TWIK in the mouse. The coding sequence as well as the untranslated sequences are contained in three exons. The twik gene (or KCNK1) has been mapped to chromosome 8, consistent with its localization to 1q42–43 in human. The twik gene is expressed in virtually all mouse tissues. It is most abundantly expressed in brain and moderately in other organs such as kidney. The level of expression is increased in brain and kidney from neonate to adult animals, but the TWIK message is also detected during embryogenesis, as early as day 7 post conception.

Chromosomal localization of 15 ion channel genes.

Several human Mendelian diseases, including the long-QT syndrome, malignant hyperthermia, and episodic ataxia/myokymia syndrome, have recently been demonstrated to be due to mutations in ion channel genes. Systematic mapping of ion channel genes may therefore reveal candidates for other heritable disorders. In this study, the GenBank and dbEST databases were used to identify members of several ion channel families (voltage-gated calcium and sodium, cardiac chloride, and all classes of potassium channels). Genes and ESTs without prior map localization were identified based on GDB and OWL database information and 15 genes and ESTs were selected for mapping. Of these 15, only the serotonin receptor 5HT3R had been previously mapped to a chromosome. A somatic cell hybrid panel (SCH) was screened with an STS from each gene and, if necessary the results verified by a second SCH panel. For three ESTs, rodent derived PCR products of the same size as the human STS precluded SCH mapping. For these three, human P1 clones were isolated and the genomic location was determined by metaphase FISH. These genes and ESTs can now be further evaluated as candidate genes for inherited cardiac, neuromuscular and psychiatric disorders mapped to these chromosomes. Furthermore, the ESTs developed in this study can be used to isolate genomic clones, enabling the determination of each transcript's genomic structure and physical map location. This approach may also be applicable to other gene families and may aid in the identification of candidate genes for groups of related heritable disorders.

New class of potassium channels

New class of potassium channels

Pattern of potassium channel expression in proliferating B lymphocytes depends upon the mode of activation.

Ionic channel expression is highly regulated during mitogenesis. But it is not clear whether these regulations only follow intrinsic programs during the course of the cell cycle or if they also depend upon the external factors used to promote cell activation. B lymphocytes express two classes of potassium channels and can be stimulated to enter the cell cycle by distinct pathways. Thus, we have analyzed, with the patch-clamp technique, if the expression of channels varies when the cells are activated by different signals that lead to cell proliferation. We found that stimulation through Ag receptors increases the expression of calcium- and voltage-activated potassium channels, whereas a bacterial mitogen, LPS, only enhances the expression of the latter. Moreover, channel expression can still be modified in proliferating cells because stimulation of LPS-activated cells through Ag receptors induces rapid expression of calcium-activated channels. The use of inhibitors of mRNA synthesis revealed that this process depends upon gene transcription. Thus, differential induction of the expression of potassium channels is not only linked to the entry into the cell cycle but depends also on pathways of stimulation.

Single potassium channel currents activated by extracellular ATP in developing chick skeletal muscle: a role for second messengers

1. In developing chick skeletal muscle, extracellular ATP activates an early excitatory current and a delayed potassium current. Previous work had shown that the potassium current elicited by ATP is sensitive to temperature and activates with a delay of nearly 1 s, suggesting that a second messenger is involved. The existence of a second messenger was confirmed by the observation that single potassium channels were activated in cell-attached patches, when ATP was applied outside of the patch pipette. 2. Two classes of ATP-activated potassium-channel currents were observed in cell-attached patches: one had a slope conductance of 23 pS, whereas the other had a slope conductance of 51 pS. 3. Pharmacological manipulations suggested that activation of the whole-cell potassium current by ATP did not require cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), inositol 1,4,5-triphosphate (IP3), nitric oxide, or a rise in internal free calcium. Additional pharmacological experiments suggested that activation of the whole-cell potassium current might not require activation of a G protein and probably did not involve intracellular protein phosphorylation. 4. The ability of arachidonic acid and its metabolites to activate potassium channels in chick skeletal muscle was also tested. Arachidonic acid, several prostaglandins and several leukotrienes activated whole-cell potassium currents. However, results with several inhibitors suggested that arachidonic acid and its metabolites are not necessary for activation of the whole-cell potassium current by ATP. 5. In excised outside-out membrane patches, ATP activated a single class of potassium channels. The slope conductance of these channels indicated that they are likely to be identical to the smaller of the two classes of second messenger activated potassium channels observed in cell-attached patches. 6. The observation that the larger class of potassium channels observed in cell-attached patches was absent in excised patches suggests that activation of these channels by ATP requires a cytosolic factor that is easily dialyzed away. In contrast, the observation that the smaller class of potassium channels could still be activated by ATP in excised patches suggests that the two classes of potassium channels are activated by different mechanisms. These results also indicate that all the molecules involved in coupling ATP receptor activation to opening of the smaller class of potassium channels remain closely associated with an excised patch. One possible explanation is that there might be an intramembranous second messenger.

Three classes of potassium channels in large monopolar cells of the blowfly Calliphora vicina

We have used single electrode voltage clamp in the intact animal and whole-cell recording from dissociated cell bodies to investigate the properties of potassium conductances in large monopolar cells (LMCs) of the first optic ganglion of the blowfly Calliphora vicina. Two classes of voltage gated potassium conductances were found: a delayed rectifier current (Kd) with slow inactivation (τinac = 1–3 sec), and an A current (Ka) showing both faster inactivation (τinac = 21 ms) and also more rapid activation. The reversal potential of both currents is ca. -90 mV with 2 mM [Ko] and 140 mM [Ki], and follows the Nernst slope with increasing [Ko]. The voltage operating range of Ka is unusually negative, with the mid point of the steady-state inactivation curve (V50) at- 101 mV. V50 for Kd is - 84 mV. Although no inward currents were detected, for technical reasons their presence cannot be excluded. In inside-out patches from LMC soma membranes the single channels underlying the currents both have a conductance of ca. 20 pS in symmetrical 140 mM K solutions and channel densities may be as high as 10/μm2. Less frequently, inside-out patches contained a large conductance (110 pS) calcium-activated potassium channel which existed almost exclusively in a rapidly flickering mode. Open probability increased with depolarization and Ca concentrations greater than 40 nM. In whole-cell recordings, dissociated LMC cell bodies fall into two classes with respect to their voltage sensitive currents: 37 % of cells only showed Kd; the remainder (63%) were dominated by Ka with a variable (0–30%) contribution from Kd. In the intact animal, intracellular recordings from LMCs, combined with dye-marking, indicate that cells expressing only Kd are type L3 cells, whilst L1 and L2 express predominantly Ka. Since L1 and L2 have resting potentials of ca. - 40 mV and maximum hyperpolarizations reaching -90 mV only transiently, inactivation of Ka is unlikely to be removed under most physiological conditions. In contrast, L3 cells have a more negative resting potential (−60 mV) and Kd should play a significant role in signal-shaping, in particular contributing to the falling phase of a prominent spike-like transient in response to dimming.